In our research we investigate molecular microbial metabolism and its linkage to ecological and evolutionary processes. We explore the distinguishing features of novel microbial metabolism and how molecular and biochemical differences in metabolism shape microbial fitness. We study novel microbial metabolism with relevance to bioremediation, bioenergy, and intestinal microbiology.

1) Microbial Electrosynthesis and Electron Transport between Microbes and Surfaces

Some microbes have the the capacity to either derive metabolic electrons from redox-active mineral surfaces or transfer such electrons to these surfaces. These processes are of great relevance to geochemical, environmental, but also bioenergy processes. We are investigating the molecular bases of such novel electron transfer to uncover the enzymes and pathways for electron uptake. More recently, we began to explore microbial electrosynthesis as a novel means to produce CO2-neutral biofuels and commodity chemicals.

2) Microbial Reductive Dehalogenation

Chloroethenes, such as PCE and TCE, are the most prevalent groundwater contaminants in the U.S. and the developed countries. Large scale remediation of contaminated aquifers relies largely on the activity of a group of unusual microbes (Dehalococcoides) that derive energy from reductive dehalogenation. We study reductive dehalogenases and the strictly anaerobic bacteria, such as Dehalococcoides mccartyj and Shewanella, on a biochemical, physiological, genomic, and population level to better understand the unprecedented biochemistry of the coenzyme B 12-containing reductive dehalogenases. We also use this information to improve chlorethene bioremediation. Population-level studies in our lab have been revealing speciation and niche adaptation in Dehalococcoides mccartyj. in response to subtle changes in physical-chemical environments.

3) Biofilms and the emergence of antibiotic tolerance and antibiotic resistance

For the last decade, we have been investigating the mechanism of biofilm formation in medically important microorganisms, including Vibrio chloreae, Pseudomonas aeruginosa, Francisella tularensis, and Shewanella oneidensis. We discovered that the stability of biofilms requires cellular energy, and that extracellular matrix material may have a supportive role. In more recent studies, we developed the first system to examine the pharmacokinetics and pharmacodynamics of Pseudomonas aeruginosa biofilms. We investigate the effect of human simulated concentrations of meropenem and tobramycin, administered singly, and in combination, on biofilms of P. aeruginosa PAO1 and clinical isolates from patients with CF, as well as the effect of human simulated concentrations of meropenem and tobramycin, administered singly, and in combination, on biofilms of P. aeruginosa PAO1 and clinical CF isolates.

4) Microbial Metabolic Processes in the Large Intestine

Irritable Bowel Syndrome (IBS) is a chronic, episodic gastrointestinal disorder that is characterized by abdominal pain and altered bowel habits. IBS prevalence is estimated to be 10-15% in Western countries comprising 25 to 50 percent of all referrals to gastroenterologists. The gastrointestinal tract harbors a complex and diverse microbial community, which plays important roles in host nutrition, immune function, health and disease, and it is hypothesized the IBS disease phenotype is associated with a change in colonic microbiota and/or host factors such as mucosal function and immunity. With our physician collaborator, we study the metabolic processes in the intestinal microbial community, and how cellular metabolism is controlled by the host mucosa.

Academic Appointments

Honors & Awards

  • Elected Fellow, American Academy of Microbiology (2013)
  • Otto Moensted Visiting Professor, Danish Technical University, Lyngby, DK (2003)
  • Research Award, Charles Lee Powell Foundation (2000)
  • CAREER Award, National Science Foundation (1998)
  • Visiting Professor, Department of Biochemistry and Biological Process Institute, University of Minnesota (1997)
  • Terman Fellowship Award, Stanford University (1995)
  • Postdoctoral Fellowship, Deutsche Forschungsgemeinschaft (1990)
  • Planetary Biology Internship Fellowship, NASA Life Sciences (Marine Biological Laboratory) (1986)

Professional Education

  • Dr. rer. nat., Philipps-University, Marburg (1989)

Current Research and Scholarly Interests

Metabolism of anaerobic microbes in diseases, bioenergy, and bioremediation

2019-20 Courses

Stanford Advisees

Graduate and Fellowship Programs

  • Biology (School of Humanities and Sciences) (Phd Program)

All Publications

  • Quasi-2D Pd/Pt nanoclams for CO2 reduction in tandem with microbial communities Wong, A., Kracke, F., Antoniuk-Pablant, A., Hahn, C., Spormann, A., Jaramillo, T. AMER CHEMICAL SOC. 2018
  • Methanococcus maripaludis Employs Three Functional Heterodisulfide Reductase Complexes for Flavin-Based Electron Bifurcation Using Hydrogen and Formate. Biochemistry Milton, R. D., Ruth, J. C., Deutzmann, J. S., Spormann, A. M. 2018


    Hydrogenotrophic methanogens oxidize molecular hydrogen to reduce carbon dioxide to methane. In methanogens without cytochromes, the initial endergonic reduction of CO2 to formylmethanofuran with H2-derived electrons is coupled to the exergonic reduction of a heterodisulfide of coenzymes B and M by flavin-based electron bifurcation (FBEB). In Methanococcus maripaludis, FBEB is performed by a heterodisulfide reductase (Hdr) enzyme complex that involves hydrogenase (Vhu), although formate dehydrogenase (Fdh) has been proposed as an alternative to Vhu. We have identified and purified three Hdr complexes of M. maripaludis, where homodimeric Hdr complexes containing (Vhu)2 or (Fdh)2 were found, in addition to a heterocomplex that contains both Vhu and Fdh. Formate was found in in vitro assays using the purified Hdr complex to act directly as the electron donor for FBEB via the associated Fdh. Furthermore, while ferredoxin was slowly reduced to 30% [-360 mV vs the standard hydrogen electrode (SHE)] by H2 and formate (0.8 atm and 30 mM, according to thermodynamics), the addition of CoB-S-S-CoM as the high-potential electron acceptor ( E°' = -140 mV vs SHE; to induce FBEB) resulted in the rapid and more complete reduction of Fd to 94% (-455 mV vs SHE).

    View details for PubMedID 30010323

  • Homoacetogenesis in Deep-Sea Chloroflexi, as Inferred by Single-Cell Genomics, Provides a Link to Reductive Dehalogenation in Terrestrial Dehalococcoidetes (vol 8, e02022-17, 2017) MBIO Sewell, H. L., Kaster, A., Spormann, A. M. 2018; 9 (2)

    View details for PubMedID 29511078

  • Mediator-free enzymatic electrosynthesis of formate by the Methanococcus maripaludis heterodisulfide reductase supercomplex. Bioresource technology Lienemann, M., Deutzmann, J. S., Milton, R. D., Sahin, M., Spormann, A. M. 2018; 254: 278–83


    Electrosynthesis of formate is a promising technology to convert CO2and electricity from renewable sources into a biocompatible, soluble, non-flammable, and easily storable compound. In the model methanogen Methanococcus maripaludis, uptake of cathodic electrons was shown to proceed indirectly via formation of formate or H2by undefined, cell-derived enzymes. Here, we identified that the multi-enzyme heterodisulfide reductase supercomplex (Hdr-SC) of M. maripaludis is capable of direct electron uptake and catalyzes rapid H2and formate formation in electrochemical reactors (-800 mV vs Ag/AgCl) and in Fe(0) corrosion assays. In Fe(0) corrosion assays and electrochemical reactors, purified Hdr-SC primarily catalyzed CO2reduction to formate with a coulombic efficiency of 90% in the electrochemical cells for 5 days. Thus, this report identified the first enzyme that stably catalyzes the mediator-free electrochemical reduction of CO2to formate, which can serve as the basis of an enzyme electrode for sustained electrochemical production of formate.

    View details for PubMedID 29413934

  • Determination of Tobramycin in M-9 Medium by LC-MS/MS: Signal Enhancement by Trichloroacetic Acid JOURNAL OF ANALYTICAL METHODS IN CHEMISTRY Huang, L., Haagensen, J., Verotta, D., Cheah, V., Spormann, A. M., Aweeka, F., Yang, K. 2018: 7965124


    It is well known that ion-pairing reagents cause ion suppression in LC-MS/MS methods. Here, we report that trichloroacetic acid increases the MS signal of tobramycin. To support studies of an in vitro pharmacokinetic/pharmacodynamic simulator for bacterial biofilms, an LC-MS/MS method for determination of tobramycin in M9 media was developed. Aliquots of 25 μL M9 media samples were mixed with the internal standard (IS) tobramycin-d5 (5 µg/mL, 25 µL) and 200 µL 2.5% trichloroacetic acid. The mixture (5 µL) was directly injected onto a PFP column (2.0 × 50 mm, 3 µm) eluted with water containing 20 mM ammonium formate and 0.14% trifluoroacetic acid and acetonitrile containing 0.1% trifluoroacetic acid in a gradient mode. ESI+ and MRM with ion m/z 468 → 324 for tobramycin and m/z 473 → 327 for the IS were used for quantification. The calibration curve concentration range was 50-25000 ng/mL. Matrix effect from M9 media was not significant when compared with injection solvents, but signal enhancement by trichloroacetic acid was significant (∼3 fold). The method is simple, fast, and reliable. Using the method, the in vitro PK/PD model was tested with one bolus dose of tobramycin.

    View details for PubMedID 29854560

    View details for PubMedCentralID PMC5944200

  • Enhanced microbial electrosynthesis by using defined co-cultures ISME JOURNAL Deutzmann, J. S., Spormann, A. M. 2017; 11 (3): 704-714


    Microbial uptake of free cathodic electrons presents a poorly understood aspect of microbial physiology. Uptake of cathodic electrons is particularly important in microbial electrosynthesis of sustainable fuel and chemical precursors using only CO2 and electricity as carbon, electron and energy source. Typically, large overpotentials (200 to 400 mV) were reported to be required for cathodic electron uptake during electrosynthesis of, for example, methane and acetate, or low electrosynthesis rates were observed. To address these limitations and to explore conceptual alternatives, we studied defined co-cultures metabolizing cathodic electrons. The Fe(0)-corroding strain IS4 was used to catalyze the electron uptake reaction from the cathode forming molecular hydrogen as intermediate, and Methanococcus maripaludis and Acetobacterium woodii were used as model microorganisms for hydrogenotrophic synthesis of methane and acetate, respectively. The IS4-M. maripaludis co-cultures achieved electromethanogenesis rates of 0.1-0.14 μmol cm(-2) h(-1) at -400 mV vs standard hydrogen electrode and 0.6-0.9 μmol cm(-2) h(-1) at -500 mV. Co-cultures of strain IS4 and A. woodii formed acetate at rates of 0.21-0.23 μmol cm(-2) h(-1) at -400 mV and 0.57-0.74 μmol cm(-2) h(-1) at -500 mV. These data show that defined co-cultures coupling cathodic electron uptake with synthesis reactions via interspecies hydrogen transfer may lay the foundation for an engineering strategy for microbial electrosynthesis.The ISME Journal advance online publication, 1 November 2016; doi:10.1038/ismej.2016.149.

    View details for DOI 10.1038/ismej.2016.149

    View details for Web of Science ID 000394542000010

  • Survival of Vinyl Chloride Respiring Dehalococcoides mccartyi under Long-Term Electron Donor Limitation ENVIRONMENTAL SCIENCE & TECHNOLOGY Mayer-Blackwell, K., Azizian, M. F., Green, J. K., Spormann, A. M., Semprini, L. 2017; 51 (3): 1635-1642


    In anoxic groundwater aquifers, the long-term survival of Dehalococcoides mccartyi populations expressing the gene vcrA (or bvcA) encoding reductive vinyl chloride dehalogenases are important to achieve complete dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE) to nonchlorinated ethene. The absence or inactivity of vcrA-containing Dehalococcoides results in the accumulation of the harmful chlorinated intermediates dichloroethene (DCE) and vinyl chloride (VC). Although vcrA-containing Dehalococcoides subpopulations depend on synergistic interaction with other organohalide-respiring populations generating their metabolic electron acceptors (DCE and VC), their survival requires successful competition for electron donor within the entire organohalide-respiring microbial community. To understand this dualism of synergy and competition under growth conditions relevant in contaminated aquifers, we investigated Dehalococcoides-level population structure when subjected to a change in the ratio of electron donor to chlorinated electron acceptor in continuously stirred tank reactors (CSTRs) operated over 7 years. When the electron donor formate was supplied in stoichiometric excess to TCE, both tceA-containing and vcrA-containing Dehalococcoides populations persisted, and near-complete dechlorination to ethene was stably maintained. When the electron donor formate was supplied at substoichiometric concentrations, the interactions between tceA-containing and vcrA-containing populations shifted toward direct competition for the same limiting catabolic electron donor substrate with subsequent niche exclusion of the vcrA-containing population. After more than 2000 days of operation under electron donor limitation, increasing the electron donor to TCE ratio facilitated a recovery of the vcrA-containing Dehalococoides population to its original frequency. We demonstrate that electron donor scarcity alone, in the absence of competing metabolic processes or inhibitory dechlorination intermediate products, is sufficient to alter the Dehalococcoides population structure. These results underscore the importance of electron donor and chloroethene stoichiometry in maintaining balanced functional performance within consortia composed of multiple D. mccartyi subpopulations, even when other competing electron acceptor processes are absent.

    View details for DOI 10.1021/acs.est.6b05050

    View details for Web of Science ID 000393738700072

    View details for PubMedID 28002948

  • Mathematical Modeling of Biofilm Structures Using COMSTAT Data. Computational and mathematical methods in medicine Verotta, D., Haagensen, J., Spormann, A. M., Yang, K. 2017; 2017: 7246286


    Mathematical modeling holds great potential for quantitatively describing biofilm growth in presence or absence of chemical agents used to limit or promote biofilm growth. In this paper, we describe a general mathematical/statistical framework that allows for the characterization of complex data in terms of few parameters and the capability to (i) compare different experiments and exposures to different agents, (ii) test different hypotheses regarding biofilm growth and interaction with different agents, and (iii) simulate arbitrary administrations of agents. The mathematical framework is divided to submodels characterizing biofilm, including new models characterizing live biofilm growth and dead cell accumulation; the interaction with agents inhibiting or stimulating growth; the kinetics of the agents. The statistical framework can take into account measurement and interexperiment variation. We demonstrate the application of (some of) the models using confocal microscopy data obtained using the computer program COMSTAT.

    View details for PubMedID 29422943

    View details for PubMedCentralID PMC5751404

  • Biochemistry of Catabolic Reductive Dehalogenation. Annual review of biochemistry Fincker, M., Spormann, A. M. 2017; 86: 357–86


    A wide range of phylogenetically diverse microorganisms couple the reductive dehalogenation of organohalides to energy conservation. Key enzymes of such anaerobic catabolic pathways are corrinoid and Fe-S cluster-containing, membrane-associated reductive dehalogenases. These enzymes catalyze the reductive elimination of a halide and constitute the terminal reductases of a short electron transfer chain. Enzymatic and physiological studies revealed the existence of quinone-dependent and quinone-independent reductive dehalogenases that are distinguishable at the amino acid sequence level, implying different modes of energy conservation in the respective microorganisms. In this review, we summarize current knowledge about catabolic reductive dehalogenases and the electron transfer chain they are part of. We review reaction mechanisms and the role of the corrinoid and Fe-S cluster cofactors and discuss physiological implications.

    View details for PubMedID 28654328

  • Spatiotemporal pharmacodynamics of meropenem- and tobramycin-treated Pseudomonas aeruginosa biofilms. The Journal of antimicrobial chemotherapy Haagensen, J., Verotta, D., Huang, L., Engel, J., Spormann, A. M., Yang, K. 2017; 72 (12): 3357–65


    The selection and dose of antibiotic therapy for biofilm-related infections are based on traditional pharmacokinetic studies using planktonic bacteria. The objective of this study was to characterize the time course and spatial activity of human exposure levels of meropenem and tobramycin against Pseudomonas aeruginosa biofilms grown in an in vitro flow-chamber model.Pharmacokinetic profiles of meropenem and tobramycin used in human therapy were administered to GFP-labelled P. aeruginosa PAO1 grown in flow chambers for 24 or 72 h. Images were acquired using confocal laser scanning microscopy throughout antibiotic treatment. Bacterial biomass was measured using COMSTAT and pharmacokinetic/pharmacodynamic models were fitted using NONMEM7.Meropenem treatment resulted in more rapid and sustained killing of both the 24 and 72 h PAO1 biofilm compared with tobramycin. Biofilm regrowth after antibiotic treatment occurred fastest with tobramycin. Meropenem preferentially killed subpopulations within the mushroom cap of the biofilms, regardless of biofilm maturity. The spatial killing by tobramycin varied with biofilm maturity. A tobramycin-treated 24 h biofilm resulted in live and dead cells detaching from the biofilm, while treatment of a 72 h biofilm preferentially killed subpopulations on the periphery of the mushroom stalk. Regrowth occurred primarily on the mushroom caps. Combination meropenem and tobramycin therapy resulted in rapid and efficient killing of biofilm cells, with a spatial pattern similar to meropenem alone.Simulated human concentrations of meropenem and tobramycin in young and mature PAO1 biofilms exhibited differences in temporal and spatial patterns of killing and antibiotic tolerance development.

    View details for PubMedID 28961810

  • Homoacetogenesis in Deep-SeaChloroflexi, as Inferred by Single-Cell Genomics, Provides a Link to Reductive Dehalogenation in TerrestrialDehalococcoidetes. mBio Sewell, H. L., Kaster, A. K., Spormann, A. M. 2017; 8 (6)


    The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylumChloroflexiIn this report, we investigated genomes of single cells obtained from deep-sea sediments of the Peruvian Margin, which are enriched in suchChloroflexi16S rRNA gene sequence analysis placed two of these single-cell-derived genomes (DscP3 and Dsc4) in a clade of subphylum IChloroflexiwhich were previously recovered from deep-sea sediment in the Okinawa Trough and a third (DscP2-2) as a member of the previously reported DscP2 population from Peruvian Margin site 1230. The presence of genes encoding enzymes of a complete Wood-Ljungdahl pathway, glycolysis/gluconeogenesis, aRhodobacternitrogen fixation (Rnf) complex, glyosyltransferases, and formate dehydrogenases in the single-cell genomes of DscP3 and Dsc4 and the presence of an NADH-dependent reduced ferredoxin:NADP oxidoreductase (Nfn) and Rnf in the genome of DscP2-2 imply a homoacetogenic lifestyle of these abundant marineChloroflexiWe also report here the first complete pathway for anaerobic benzoate oxidation to acetyl coenzyme A (CoA) in the phylumChloroflexi(DscP3 and Dsc4), including a class I benzoyl-CoA reductase. Of remarkable evolutionary significance, we discovered a gene encoding a formate dehydrogenase (FdnI) with reciprocal closest identity to the formate dehydrogenase-like protein (complex iron-sulfur molybdoenzyme [CISM], DET0187) of terrestrialDehalococcoides/Dehalogenimonasspp. This formate dehydrogenase-like protein has been shown to lack formate dehydrogenase activity inDehalococcoides/Dehalogenimonasspp. and is instead hypothesized to couple HupL hydrogenase to a reductive dehalogenase in the catabolic reductive dehalogenation pathway. This finding of a close functional homologue provides an important missing link for understanding the origin and the metabolic core of terrestrialDehalococcoides/Dehalogenimonasspp. and of reductive dehalogenation, as well as the biology of abundant deep-seaChloroflexiIMPORTANCEThe deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylumChloroflexiIn this report, we investigated genomes of single cells obtained from deep-sea sediments and provide evidence for a homacetogenic lifestyle of these abundant marineChloroflexiMoreover, genome signature and key metabolic genes indicate an evolutionary relationship between these deep-sea sediment microbes and terrestrial, reductively dehalogenatingDehalococcoides.

    View details for PubMedID 29259088

    View details for PubMedCentralID PMC5736913

  • 1,2-Dichloroethane Exposure Alters the Population Structure, Metabolism, and Kinetics of a Trichloroethene-Dechlorinating Dehalococcoides mccartyi Consortium. Environmental science & technology Mayer-Blackwell, K., Fincker, M., Molenda, O., Callahan, B., Sewell, H., Holmes, S., Edwards, E. A., Spormann, A. M. 2016: -?


    Bioremediation of groundwater contaminated with chlorinated aliphatic hydrocarbons such as perchloroethene and trichloroethene can result in the accumulation of the undesirable intermediate vinyl chloride. Such accumulation can either be due to the absence of specific vinyl chloride respiring Dehalococcoides mccartyi or to the inhibition of such strains by the metabolism of other microorganisms. The fitness of vinyl chloride respiring Dehalococcoides mccartyi subpopulations is particularly uncertain in the presence of chloroethene/chloroethane cocontaminant mixtures, which are commonly found in contaminated groundwater. Therefore, we investigated the structure of Dehalococcoides populations in a continuously fed reactor system under changing chloroethene/ethane influent conditions. We observed that increasing the influent ratio of 1,2-dichloroethane to trichloroethene was associated with ecological selection of a tceA-containing Dehalococcoides population relative to a vcrA-containing Dehalococcoides population. Although both vinyl chloride and 1,2-dichloroethane could be simultaneously transformed to ethene, prolonged exposure to 1,2-dichloroethane diminished the vinyl chloride transforming capacity of the culture. Kinetic tests revealed that dechlorination of 1,2-dichloroethane by the consortium was strongly inhibited by cis-dichloroethene but not vinyl chloride. Native polyacrylamide gel electrophoresis and mass spectrometry revealed that a trichloroethene reductive dehalogenase (TceA) homologue was the most consistently expressed of four detectable reductive dehalogenases during 1,2-dichloroethane exposure, suggesting that it catalyzes the reductive dihaloelimination of 1,2-dichloroethane to ethene.

    View details for PubMedID 27809491

  • Enhanced microbial electrosynthesis by using defined co-cultures. ISME journal Deutzmann, J. S., Spormann, A. M. 2016


    Microbial uptake of free cathodic electrons presents a poorly understood aspect of microbial physiology. Uptake of cathodic electrons is particularly important in microbial electrosynthesis of sustainable fuel and chemical precursors using only CO2 and electricity as carbon, electron and energy source. Typically, large overpotentials (200 to 400 mV) were reported to be required for cathodic electron uptake during electrosynthesis of, for example, methane and acetate, or low electrosynthesis rates were observed. To address these limitations and to explore conceptual alternatives, we studied defined co-cultures metabolizing cathodic electrons. The Fe(0)-corroding strain IS4 was used to catalyze the electron uptake reaction from the cathode forming molecular hydrogen as intermediate, and Methanococcus maripaludis and Acetobacterium woodii were used as model microorganisms for hydrogenotrophic synthesis of methane and acetate, respectively. The IS4-M. maripaludis co-cultures achieved electromethanogenesis rates of 0.1-0.14 μmol cm(-2) h(-1) at -400 mV vs standard hydrogen electrode and 0.6-0.9 μmol cm(-2) h(-1) at -500 mV. Co-cultures of strain IS4 and A. woodii formed acetate at rates of 0.21-0.23 μmol cm(-2) h(-1) at -400 mV and 0.57-0.74 μmol cm(-2) h(-1) at -500 mV. These data show that defined co-cultures coupling cathodic electron uptake with synthesis reactions via interspecies hydrogen transfer may lay the foundation for an engineering strategy for microbial electrosynthesis.The ISME Journal advance online publication, 1 November 2016; doi:10.1038/ismej.2016.149.

    View details for DOI 10.1038/ismej.2016.149

    View details for PubMedID 27801903

  • Effect of biofilm coatings at metal-oxide/water interfaces II: Competitive sorption between Pb(II) and Zn(II) at Shewanella oneidensis/metal-oxide/water interfaces GEOCHIMICA ET COSMOCHIMICA ACTA Wang, Y., Gelabert, A., Michel, F. M., Choi, Y., Eng, P. J., Spormann, A. M., Brown, G. E. 2016; 188: 393-406
  • Pf4 bacteriophage produced by Pseudomonas aeruginosa inhibits Aspergillus fumigatus metabolism via iron sequestration MICROBIOLOGY-SGM Penner, J. C., Ferreira, J. A., Secor, P. R., Sweere, J. M., Birukova, M. K., Joubert, L., Haagensen, J. A., Garcia, O., Malkovskiy, A. V., Kaber, G., Nazik, H., Manasherob, R., Spormann, A. M., Clemons, K. V., Stevens, D. A., Bollyky, P. L. 2016; 162 (9): 1583-1594


    Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) are major human pathogens known to interact in a variety of disease settings, including airway infections in cystic fibrosis. We recently reported that clinical CF isolates of Pa inhibit the formation and growth of Af biofilms. Here, we report that the bacteriophage Pf4, produced by Pa, can inhibit the metabolic activity of Af biofilms. This phage-mediated inhibition was dose dependent, ablated by phage denaturation, and was more pronounced against preformed Af biofilm rather than biofilm formation. In contrast, planktonic conidial growth was unaffected. Two other phages, Pf1 and fd, did not inhibit Af, nor did supernatant from a Pa strain incapable of producing Pf4. Pf4, but not Pf1, attaches to Af hyphae in an avid and prolonged manner, suggesting that Pf4-mediated inhibition of Af may occur at the biofilm surface. We show that Pf4 binds iron, thus denying Af a crucial resource. Consistent with this, the inhibition of Af metabolism by Pf4 could be overcome with supplemental ferric iron, with preformed biofilm more resistant to reversal. To our knowledge, this is the first report of a bacterium producing a phage that inhibits the growth of a fungus and the first description of a phage behaving as an iron chelator in a biological system.

    View details for DOI 10.1099/mic.0.000344

    View details for Web of Science ID 000385273100008

    View details for PubMedID 27473221

  • Effect of biofilm coatings at metal-oxide/water interfaces I: Pb(II) and Zn(II) partitioning and speciation at Shewanella oneidensis/metal-oxide/water interfaces GEOCHIMICA ET COSMOCHIMICA ACTA Wang, Y., Gelabert, A., Michel, F. M., Choi, Y., Gescher, J., Ona-Nguema, G., Eng, P. J., Bargar, J. R., Farges, F., Spormann, A. M., Brown, G. E. 2016; 188: 368-392
  • Effects of Iron Chelators on the Formation and Development of Aspergillus fumigatus Biofilm ANTIMICROBIAL AGENTS AND CHEMOTHERAPY Nazik, H., Penner, J. C., Ferreira, J. A., Haagensen, J. A., Cohen, K., Spormann, A. M., Martinez, M., Chen, V., Hsu, J. L., Clemons, K. V., Stevens, D. A. 2015; 59 (10): 6514-6520


    Iron acquisition is crucial for the growth of Aspergillus fumigatus. A. fumigatus biofilm formation occurs in vitro and in vivo and is associated with physiological changes. In this study, we assessed the effects of Fe chelators on biofilm formation and development. Deferiprone (DFP), deferasirox (DFS), and deferoxamine (DFM) were tested for MIC against a reference isolate via a broth macrodilution method. The metabolic effects (assessed by XTT [2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt]) on biofilm formation by conidia were studied upon exposure to DFP, DFM, DFP plus FeCl3, or FeCl3 alone. A preformed biofilm was exposed to DFP with or without FeCl3. The DFP and DFS MIC50 against planktonic A. fumigatus was 1,250 μM, and XTT gave the same result. DFM showed no planktonic inhibition at concentrations of ≤2,500 μM. By XTT testing, DFM concentrations of <1,250 μM had no effect, whereas DFP at 2,500 μM increased biofilms forming in A. fumigatus or preformed biofilms (P < 0.01). DFP at 156 to 2,500 μM inhibited biofilm formation (P < 0.01 to 0.001) in a dose-responsive manner. Biofilm formation with 625 μM DFP plus any concentration of FeCl3 was lower than that in the controls (P < 0.05 to 0.001). FeCl3 at ≥625 μM reversed the DFP inhibitory effect (P < 0.05 to 0.01), but the reversal was incomplete compared to the controls (P < 0.05 to 0.01). For preformed biofilms, DFP in the range of ≥625 to 1,250 μM was inhibitory compared to the controls (P < 0.01 to 0.001). FeCl3 at ≥625 μM overcame inhibition by 625 μM DFP (P < 0.001). FeCl3 alone at ≥156 μM stimulated biofilm formation (P < 0.05 to 0.001). Preformed A. fumigatus biofilm increased with 2,500 μM FeCl3 only (P < 0.05). In a strain survey, various susceptibilities of biofilms of A. fumigatus clinical isolates to DFP were noted. In conclusion, iron stimulates biofilm formation and preformed biofilms. Chelators can inhibit or enhance biofilms. Chelation may be a potential therapy for A. fumigatus, but we show here that chelators must be chosen carefully. Individual isolate susceptibility assessments may be needed.

    View details for DOI 10.1128/AAC.01684-15

    View details for Web of Science ID 000367591800078

    View details for PubMedCentralID PMC4576070

  • Inhibition of Aspergillus fumigatus and Its Biofilm by Pseudomonas aeruginosa Is Dependent on the Source, Phenotype and Growth Conditions of the Bacterium PLOS ONE Ferreira, J. A., Penner, J. C., Moss, R. B., Haagensen, J. A., Clemons, K. V., Spormann, A. M., Nazik, H., Cohen, K., Banaei, N., Carolino, E., Stevens, D. A. 2015; 10 (8)

    View details for DOI 10.1371/journal.pone.0134692

    View details for Web of Science ID 000359121100068

    View details for PubMedID 26252384

  • New In Vitro Model To Study the Effect of Human Simulated Antibiotic Concentrations on Bacterial Biofilms. Antimicrobial agents and chemotherapy Haagensen, J. A., Verotta, D., Huang, L., Spormann, A., Yang, K. 2015; 59 (7): 4074-4081


    A new in vitro pharmacokinetic/pharmacodynamic simulator for bacterial biofilms utilizing flow cell technology and confocal laser scanning microscopy is described. The device has the ability to simulate the changing antibiotic concentrations in humans associated with intravenous dosing on bacterial biofilms grown under continuous culture conditions. The free drug concentrations of a single 2-g meropenem intravenous bolus dose and first-order elimination utilizing a half-life of 0.895 h (elimination rate constant, 0.776 h(-1)) were simulated. The antibacterial activity of meropenem against biofilms of Pseudomonas aeruginosa PAO1 and three clinical strains isolated from patients with cystic fibrosis was investigated. Additionally, the effect of meropenem on PAO1 biofilms cultured for 24 h versus that on biofilms cultured for 72 h was examined. Using confocal laser scanning microscopy, rapid biofilm killing was observed in the first hour of the dosing interval for all biofilms. However, for PAO1 biofilms cultured for 72 h, only bacterial subpopulations at the periphery of the biofilm were affected, with subpopulations at the substratum remaining viable, even at the conclusion of the dosing interval. The described model is a novel method to investigate antimicrobial killing of bacterial biofilms using human simulated concentrations.

    View details for DOI 10.1128/AAC.05037-14

    View details for PubMedID 25918138

    View details for PubMedCentralID PMC4468651

  • Methanobacterium Dominates Biocathodic Archaeal Communities in Methanogenic Microbial Electrolysis Cells ACS SUSTAINABLE CHEMISTRY & ENGINEERING Siegert, M., Yates, M. D., Spormann, A. M., Logan, B. E. 2015; 3 (7): 1668-1676
  • Biochemical and EPR-Spectroscopic Investigation into Heterologously Expressed Vinyl Chloride Reductive Dehalogenase (VcrA) from Dehalococcoides mccartyi Strain VS JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Parthasarathy, A., Stich, T. A., Lohner, S. T., Lesnefsky, A., Britt, R. D., Spormann, A. M. 2015; 137 (10): 3525-3532


    Reductive dehalogenases play a critical role in the microbial detoxification of aquifers contaminated with chloroethenes and chlorethanes by catalyzing the reductive elimination of a halogen. We report here the first heterologous production of vinyl chloride reductase VcrA from Dehalococcoides mccartyi strain VS. Heterologously expressed VcrA was reconstituted to its active form by addition of hydroxocobalamin/adenosylcobalamin, Fe(3+), and sulfide in the presence of mercaptoethanol. The kinetic properties of reconstituted VcrA catalyzing vinyl chloride reduction with Ti(III)-citrate as reductant and methyl viologen as mediator were similar to those obtained previously for VcrA as isolated from D. mccartyi strain VS. VcrA was also found to catalyze a novel reaction, the environmentally important dihaloelimination of 1,2-dichloroethane to ethene. Electron paramagnetic resonance (EPR) spectroscopic studies with reconstituted VcrA in the presence of mercaptoethanol revealed the presence of Cob(II)alamin. Addition of Ti(III)-citrate resulted in the appearance of a new signal characteristic of a reduced [4Fe-4S] cluster and the disappearance of the Cob(II)alamin signal. UV-vis absorption spectroscopy of Ti(III)citrate-treated samples revealed the formation of two new absorption maxima characteristic of Cob(I)alamin. No evidence for the presence of a [3Fe-4S] cluster was found. We postulate that during the reaction cycle of VcrA, a reduced [4Fe-4S] cluster reduces Co(II) to Co(I) of the enzyme-bound cobalamin. Vinyl chloride reduction to ethene would be initiated when Cob(I)alamin transfers an electron to the substrate, generating a vinyl radical as a potential reaction intermediate.

    View details for DOI 10.1021/ja511653d

    View details for Web of Science ID 000351420800020

    View details for PubMedID 25686300

  • Extracellular Enzymes Facilitate Electron Uptake in Biocorrosion and Bioelectrosynthesis MBIO Deutzmann, J. S., Sahin, M., Spormann, A. M. 2015; 6 (2)


    Direct, mediator-free transfer of electrons between a microbial cell and a solid phase in its surrounding environment has been suggested to be a widespread and ecologically significant process. The high rates of microbial electron uptake observed during microbially influenced corrosion of iron [Fe(0)] and during microbial electrosynthesis have been considered support for a direct electron uptake in these microbial processes. However, the underlying molecular mechanisms of direct electron uptake are unknown. We investigated the electron uptake characteristics of the Fe(0)-corroding and electromethanogenic archaeon Methanococcus maripaludis and discovered that free, surface-associated redox enzymes, such as hydrogenases and presumably formate dehydrogenases, are sufficient to mediate an apparent direct electron uptake. In genetic and biochemical experiments, we showed that these enzymes, which are released from cells during routine culturing, catalyze the formation of H2 or formate when sorbed to an appropriate redox-active surface. These low-molecular-weight products are rapidly consumed by M. maripaludis cells when present, thereby preventing their accumulation to any appreciable or even detectable level. Rates of H2 and formate formation by cell-free spent culture medium were sufficient to explain the observed rates of methane formation from Fe(0) and cathode-derived electrons by wild-type M. maripaludis as well as by a mutant strain carrying deletions in all catabolic hydrogenases. Our data collectively show that cell-derived free enzymes can mimic direct extracellular electron transfer during Fe(0) corrosion and microbial electrosynthesis and may represent an ecologically important but so far overlooked mechanism in biological electron transfer.The intriguing trait of some microbial organisms to engage in direct electron transfer is thought to be widespread in nature. Consequently, direct uptake of electrons into microbial cells from solid surfaces is assumed to have a significant impact not only on fundamental microbial and biogeochemical processes but also on applied bioelectrochemical systems, such as microbial electrosynthesis and biocorrosion. This study provides a simple mechanistic explanation for frequently observed fast electron uptake kinetics in microbiological systems without a direct transfer: free, cell-derived enzymes can interact with cathodic surfaces and catalyze the formation of intermediates that are rapidly consumed by microbial cells. This electron transfer mechanism likely plays a significant role in various microbial electron transfer reactions in the environment.

    View details for DOI 10.1128/mBio.00496-15

    View details for Web of Science ID 000355312400022

    View details for PubMedID 25900658

    View details for PubMedCentralID PMC4453541

  • Revisiting N2 fixation in Guerrero Negro intertidal microbial mats with a functional single-cell approach. ISME journal Woebken, D., Burow, L. C., Behnam, F., Mayali, X., Schintlmeister, A., Fleming, E. D., Prufert-Bebout, L., Singer, S. W., Cortés, A. L., Hoehler, T. M., Pett-Ridge, J., Spormann, A. M., Wagner, M., Weber, P. K., Bebout, B. M. 2015; 9 (2): 485-496


    Photosynthetic microbial mats are complex, stratified ecosystems in which high rates of primary production create a demand for nitrogen, met partially by N2 fixation. Dinitrogenase reductase (nifH) genes and transcripts from Cyanobacteria and heterotrophic bacteria (for example, Deltaproteobacteria) were detected in these mats, yet their contribution to N2 fixation is poorly understood. We used a combined approach of manipulation experiments with inhibitors, nifH sequencing and single-cell isotope analysis to investigate the active diazotrophic community in intertidal microbial mats at Laguna Ojo de Liebre near Guerrero Negro, Mexico. Acetylene reduction assays with specific metabolic inhibitors suggested that both sulfate reducers and members of the Cyanobacteria contributed to N2 fixation, whereas (15)N2 tracer experiments at the bulk level only supported a contribution of Cyanobacteria. Cyanobacterial and nifH Cluster III (including deltaproteobacterial sulfate reducers) sequences dominated the nifH gene pool, whereas the nifH transcript pool was dominated by sequences related to Lyngbya spp. Single-cell isotope analysis of (15)N2-incubated mat samples via high-resolution secondary ion mass spectrometry (NanoSIMS) revealed that Cyanobacteria were enriched in (15)N, with the highest enrichment being detected in Lyngbya spp. filaments (on average 4.4 at% (15)N), whereas the Deltaproteobacteria (identified by CARD-FISH) were not significantly enriched. We investigated the potential dilution effect from CARD-FISH on the isotopic composition and concluded that the dilution bias was not substantial enough to influence our conclusions. Our combined data provide evidence that members of the Cyanobacteria, especially Lyngbya spp., actively contributed to N2 fixation in the intertidal mats, whereas support for significant N2 fixation activity of the targeted deltaproteobacterial sulfate reducers could not be found.

    View details for DOI 10.1038/ismej.2014.144

    View details for PubMedID 25303712

    View details for PubMedCentralID PMC4303640

  • Bacteria from Diverse Habitats Colonize and Compete in the Mouse Gut CELL Seedorf, H., Griffin, N. W., Ridaura, V. K., Reyes, A., Cheng, J., Rey, F. E., Smith, M. I., Simon, G. M., Scheffrahn, R. H., Woebken, D., Spormann, A. M., Van Treuren, W., Ursell, L. K., Pirrung, M., Robbins-Pianka, A., Cantarel, B. L., Lombard, V., Henrissat, B., Knight, R., Gordon, J. I. 2014; 159 (2): 253-266


    To study how microbes establish themselves in a mammalian gut environment, we colonized germ-free mice with microbial communities from human, zebrafish, and termite guts, human skin and tongue, soil, and estuarine microbial mats. Bacteria from these foreign environments colonized and persisted in the mouse gut; their capacity to metabolize dietary and host carbohydrates and bile acids correlated with colonization success. Cohousing mice harboring these xenomicrobiota or a mouse cecal microbiota, along with germ-free "bystanders," revealed the success of particular bacterial taxa in invading guts with established communities and empty gut habitats. Unanticipated patterns of ecological succession were observed; for example, a soil-derived bacterium dominated even in the presence of bacteria from other gut communities (zebrafish and termite), and human-derived bacteria colonized germ-free bystander mice before mouse-derived organisms. This approach can be generalized to address a variety of mechanistic questions about succession, including succession in the context of microbiota-directed therapeutics.

    View details for DOI 10.1016/j.cell.2014.09.008

    View details for Web of Science ID 000343095600009

    View details for PubMedCentralID PMC4194163

  • Single cell genomic study of Dehalococcoidetes species from deep-sea sediments of the Peruvian Margin. ISME journal Kaster, A., Mayer-Blackwell, K., Pasarelli, B., Spormann, A. M. 2014; 8 (9): 1831-1842


    The phylum Chloroflexi is one of the most frequently detected phyla in the subseafloor of the Pacific Ocean margins. Dehalogenating Chloroflexi (Dehalococcoidetes) was originally discovered as the key microorganisms mediating reductive dehalogenation via their key enzymes reductive dehalogenases (Rdh) as sole mode of energy conservation in terrestrial environments. The frequent detection of Dehalococcoidetes-related 16S rRNA and rdh genes in the marine subsurface implies a role for dissimilatory dehalorespiration in this environment; however, the two genes have never been linked to each other. To provide fundamental insights into the metabolism, genomic population structure and evolution of marine subsurface Dehalococcoidetes sp., we analyzed a non-contaminated deep-sea sediment core sample from the Peruvian Margin Ocean Drilling Program (ODP) site 1230, collected 7.3 m below the seafloor by a single cell genomic approach. We present for the first time single cell genomic data on three deep-sea Chloroflexi (Dsc) single cells from a marine subsurface environment. Two of the single cells were considered to be part of a local Dehalococcoidetes population and assembled together into a 1.38-Mb genome, which appears to be at least 85% complete. Despite a high degree of sequence-level similarity between the shared proteins in the Dsc and terrestrial Dehalococcoidetes, no evidence for catabolic reductive dehalogenation was found in Dsc. The genome content is however consistent with a strictly anaerobic organotrophic or lithotrophic lifestyle.

    View details for DOI 10.1038/ismej.2014.24

    View details for PubMedID 24599070

    View details for PubMedCentralID PMC4139717

  • Nanoliter qPCR Platform for Highly Parallel, Quantitative Assessment of Reductive Dehalogenase Genes and Populations of Dehalogenating Microorganisms in Complex Environments. Environmental science & technology Mayer-Blackwell, K., Azizian, M. F., Machak, C., Vitale, E., Carpani, G., de Ferra, F., Semprini, L., Spormann, A. M. 2014; 48 (16): 9659-9667


    Idiosyncratic combinations of reductive dehalogenase (rdh) genes are a distinguishing genomic feature of closely related organohalogen-respiring bacteria. This feature can be used to deconvolute the population structure of organohalogen-respiring bacteria in complex environments and to identify relevant subpopulations, which is important for tracking interspecies dynamics needed for successful site remediation. Here we report the development of a nanoliter qPCR platform to identify organohalogen-respiring bacteria and populations by quantifying major orthologous reductive dehalogenase gene groups. The qPCR assays can be operated in parallel within a 5184-well nanoliter qPCR (nL-qPCR) chip at a single annealing temperature and buffer condition. We developed a robust bioinformatics approach to select from thousands of computationally proposed primer pairs those that are specific to individual rdh gene groups and compatible with a single amplification condition. We validated hundreds of the most selective qPCR assays and examined their performance in a trichloroethene-degrading bioreactor, revealing population structures as well as their unexpected shifts in abundance and community dynamics.

    View details for DOI 10.1021/es500918w

    View details for PubMedID 25046033

  • Hydrogenase-independent uptake and metabolism of electrons by the archaeon Methanococcus maripaludis. ISME journal Lohner, S. T., Deutzmann, J. S., Logan, B. E., Leigh, J., Spormann, A. M. 2014; 8 (8): 1673-1681


    Direct, shuttle-free uptake of extracellular, cathode-derived electrons has been postulated as a novel mechanism of electron metabolism in some prokaryotes that may also be involved in syntrophic electron transport between two microorganisms. Experimental proof for direct uptake of cathodic electrons has been mostly indirect and has been based on the absence of detectable concentrations of molecular hydrogen. However, hydrogen can be formed as a transient intermediate abiotically at low cathodic potentials (<-414 mV) under conditions of electromethanogenesis. Here we provide genetic evidence for hydrogen-independent uptake of extracellular electrons. Methane formation from cathodic electrons was observed in a wild-type strain of the methanogenic archaeon Methanococcus maripaludis as well as in a hydrogenase-deletion mutant lacking all catabolic hydrogenases, indicating the presence of a hydrogenase-independent mechanism of electron catabolism. In addition, we discovered a new route for hydrogen or formate production from cathodic electrons: Upon chemical inhibition of methanogenesis with 2-bromo-ethane sulfonate, hydrogen or formate accumulated in the bioelectrochemical cells instead of methane. These results have implications for our understanding on the diversity of microbial electron uptake and metabolism.

    View details for DOI 10.1038/ismej.2014.82

    View details for PubMedID 24844759

  • Influence of setup and carbon source on the bacterial community of biocathodes in microbial electrolysis cells ENZYME AND MICROBIAL TECHNOLOGY Croese, E., Jeremiasse, A. W., Marshall, I. P., Spormann, A. M., Euveritik, G. W., Geelhoed, J. S., Stams, A. J., Plugge, C. M. 2014; 61-62: 67-75
  • Influence of setup and carbon source on the bacterial community of biocathodes in microbial electrolysis cells. Enzyme and microbial technology Croese, E., Jeremiasse, A. W., Marshall, I. P., Spormann, A. M., Euverink, G. W., Geelhoed, J. S., Stams, A. J., Plugge, C. M. 2014; 61-62: 67-75


    The microbial electrolysis cell (MEC) biocathode has shown great potential as alternative for expensive metals as catalyst for H2 synthesis. Here, the bacterial communities at the biocathode of five hydrogen producing MECs using molecular techniques were characterized. The setups differed in design (large versus small) including electrode material and flow path and in carbon source provided at the cathode (bicarbonate or acetate). A hydrogenase gene-based DNA microarray (Hydrogenase Chip) was used to analyze hydrogenase genes present in the three large setups. The small setups showed dominant groups of Firmicutes and two of the large setups showed dominant groups of Proteobacteria and Bacteroidetes. The third large setup received acetate but no sulfate (no sulfur source). In this setup an almost pure culture of a Promicromonospora sp. developed. Most of the hydrogenase genes detected were coding for bidirectional Hox-type hydrogenases, which have shown to be involved in cytoplasmatic H2 production.

    View details for DOI 10.1016/j.enzmictec.2014.04.019

    View details for PubMedID 24910339

  • Identification of Desulfobacterales as primary hydrogenotrophs in a complex microbial mat community GEOBIOLOGY Burow, L. C., Woebken, D., Marshall, I. P., Singer, S. W., Pett-Ridge, J., Prufert-Bebout, L., Spormann, A. M., Bebout, B. M., Weber, P. K., Hoehler, T. M. 2014; 12 (3): 221-230


    Hypersaline microbial mats have been shown to produce significant quantities of H2 under dark, anoxic conditions via cyanobacterial fermentation. This flux of a widely accessible microbial substrate has potential to significantly influence the ecology of the mat, and any consumption will affect the net efflux of H2 that might otherwise be captured as a resource. Here, we focus on H2 consumption in a microbial mat from Elkhorn Slough, California, USA, for which H2 production has been previously characterized. Active biologic H2 consumption in this mat is indicated by a significant time-dependent decrease in added H2 compared with a killed control. Inhibition of sulfate reduction, as indicated by a decrease in hydrogen sulfide production relative to controls, resulted in a significant increase in H2 efflux, suggesting that sulfate-reducing bacteria (SRB) are important hydrogenotrophs. Low methane efflux under these same conditions indicated that methanogens are likely not important hydrogenotrophs. Analyses of genes and transcripts that encode for rRNA or dissimilatory sulfite reductase, using both PCR-dependent and PCR-independent metatranscriptomic sequencing methods, demonstrated that Desulfobacterales are the dominant, active SRB in the upper, H2-producing layer of the mat (0-2 mm). This hypothesis was further supported by the identification of transcripts encoding hydrogenases derived from Desulfobacterales capable of H2 oxidation. Analysis of molecular data provided no evidence for the activity of hydrogenotrophic methanogens. The combined biogeochemical and molecular data strongly indicate that SRB belonging to the Desulfobacterales are the quantitatively important hydrogenotrophs in the Elkhorn Slough mat.

    View details for DOI 10.1111/gbi.12080

    View details for Web of Science ID 000334361800004

    View details for PubMedID 24730641

  • Comparison of Nonprecious Metal Cathode Materials for Methane Production by Electromethanogenesis ACS SUSTAINABLE CHEMISTRY & ENGINEERING Siegert, M., Yates, M. D., Call, D. F., Zhu, X., Spormann, A., Logan, B. E. 2014; 2 (4): 910-917

    View details for DOI 10.1021/sc400520x

    View details for Web of Science ID 000334092600046

  • Inferring community dynamics of organohalide-respiring bacteria in chemostats by covariance of rdhA gene abundance FEMS MICROBIOLOGY ECOLOGY Marshall, I. P., Azizian, M. F., Semprini, L., Spormann, A. M. 2014; 87 (2): 428-440
  • Inferring community dynamics of organohalide-respiring bacteria in chemostats by covariance of rdhA gene abundance. FEMS microbiology ecology Marshall, I. P., Azizian, M. F., Semprini, L., Spormann, A. M. 2014; 87 (2): 428-440


    We have developed a novel approach to identifying and quantifying closely related organohalide-respiring bacteria. Our approach made use of the unique genomic associations of specific reductive dehalogenase subunit A encoding genes (rdhA) that exist in known strains of Dehalococcoides mccartyi and Desulfitobacterium and the distinguishing covariance pattern of observed rdhA genes to assign genes to unknown strains. To test this approach, we operated five anaerobic reductively dechlorinating chemostats for 3-4 years with tetrachloroethene and trichloroethene as terminal electron acceptors and lactate/formate as electron donors. The presence and abundance of rdhA genes were determined comprehensively at the community level using a custom-developed Reductive Dehalogenase Chip (RDH Chip) DNA microarray and used to define putative strains of Dehalococcoides mccartyi and Desulfitobacterium sp. This monitoring revealed that stable chemical performance of chemostats was reflected by a stable community of reductively dechlorinating bacteria. However, perturbations introduced by, for example, electron donor limitation or addition of the competing electron acceptor sulfate led to overall changes in the chemostat performance, including incomplete reduction in the chloroethene substrates, and in the population composition of reductively dehalogenating bacteria. Interestingly, there was a high diversity of operationally defined D. mccartyi strains between the chemostats with almost all strains unique to their specific chemostats in spite of similar selective pressure and similar inocula shared between chemostats.

    View details for DOI 10.1111/1574-6941.12235

    View details for PubMedID 24118060

  • Single Cell Genomic study of Dehalococcoidetes species from Deep-sea Sediments of the Peruvian Margin ISME Journal Kasters, A. K., Mayer-Blackwell, K., Pasarelli, B., Spormann, A. M. 2014
  • Microbially enhanced dissolution of HgS in an acid mine drainage system in the California Coast Range GEOBIOLOGY Jew, A. D., Behrens, S. F., Rytuba, J. J., Kappler, A., Spormann, A. M., Brown, G. E. 2014; 12 (1): 20-33

    View details for DOI 10.1111/gbi.12066

    View details for Web of Science ID 000328088500002

    View details for PubMedID 24224806

  • Fermentation couples Chloroflexi and sulfate-reducing bacteria to Cyanobacteria in hypersaline microbial mats. Frontiers in microbiology Lee, J. Z., Burow, L. C., Woebken, D., Everroad, R. C., Kubo, M. D., Spormann, A. M., Weber, P. K., Pett-Ridge, J., Bebout, B. M., Hoehler, T. M. 2014; 5: 61-?


    Past studies of hydrogen cycling in hypersaline microbial mats have shown an active nighttime cycle, with production largely from Cyanobacteria and consumption from sulfate-reducing bacteria (SRB). However, the mechanisms and magnitude of hydrogen cycling have not been extensively studied. Two mats types near Guerrero Negro, Mexico-permanently submerged Microcoleus microbial mat (GN-S), and intertidal Lyngbya microbial mat (GN-I)-were used in microcosm diel manipulation experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), molybdate, ammonium addition, and physical disruption to understand the processes responsible for hydrogen cycling between mat microbes. Across microcosms, H2 production occurred under dark anoxic conditions with simultaneous production of a suite of organic acids. H2 production was not significantly affected by inhibition of nitrogen fixation, but rather appears to result from constitutive fermentation of photosynthetic storage products by oxygenic phototrophs. Comparison to accumulated glycogen and to CO2 flux indicated that, in the GN-I mat, fermentation released almost all of the carbon fixed via photosynthesis during the preceding day, primarily as organic acids. Across mats, although oxygenic and anoxygenic phototrophs were detected, cyanobacterial [NiFe]-hydrogenase transcripts predominated. Molybdate inhibition experiments indicated that SRBs from a wide distribution of DsrA phylotypes were responsible for H2 consumption. Incubation with (13)C-acetate and NanoSIMS (secondary ion mass-spectrometry) indicated higher uptake in both Chloroflexi and SRBs relative to other filamentous bacteria. These manipulations and diel incubations confirm that Cyanobacteria were the main fermenters in Guerrero Negro mats and that the net flux of nighttime fermentation byproducts (not only hydrogen) was largely regulated by the interplay between Cyanobacteria, SRBs, and Chloroflexi.

    View details for DOI 10.3389/fmicb.2014.00061

    View details for PubMedID 24616716

    View details for PubMedCentralID PMC3935151

  • Microbially enhanced dissolution of HgS in an acid mine drainage system in the California Coast Range. Geobiology Jew, A. D., Behrens, S. F., Rytuba, J. J., Kappler, A., Spormann, A. M., BROWN, G. E. 2014; 12 (1): 20-33


    Mercury sulfides (cinnabar and metacinnabar) are the main ores of Hg and are relatively stable under oxic conditions (Ksp = 10⁻⁵⁴ and 10⁻⁵² , respectively). However, until now their stability in the presence of micro-organisms inhabiting acid mine drainage (AMD) systems was unknown. We tested the effects of the AMD microbial community from the inoperative Hg mine at New Idria, CA, present in sediments of an AMD settling pond adjacent to the main waste pile and in a microbial biofilm on the surface of this pond, on the solubility of crystalline HgS. A 16S rRNA gene clone library revealed that the AMD microbial community was dominated by Fe-oxidizing (orders Ferritrophicales and Gallionellas) and S-oxidizing bacteria (Thiomonas sp.), with smaller amounts (≤ 6%) being comprised of the orders Xanthomondales and Rhodospirillales. Though the order Ferritrophicales dominate the 16S rRNA clones (>60%), qPCR results of the microbial community indicate that the Thiomonas sp. represents ~55% of the total micro-organisms in the top 1 cm of the AMD microbial community. Although supersaturated with respect to cinnabar and metacinnabar, microcosms inoculated with the AMD microbial community were capable of releasing significantly more Hg into solution compared to inactivated or abiotic controls. Four different Hg-containing materials were tested for bacterially enhanced HgS dissolution: pure cinnabar, pure metacinnabar, mine tailings, and calcine material (processed ore). In the microcosm with metacinnabar, the presence of the AMD microbial community resulted in an increase of dissolved Hg concentrations up to 500 μg L ⁻¹during the first 30 days of incubation. In abiotic control microcosms, dissolved Hg concentrations did not increase above 100 ng L⁻¹ . When Hg concentrations were below 50 μg L⁻¹ , the Fe-oxidizing bacteria in the AMD microbial community were still capable of oxidizing Fe(II) to Fe(III) in the AMD solution, whereas concentrations above 50 μg L⁻¹ resulted in inhibition of microbial iron oxidation. Our experiments show that the AMD microbial community contributes to the dissolution of mercury sulfide minerals. These findings have major implications for risk assessment and future management of inoperative Hg mines worldwide.

    View details for DOI 10.1111/gbi.12066

    View details for PubMedID 24224806

  • A metabolomic view of how the human gut microbiota impacts the host metabolome using humanized and gnotobiotic mice. ISME journal Marcobal, A., Kashyap, P. C., Nelson, T. A., Aronov, P. A., Donia, M. S., Spormann, A., Fischbach, M. A., Sonnenburg, J. L. 2013; 7 (10): 1933-1943


    Defining the functional status of host-associated microbial ecosystems has proven challenging owing to the vast number of predicted genes within the microbiome and relatively poor understanding of community dynamics and community-host interaction. Metabolomic approaches, in which a large number of small molecule metabolites can be defined in a biological sample, offer a promising avenue to 'fingerprint' microbiota functional status. Here, we examined the effects of the human gut microbiota on the fecal and urinary metabolome of a humanized (HUM) mouse using an optimized ultra performance liquid chromatography-mass spectrometry-based method. Differences between HUM and conventional mouse urine and fecal metabolomic profiles support host-specific aspects of the microbiota's metabolomic contribution, consistent with distinct microbial compositions. Comparison of microbiota composition and metabolome of mice humanized with different human donors revealed that the vast majority of metabolomic features observed in donor samples are produced in the corresponding HUM mice, and individual-specific features suggest 'personalized' aspects of functionality can be reconstituted in mice. Feeding the mice a defined, custom diet resulted in modification of the metabolite signatures, illustrating that host diet provides an avenue for altering gut microbiota functionality, which in turn can be monitored via metabolomics. Using a defined model microbiota consisting of one or two species, we show that simplified communities can drive major changes in the host metabolomic profile. Our results demonstrate that metabolomics constitutes a powerful avenue for functional characterization of the intestinal microbiota and its interaction with the host.The ISME Journal advance online publication, 6 June 2013; doi:10.1038/ismej.2013.89.

    View details for DOI 10.1038/ismej.2013.89

    View details for PubMedID 23739052

  • PdeB, a Cyclic Di-GMP-Specific Phosphodiesterase That Regulates Shewanella oneidensis MR-1 Motility and Biofilm Formation JOURNAL OF BACTERIOLOGY Chao, L., Rakshe, S., Leff, M., Spormann, A. M. 2013; 195 (17): 3827-3833


    Shewanella oneidensis MR-1, a gammaproteobacterium with respiratory versatility, forms biofilms on mineral surfaces through a process controlled by the cyclic dinucleotide messenger c-di-GMP. Cellular concentrations of c-di-GMP are maintained by proteins containing GGDEF and EAL domains, which encode diguanylate cyclases for c-di-GMP synthesis and phosphodiesterases for c-di-GMP hydrolysis, respectively. The S. oneidensis MR-1 genome encodes several GGDEF and EAL domain proteins (50 and 31, respectively), with a significant fraction (∼10) predicted to be multidomain (e.g., GGDEF-EAL) enzymes containing an additional Per-Arnt-Sim (PAS) sensor domain. However, the biochemical activities and physiological functions of these multidomain enzymes remain largely unknown. Here, we present genetic and biochemical analyses of a predicted PAS-GGDEF-EAL domain-containing protein, SO0437, here named PdeB. A pdeB deletion mutant exhibited decreased swimming motility and increased biofilm formation under rich growth medium conditions, which was consistent with an increase in intracellular c-di-GMP. A mutation inactivating the EAL domain also produced similar swimming and biofilm phenotypes, indicating that the increase in c-di-GMP was likely due to a loss in phosphodiesterase activity. Therefore, we also examined the enzymatic activity of purified PdeB and found that the protein exhibited phosphodiesterase activity via the EAL domain. No diguanylate cyclase activity was observed. In addition to the motility and biofilm phenotypes, transcriptional profiling by DNA microarray analysis of biofilms of pdeB (in-frame deletion and EAL) mutant cells revealed that expression of genes involved in sulfate uptake and assimilation were repressed. Addition of sulfate to the growth medium resulted in significantly less motile pdeB mutants. Together, these results indicate a link between c-di-GMP metabolism, S. oneidensis MR-1 biofilm development, and sulfate uptake/assimilation.

    View details for DOI 10.1128/JB.00498-13

    View details for Web of Science ID 000323047900004

    View details for PubMedID 23794617

    View details for PubMedCentralID PMC3754596

  • The mxd operon in Shewanella oneidensis MR-1 is induced in response to starvation and regulated by ArcS/ArcA and BarA/UvrY BMC MICROBIOLOGY Mueller, J., Shukla, S., Jost, K. A., Spormann, A. M. 2013; 13


    S. oneidensis MR-1 is a dissimilatory metal-reducing bacterium. Under anoxic conditions S. oneidensis MR-1 attaches to and uses insoluble minerals such as Fe(III) and Mn(IV) oxides as electron acceptors. In the laboratory, S. oneidensis MR-1 forms biofilms under hydrodynamic flow conditions on a borosilicate glass surface; formation of biofilms was previously found to be dependent on the mxd gene cluster (mxdABCD).This study revealed environmental and genetic factors regulating expression of the mxd genes in S. oneidensis MR-1. Physiological experiments conducted with a S. oneidensis MR-1 strain carrying a transcriptional lacZ fusion to the mxd promoter identified electron donor starvation as a key factor inducing mxd gene expression. Tn5 mutagenesis identified the ArcS/ArcA two-component signaling system as a repressor of mxd expression in S. oneidensis MR-1 under planktonic conditions. Biofilms of ∆arcS and ∆arcA strains carrying a transcriptional gfp -reporter fused to the mxd promoter revealed a reduced mxd expression, suggesting that ArcS/ArcA are necessary for activation of mxd expression under biofilm conditions. Biofilms of ∆arcS and ∆arcA mutants were unable to form a compact three-dimensional structure consistent with a low level of mxd expression. In addition, BarA/UvrY was identified as a major regulator of mxd expression under planktonic conditions. Interestingly, biofilms of ∆barA and ∆uvrY mutants were able to form three-dimensional structures that were, however, less compact compared to wild type biofilms.We have shown here that the mxd genes in S. oneidensis MR-1 are controlled transcriptionally in response to carbon starvation and by the ArcS/ArcA and the BarA/UvrY signaling system. BarA might function as a sensor to assess the metabolic state of the cell, including carbon starvation, leading to expression of the mxd operon and therefore control biofilm formation.

    View details for DOI 10.1186/1471-2180-13-119

    View details for Web of Science ID 000320915100001

    View details for PubMedID 23705927

    View details for PubMedCentralID PMC3691769

  • Identification of a reductive tetrachloroethene dehalogenase in Shewanella sediminis PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES Lohner, S. T., Spormann, A. M. 2013; 368 (1616)


    The genome sequence of psychrophilic Shewanella sediminis revealed the presence of five putative reductive dehalogenases (Rdhs). We found that cell extracts of pyruvate/fumarate-grown S. sediminis cells catalysed reduced methyl viologen-dependent reductive dechlorination of tetrachloroethene (PCE) to trichloroethene (TCE) at a specific activity of approximately 1 nmol TCE min(-1) (mg protein)(-1). Dechlorination of PCE followed Michaelis-Menten kinetics with an apparent Km of 120 μM PCE. No PCE dechlorination was observed with heat-denatured extract or when cyanocobalamin was omitted from the growth medium; however, the presence of PCE in the growth medium increased PCE transformation rates. Analysis of mutants carrying in-frame deletions of all five Rdhs encoding genes showed that only deletion of Ssed_3769 resulted in the loss of PCE dechlorination activity suggesting that Ssed_3769 is a functional Rdh. This is the first study to show reductive dechlorination activity of PCE in a sediment-dwelling Shewanella species that may be important for linking the flux of organohalogens to organic carbon via reductive dehalogenation in marine sediments.

    View details for DOI 10.1098/rstb.2012.0326

    View details for Web of Science ID 000315995300011

    View details for PubMedID 23479755

    View details for PubMedCentralID PMC3638466

  • Anoxic carbon flux in photosynthetic microbial mats as revealed by metatranscriptomics ISME JOURNAL Burow, L. C., Woebken, D., Marshall, I. P., Lindquist, E. A., Bebout, B. M., Prufert-Bebout, L., Hoehler, T. M., Tringe, S. G., Pett-Ridge, J., Weber, P. K., Spormann, A. M., Singer, S. W. 2013; 7 (4): 817-829


    Photosynthetic microbial mats possess extraordinary phylogenetic and functional diversity that makes linking specific pathways with individual microbial populations a daunting task. Close metabolic and spatial relationships between Cyanobacteria and Chloroflexi have previously been observed in diverse microbial mats. Here, we report that an expressed metabolic pathway for the anoxic catabolism of photosynthate involving Cyanobacteria and Chloroflexi in microbial mats can be reconstructed through metatranscriptomic sequencing of mats collected at Elkhorn Slough, Monterey Bay, CA, USA. In this reconstruction, Microcoleus spp., the most abundant cyanobacterial group in the mats, ferment photosynthate to organic acids, CO2 and H2 through multiple pathways, and an uncultivated lineage of the Chloroflexi take up these organic acids to store carbon as polyhydroxyalkanoates. The metabolic reconstruction is consistent with metabolite measurements and single cell microbial imaging with fluorescence in situ hybridization and NanoSIMS.

    View details for DOI 10.1038/ismej.2012.150

    View details for Web of Science ID 000316727800012

    View details for PubMedID 23190731

    View details for PubMedCentralID PMC3603402

  • Effects of Sulfate Reduction on the Bacterial Community and Kinetic Parameters of a Dechlorinating Culture under Chemostat Growth Conditions ENVIRONMENTAL SCIENCE & TECHNOLOGY Berggren, D. R., Marshall, I. P., Azizian, M. F., Spormann, A. M., Semprini, L. 2013; 47 (4): 1879-1886


    Results are presented from a chemostat study where the reductive dehalogenation of PCE was evaluated in the absence and presence of sulfate. Two chemostats inoculated with the Point Mugu culture, which contains strains of Dehalococcoides mccartyi, were operated at a 50 day HRT and fed PCE (1.12 mM) and lactate (4.3 mM). The control chemostat (PM-5L, no sulfate), achieved pseudo-steady-state transformation of PCE to ethene (98%) and VC (2%) at 2.4 nM of H(2). Batch kinetic tests with chemostat harvested cells showed the maximum rate (k(max)X) value for each dehalogenation step remained fairly constant, while hupL clone library analyses showed maintenance of a diverse D. mccartyi community. Sulfate (1 mM) was introduced to the second chemostat, PM-2L. Effective sulfate reduction was achieved 110 days later, resulting in 600 μM of total sulfide. PCE dechlorination efficiency decreased following complete sulfate reduction, yielding ethene (25%), VC (67%), and cis-DCE (8%). VC dechlorination was most affected, with k(max)X values decreasing by a factor of 50. The decrease was associated with the enrichment of the Cornell group of D. mccartyi and decline of the Pinellas group. Long-term exposure to sulfides and/or competition for H(2) may have been responsible for the community shift.

    View details for DOI 10.1021/es304244z

    View details for Web of Science ID 000315326700013

    View details for PubMedID 23316874

  • Dehalococcoides mccartyi gen. nov., sp. nov., obligately organohalide-respiring anaerobic bacteria relevant to halogen cycling and bioremediation, belong to a novel bacterial class, Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov., within the phylum Chloroflexi. International journal of systematic and evolutionary microbiology Löffler, F. E., Yan, J., Ritalahti, K. M., Adrian, L., Edwards, E. A., Konstantinidis, K. T., Müller, J. A., Fullerton, H., Zinder, S. H., Spormann, A. M. 2013; 63: 625-635


    Six obligately anaerobic bacterial isolates (195(T), CBDB1, BAV1, VS, FL2 and GT) with strictly organohalide-respiring metabolisms were obtained from chlorinated solvent-contaminated aquifers, contaminated and uncontaminated river sediments or anoxic digester sludge. Cells were non-motile with a disc-shaped morphology, 0.3-1 µm in diameter and 0.1-0.2 µm thick, and characteristic indentations on opposite flat sides of the cell. Growth occurred in completely synthetic, reduced medium amended with a haloorganic electron acceptor (mostly chlorinated but also some brominated compounds), hydrogen as electron donor, acetate as carbon source, and vitamins. No other growth-supporting redox couples were identified. Aqueous hydrogen consumption threshold concentrations were <1 nM. Growth ceased when vitamin B(12) was omitted from the medium. Addition of sterile cell-free supernatant of Dehalococcoides-containing enrichment cultures enhanced dechlorination and growth of strains 195 and FL2, suggesting the existence of so-far unidentified stimulants. Dechlorination occurred between pH 6.5 and 8.0 and over a temperature range of 15-35 °C, with an optimum growth temperature between 25 and 30 °C. The major phospholipid fatty acids were 14 : 0 (15.7 mol%), br15 : 0 (6.2 mol%), 16 : 0 (22.7 mol%), 10-methyl 16 : 0 (25.8 mol%) and 18 : 0 (16.6 mol%). Unusual furan fatty acids including 9-(5-pentyl-2-furyl)-nonanoate and 8-(5-hexyl-2-furyl)-octanoate were detected in strains FL2, BAV1 and GT, but not in strains 195(T) and CBDB1. The 16S rRNA gene sequences of the six isolates shared more than 98 % identity, and phylogenetic analysis revealed an affiliation with the phylum Chloroflexi and more than 10 % sequence divergence from other described isolates. The genome sizes and G+C contents ranged from 1.34 to 1.47 Mbp and 47 to 48.9 mol% G+C, respectively. Based on 16S rRNA gene sequence comparisons, genome-wide average nucleotide identity and phenotypic characteristics, the organohalide-respiring isolates represent a new genus and species, for which the name Dehalococcoides mccartyi gen. nov., sp. nov. is proposed. Isolates BAV1 ( = ATCC BAA-2100  = JCM 16839  = KCTC 5957), FL2 ( = ATCC BAA-2098  = DSM 23585  = JCM 16840  = KCTC 5959), GT ( = ATCC BAA-2099  = JCM 16841  = KCTC 5958), CBDB1, 195(T) ( = ATCC BAA-2266(T)  = KCTC 15142(T)) and VS are considered strains of Dehalococcoides mccartyi, with strain 195(T) as the type strain. The new class Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov. are described to accommodate the new taxon.

    View details for DOI 10.1099/ijs.0.034926-0

    View details for PubMedID 22544797

  • Dehalococcoides mccartyi gen. nov., sp nov., obligately organohalide-respiring anaerobic bacteria relevant to halogen cycling and bioremediation, belong to a novel bacterial class, Dehalococcoidia classis nov., order Dehalococcoidales ord. nov and family Dehalococcoidaceae fam. nov., within the phylum Chloroflexi INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY Loeffler, F. E., Yan, J., Ritalahti, K. M., Adrian, L., Edwards, E. A., Konstantinidis, K. T., Mueller, J. A., Fullerton, H., Zinder, S. H., Spormann, A. M. 2013; 63: 625-635
  • Inferring community dynamics of organohalide-respiring bacteria in chemostats by covariance of rdhA gene abundance FEMS Microbiol Ecol. Marshall , I. P., Azizian , M. F., Semprini , L., Spormann , A. M. 2013
  • A Single-Cell Genome for Thiovulum sp. APPLIED AND ENVIRONMENTAL MICROBIOLOGY Marshall, I. P., Blainey, P. C., Spormann, A. M., Quake, S. R. 2012; 78 (24): 8555-8563


    We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including 2,162 genes. This single-cell genome represents a genomic view of the physiological capabilities of isolated Thiovulum cells. Thiovulum is the second-fastest bacterium ever observed, swimming at 615 μm/s, and this genome shows that this rapid swimming motility is a result of a standard flagellar machinery that has been extensively characterized in other bacteria. This suggests that standard flagella are capable of propelling bacterial cells at speeds much faster than typically thought. Analysis of the genome suggests that naturally occurring Thiovulum populations are more diverse than previously recognized and that studies performed in the past probably address a wide range of unrecognized genotypic and phenotypic diversities of Thiovulum. The genome presented in this article provides a basis for future isolation-independent studies of Thiovulum, where single-cell and metagenomic tools can be used to differentiate between different Thiovulum genotypes.

    View details for DOI 10.1128/AEM.02314-12

    View details for Web of Science ID 000311213200007

    View details for PubMedID 23023751

    View details for PubMedCentralID PMC3502928

  • Identification of a novel cyanobacterial group as active diazotrophs in a coastal microbial mat using NanoSIMS analysis ISME JOURNAL Woebken, D., Burow, L. C., Prufert-Bebout, L., Bebout, B. M., Hoehler, T. M., Pett-Ridge, J., Spormann, A. M., Weber, P. K., Singer, S. W. 2012; 6 (7): 1427-1439


    N(2) fixation is a key process in photosynthetic microbial mats to support the nitrogen demands associated with primary production. Despite its importance, groups that actively fix N(2) and contribute to the input of organic N in these ecosystems still remain largely unclear. To investigate the active diazotrophic community in microbial mats from the Elkhorn Slough estuary, Monterey Bay, CA, USA, we conducted an extensive combined approach, including biogeochemical, molecular and high-resolution secondary ion mass spectrometry (NanoSIMS) analyses. Detailed analysis of dinitrogenase reductase (nifH) transcript clone libraries from mat samples that fixed N(2) at night indicated that cyanobacterial nifH transcripts were abundant and formed a novel monophyletic lineage. Independent NanoSIMS analysis of (15)N(2)-incubated samples revealed significant incorporation of (15)N into small, non-heterocystous cyanobacterial filaments. Mat-derived enrichment cultures yielded a unicyanobacterial culture with similar filaments (named Elkhorn Slough Filamentous Cyanobacterium-1 (ESFC-1)) that contained nifH gene sequences grouping with the novel cyanobacterial lineage identified in the transcript clone libraries, displaying up to 100% amino-acid sequence identity. The 16S rRNA gene sequence recovered from this enrichment allowed for the identification of related sequences from Elkhorn Slough mats and revealed great sequence diversity in this cluster. Furthermore, by combining (15)N(2) tracer experiments, fluorescence in situ hybridization and NanoSIMS, in situ N(2) fixation activity by the novel ESFC-1 group was demonstrated, suggesting that this group may be the most active cyanobacterial diazotroph in the Elkhorn Slough mat. Pyrotag sequences affiliated with ESFC-1 were recovered from mat samples throughout 2009, demonstrating the prevalence of this group. This work illustrates that combining standard and single-cell analyses can link phylogeny and function to identify previously unknown key functional groups in complex ecosystems.

    View details for DOI 10.1038/ismej.2011.200

    View details for Web of Science ID 000305631100016

    View details for PubMedID 22237543

    View details for PubMedCentralID PMC3379636

  • Hydrogen production in photosynthetic microbial mats in the Elkhorn Slough estuary, Monterey Bay ISME JOURNAL Burow, L. C., Woebken, D., Bebout, B. M., McMurdie, P. J., Singer, S. W., Pett-Ridge, J., Prufert-Bebout, L., Spormann, A. M., Weber, P. K., Hoehler, T. M. 2012; 6 (4): 863-874


    Hydrogen (H(2)) release from photosynthetic microbial mats has contributed to the chemical evolution of Earth and could potentially be a source of renewable H(2) in the future. However, the taxonomy of H(2)-producing microorganisms (hydrogenogens) in these mats has not been previously determined. With combined biogeochemical and molecular studies of microbial mats collected from Elkhorn Slough, Monterey Bay, California, we characterized the mechanisms of H(2) production and identified a dominant hydrogenogen. Net production of H(2) was observed within the upper photosynthetic layer (0-2 mm) of the mats under dark and anoxic conditions. Pyrosequencing of rRNA gene libraries generated from this layer demonstrated the presence of 64 phyla, with Bacteriodetes, Cyanobacteria and Proteobacteria dominating the sequences. Sequencing of rRNA transcripts obtained from this layer demonstrated that Cyanobacteria dominated rRNA transcript pyrotag libraries. An OTU affiliated to Microcoleus spp. was the most abundant OTU in both rRNA gene and transcript libraries. Depriving mats of sunlight resulted in an order of magnitude decrease in subsequent nighttime H(2) production, suggesting that newly fixed carbon is critical to H(2) production. Suppression of nitrogen (N(2))-fixation in the mats did not suppress H(2) production, which indicates that co-metabolic production of H(2) during N(2)-fixation is not an important contributor to H(2) production. Concomitant production of organic acids is consistent with fermentation of recently produced photosynthate as the dominant mode of H(2) production. Analysis of rRNA % transcript:% gene ratios and H(2)-evolving bidirectional [NiFe] hydrogenase % transcript:% gene ratios indicated that Microcoelus spp. are dominant hydrogenogens in the Elkhorn Slough mats.

    View details for DOI 10.1038/ismej.2011.142

    View details for Web of Science ID 000301945500015

    View details for PubMedID 22011721

    View details for PubMedCentralID PMC3309353

  • The Hydrogenase Chip: a tiling oligonucleotide DNA microarray technique for characterizing hydrogen-producing and -consuming microbes in microbial communities ISME JOURNAL Marshall, I. P., Berggren, D. R., Azizian, M. F., Burow, L. C., Semprini, L., Spormann, A. M. 2012; 6 (4): 814-826


    We developed a broad-ranging method for identifying key hydrogen-producing and consuming microorganisms through analysis of hydrogenase gene content and expression in complex anaerobic microbial communities. The method is based on a tiling hydrogenase gene oligonucleotide DNA microarray (Hydrogenase Chip), which implements a high number of probes per gene by tiling probe sequences across genes of interest at 1.67 × -2 × coverage. This design favors the avoidance of false positive gene identification in samples of DNA or RNA extracted from complex microbial communities. We applied this technique to interrogate interspecies hydrogen transfer in complex communities in (i) lab-scale reductive dehalogenating microcosms enabling us to delineate key H(2)-consuming microorganisms, and (ii) hydrogen-generating microbial mats where we found evidence for significant H(2) production by cyanobacteria. Independent quantitative PCR analysis on selected hydrogenase genes showed that this Hydrogenase Chip technique is semiquantitative. We also determined that as microbial community complexity increases, specificity must be traded for sensitivity in analyzing data from tiling DNA microarrays.

    View details for DOI 10.1038/ismej.2011.136

    View details for Web of Science ID 000301945500011

    View details for PubMedID 21993396

    View details for PubMedCentralID PMC3309348

  • Energy-Dependent Stability of Shewanella oneidensis MR-1 Biofilms JOURNAL OF BACTERIOLOGY Saville, R. M., Rakshe, S., Haagensen, J. A., Shukla, S., Spormann, A. M. 2011; 193 (13): 3257-3264


    Stability and resistance to dissolution are key features of microbial biofilms. How these macroscopic properties are determined by the physiological state of individual biofilm cells in their local physical-chemical and cellular environment is largely unknown. In order to obtain molecular and energetic insight into biofilm stability, we investigated whether maintenance of biofilm stability is an energy-dependent process and whether transcription and/or translation is required for biofilm dissolution. We found that in 12-hour-old Shewanella oneidensis MR-1 biofilms, a reduction in cellular ATP concentration, induced either by oxygen deprivation or by addition of the inhibitor of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), dinitrophenol (DNP), or CN(-), resulted in massive dissolution. In 60-hour-old biofilms, the extent of uncoupler-induced cell loss was strongly attenuated, indicating that the integrity of older biofilms is maintained by means other than those operating in younger biofilms. In experiments with 12-hour-old biofilms, the transcriptional and translational inhibitors rifampin, tetracycline, and erythromycin were found to be ineffective in preventing energy starvation-induced detachment, suggesting that neither transcription nor translation is required for this process. Biofilms of Vibrio cholerae were also induced to dissolve upon CCCP addition to an extent similar to that in S. oneidensis. However, Pseudomonas aeruginosa and P. putida biofilms remained insensitive to CCCP addition. Collectively, our data show that metabolic energy is directly or indirectly required for maintaining cell attachment, and this may represent a common but not ubiquitous mechanism for stability of microbial biofilms.

    View details for DOI 10.1128/JB.00251-11

    View details for Web of Science ID 000291592600010

    View details for PubMedID 21572002

    View details for PubMedCentralID PMC3133257

  • Site-Specific Mobilization of Vinyl Chloride Respiration Islands by a Mechanism Common in Dehalococcoides BMC GENOMICS McMurdie, P. J., Hug, L. A., Edwards, E. A., Holmes, S., Spormann, A. M. 2011; 12


    Vinyl chloride is a widespread groundwater pollutant and Group 1 carcinogen. A previous comparative genomic analysis revealed that the vinyl chloride reductase operon, vcrABC, of Dehalococcoides sp. strain VS is embedded in a horizontally-acquired genomic island that integrated at the single-copy tmRNA gene, ssrA.We targeted conserved positions in available genomic islands to amplify and sequence four additional vcrABC -containing genomic islands from previously-unsequenced vinyl chloride respiring Dehalococcoides enrichments. We identified a total of 31 ssrA-specific genomic islands from Dehalococcoides genomic data, accounting for 47 reductive dehalogenase homologous genes and many other non-core genes. Sixteen of these genomic islands contain a syntenic module of integration-associated genes located adjacent to the predicted site of integration, and among these islands, eight contain vcrABC as genetic 'cargo'. These eight vcrABC -containing genomic islands are syntenic across their ~12 kbp length, but have two phylogenetically discordant segments that unambiguously differentiate the integration module from the vcrABC cargo. Using available Dehalococcoides phylogenomic data we estimate that these ssrA-specific genomic islands are at least as old as the Dehalococcoides group itself, which in turn is much older than human civilization.The vcrABC -containing genomic islands are a recently-acquired subset of a diverse collection of ssrA-specific mobile elements that are a major contributor to strain-level diversity in Dehalococcoides, and may have been throughout its evolution. The high similarity between vcrABC sequences is quantitatively consistent with recent horizontal acquisition driven by ~100 years of industrial pollution with chlorinated ethenes.

    View details for DOI 10.1186/1471-2164-12-287

    View details for Web of Science ID 000293280200001

    View details for PubMedID 21635780

    View details for PubMedCentralID PMC3146451

  • Antimicrobial Peptoids Are Effective against Pseudomonas aeruginosa Biofilms ANTIMICROBIAL AGENTS AND CHEMOTHERAPY Kapoor, R., Wadman, M. W., Dohm, M. T., Czyzewski, A. M., Spormann, A. M., Barron, A. E. 2011; 55 (6): 3054-3057


    The resistance of biofilms to conventional antibiotics complicates the treatment of chronic cystic fibrosis (CF). We investigated the effects of peptoids, peptides, and conventional antibiotics on the biomass and cell viability within Pseudomonas aeruginosa biofilms. At their MICs, peptoids 1 and 1-C13(4mer) caused maximum reductions in biomass and cell viability, respectively. These results suggest that peptoids of this class could be worth exploring for the treatment of pulmonary infections occurring in CF patients.

    View details for DOI 10.1128/AAC.01516-10

    View details for Web of Science ID 000290713400090

    View details for PubMedID 21422218

    View details for PubMedCentralID PMC3101385

  • Partial Functional Replacement of CymA by SirCD in Shewanella oneidensis MR-1 JOURNAL OF BACTERIOLOGY Cordova, C. D., Schicklberger, M. F., Yu, Y., Spormann, A. M. 2011; 193 (9): 2312-2321


    The gammaproteobacterium Shewanella oneidensis MR-1 utilizes a complex electron transfer network composed primarily of c-type cytochromes to respire under anoxic conditions a variety of compounds, including fumarate, nitrate, and dimethyl sulfoxide (DMSO), in addition to the minerals Fe(III) and Mn(IV). Central to several respiratory pathways is CymA, a cytoplasmic membrane-bound tetraheme c-type cytochrome that functions as the major hydroquinone dehydrogenase. To investigate functional redundancy and plasticity in S. oneidensis MR-1 electron transport, we isolated ΔcymA suppressor mutants and characterized one biochemically and genetically. Interestingly, in the characterized ΔcymA suppressor mutant, respiration of fumarate, ferric citrate, and DMSO was restored but that of nitrate was not. The suppression was found to be due to transcriptional activation of sirC and sirD, encoding a periplasmic iron sulfur protein and an integral membrane hydroquinone dehydrogenase, respectively. Biochemical in vitro reconstitution experiments confirmed electron transport between formate and fumarate via fumarate reductase by suppressor membrane fractions. The suppression was found to be caused by insertion of an ISSod1 element upstream of the sirCD transcriptional start site, generating a novel, constitutively active hybrid promoter. This work revealed that adaptation of an alternative electron transfer pathway from quinol to terminal oxidoreductases independent of CymA occurs rapidly in S. oneidensis MR-1.

    View details for DOI 10.1128/JB.01355-10

    View details for Web of Science ID 000289670000026

    View details for PubMedID 21378180

    View details for PubMedCentralID PMC3133100

  • Indirect Modulation of the Intracellular c-Di-GMP Level in Shewanella oneidensis MR-1 by MxdA APPLIED AND ENVIRONMENTAL MICROBIOLOGY Rakshe, S., Leff, M., Spormann, A. M. 2011; 77 (6): 2196-2198


    The GGDEF domain protein MxdA, which is important for biofilm formation in Shewanella oneidensis MR-1, was hypothesized to possess diguanylate cyclase activity. Here, we demonstrate that while MxdA controls the cellular level of c-di-GMP in S. oneidensis, it modulates the c-di-GMP pool indirectly.

    View details for DOI 10.1128/AEM.01985-10

    View details for Web of Science ID 000288203500035

    View details for PubMedID 21278272

    View details for PubMedCentralID PMC3067315

  • PhyloChip microarray analysis reveals altered gastrointestinal microbial communities in a rat model of colonic hypersensitivity NEUROGASTROENTEROLOGY AND MOTILITY Nelson, T. A., Holmes, S., ALEKSEYENKO, A. V., Shenoy, M., DeSantis, T., Wu, C. H., Andersen, G. L., Winston, J., Sonnenburg, J., Pasricha, P. J., Spormann, A. 2011; 23 (2)


    Irritable bowel syndrome (IBS) is a chronic, episodic gastrointestinal disorder that is prevalent in a significant fraction of western human populations; and changes in the microbiota of the large bowel have been implicated in the pathology of the disease.Using a novel comprehensive, high-density DNA microarray (PhyloChip) we performed a phylogenetic analysis of the microbial community of the large bowel in a rat model in which intracolonic acetic acid in neonates was used to induce long lasting colonic hypersensitivity and decreased stool water content and frequency, representing the equivalent of human constipation-predominant IBS.Our results revealed a significantly increased compositional difference in the microbial communities in rats with neonatal irritation as compared with controls. Even more striking was the dramatic change in the ratio of Firmicutes relative to Bacteroidetes, where neonatally irritated rats were enriched more with Bacteroidetes and also contained a different composition of species within this phylum. Our study also revealed differences at the level of bacterial families and species.The PhyloChip is a useful and convenient method to study enteric microflora. Further, this rat model system may be a useful experimental platform to study the causes and consequences of changes in microbial community composition associated with IBS.

    View details for DOI 10.1111/j.1365-2982.2010.01637.x

    View details for Web of Science ID 000286211600017

    View details for PubMedID 21129126

    View details for PubMedCentralID PMC3353725

  • Visualization and statistical comparisons of microbial communities using R packages on Phylochip data. Pacific Symposium on Biocomputing. Pacific Symposium on Biocomputing Holmes, S., Alekseyenko, A., Timme, A., Nelson, T., Pasricha, P. J., Spormann, A. 2011: 142-153


    This article explains the statistical and computational methodology used to analyze species abundances collected using the LNBL Phylochip in a study of Irritable Bowel Syndrome (IBS) in rats. Some tools already available for the analysis of ordinary microarray data are useful in this type of statistical analysis. For instance in correcting for multiple testing we use Family Wise Error rate control and step-down tests (available in the multtest package). Once the most significant species are chosen we use the hypergeometric tests familiar for testing GO categories to test specific phyla and families. We provide examples of normalization, multivariate projections, batch effect detection and integration of phylogenetic covariation, as well as tree equalization and robustification methods.

    View details for PubMedID 21121042

  • Evaluation in a continuous-flow column of different fermenting substrates for the reductive dehalogenation of trichloroethene 7th International Groundwater Quality Conference Azizian, M., Marshall, I., Behrens, S., Spormann, A., Semprini, L. INT ASSOC HYDROLOGICAL SCIENCES. 2011: 209–212
  • Spatiotemporal activity of the mshA gene system in Shewanella oneidensis MR-1 biofilms FEMS MICROBIOLOGY LETTERS Saville, R. M., Dieckmann, N., Spormann, A. M. 2010; 308 (1): 76-83


    Type IV pili and a putative EPS biosynthetic gene cluster (mxdABCD) have been implicated previously in biofilm formation in Shewanella oneidensis MR-1. Here, we report that the mannose-sensitive hemagglutinin (MSHA) pilus mediates a reversible, d-mannose-sensitive association of cells to the substratum surface or to other cells that is critical within the first 5 microm of the biofilm from the substratum. The presence of the MSHA pilus alone is insufficient to confer biofilm-forming capacity; its activity, as mediated by the putative pilus retraction motor protein, PilT, is also required. Deletion of pilD, encoding the type IV pili prepilin peptidase, revealed that additional PilD substrate(s) may be involved in biofilm formation beyond the major structural pilin of the MSHA pilus. We also present data showing that the MSHA pilus and mxd genes encode for a complementary set of molecular machineries that constitute the dominant mechanisms enabling biofilm formation in this microorganism under hydrodynamic conditions.

    View details for DOI 10.1111/j.1574-6968.2010.01995.x

    View details for Web of Science ID 000278308000012

    View details for PubMedID 20487019

  • NanoSIP: Combining stable isotope probing and high resolution secondary ion mass spectrometry to identify diazotrophs in stratified marine microbial communities Conference on Goldschmidt 2010 - Earth, Energy, and the Environment Singer, S. W., Woebken, D., Burow, L. C., Prufert-Bebout, L., Bebout, B. M., Pett-Ridge, J., Spormann, A. M., Weber, P. K. PERGAMON-ELSEVIER SCIENCE LTD. 2010: A966–A966
  • Comparison of lactate, formate, and propionate as hydrogen donors for the reductive dehalogenation of trichloroethene in a continuous-flow column JOURNAL OF CONTAMINANT HYDROLOGY Azizian, M. F., Marshall, I. P., Behrens, S., Spormann, A. M., Semprini, L. 2010; 113 (1-4): 77-92


    A continuous-flow column study was conducted to analyze the reductive dehalogenation of trichloroethene (TCE) with aquifer material with high content of iron oxides. The column was bioaugmented with the Point Mugu (PM) culture, which is a mixed microbial enrichment culture capable of completely transforming TCE to ethene (ETH). We determined whether lactate, formate, or propionate fermentation resulted in more effective dehalogenation. Reductive dehalogenation, fermentation, and sulfate, Fe(III), and Mn(IV) reduction were all exhibited within the column. Different steady-states of dehalogenation were achieved based on the concentration of substrates added, with effective transformation to ETH obtained when ample electron donor equivalents were provided. Most of the metabolic reducing equivalents were channeled to sulfate, Fe(III), and Mn(IV) reduction. When similar electron reducing equivalents were added, the most effective dehalogenation was achieved with formate, with 14% of the electron equivalents going towards dehalogenation reactions, compared to 6.5% for lactate and 9.6% for propionate. Effective dehalogenation was maintained over 1000 days of column operation. Over 90% of electron equivalents added could be accounted for by the different electron accepting processes in the column, with 50% associated with soluble and precipitated Fe(II) and Mn(II). Bulk Fe(III) and Mn(IV) reduction was rather associated with lactate and propionate addition than formate addition. Sulfate reduction was a competing electron acceptor reaction with all three electron donors. DNA was extracted from solid coupon samples obtained during the course of the experiment and analyzed using 16S rRNA gene clone libraries and quantitative PCR. Lactate and propionate addition resulted in a significant increase in Geobacter, Spirochaetes, and Desulfitobacterium phylotypes relative to "Dehalococcoides" when compared to formate addition. Results from the molecular biological analyses support chemical observations that a greater percentage of the electron donor addition was channeled to Fe(III) reduction when lactate and propionate were added compared to formate, and formate was more effective than lactate in supporting dehalogenation. The results demonstrate the importance of electron donor selection and competing electron acceptor reactions when implementing reductive dehalogenation remediation technologies.

    View details for DOI 10.1016/j.jconhyd.2010.02.004

    View details for Web of Science ID 000276766000006

    View details for PubMedID 20202715

  • Contributions of Francisella tularensis subsp novicida Chitinases and Sec Secretion System to Biofilm Formation on Chitin APPLIED AND ENVIRONMENTAL MICROBIOLOGY Margolis, J. J., El-Etr, S., Joubert, L., Moore, E., Robison, R., Rasley, A., Spormann, A. M., Monack, D. M. 2010; 76 (2): 596-608


    Francisella tularensis, the zoonotic cause of tularemia, can infect numerous mammals and other eukaryotes. Although studying F. tularensis pathogenesis is essential to comprehending disease, mammalian infection is just one step in the ecology of Francisella species. F. tularensis has been isolated from aquatic environments and arthropod vectors, environments in which chitin could serve as a potential carbon source and as a surface for attachment and growth. We show that F. tularensis subsp. novicida forms biofilms during the colonization of chitin surfaces. The ability of F. tularensis to persist using chitin as a sole carbon source is dependent on chitinases, since mutants lacking chiA or chiB are attenuated for chitin colonization and biofilm formation in the absence of exogenous sugar. A genetic screen for biofilm mutants identified the Sec translocon export pathway and 14 secreted proteins. We show that these genes are important for initial attachment during biofilm formation. We generated defined deletion mutants by targeting two chaperone genes (secB1 and secB2) involved in Sec-dependent secretion and four genes that encode putative secreted proteins. All of the mutants were deficient in attachment to polystyrene and chitin surfaces and for biofilm formation compared to wild-type F. novicida. In contrast, mutations in the Sec translocon and secreted factors did not affect virulence. Our data suggest that biofilm formation by F. tularensis promotes persistence on chitin surfaces. Further study of the interaction of F. tularensis with the chitin microenvironment may provide insight into the environmental survival and transmission mechanisms of this pathogen.

    View details for DOI 10.1128/AEM.02037-09

    View details for Web of Science ID 000273354200027

    View details for PubMedID 19948864

    View details for PubMedCentralID PMC2805214

  • Role of extracellular polymeric substances in metal ion complexation on Shewanella oneidensis: Batch uptake, thermodynamic modeling, ATR-FTIR, and EXAFS study GEOCHIMICA ET COSMOCHIMICA ACTA Ha, J., Gelabert, A., Spormann, A. M., Brown, G. E. 2010; 74 (1): 1-15
  • Periplasmic Electron Transfer via the c-Type Cytochromes MtrA and FccA of Shewanella oneidensis MR-1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY Schuetz, B., Schicklberger, M., Kuermann, J., Spormann, A. M., Gescher, J. 2009; 75 (24): 7789-7796


    Dissimilatory microbial reduction of insoluble Fe(III) oxides is a geochemically and ecologically important process which involves the transfer of cellular, respiratory electrons from the cytoplasmic membrane to insoluble, extracellular, mineral-phase electron acceptors. In this paper evidence is provided for the function of the periplasmic fumarate reductase FccA and the decaheme c-type cytochrome MtrA in periplasmic electron transfer reactions in the gammaproteobacterium Shewanella oneidensis. Both proteins are abundant in the periplasm of ferric citrate-reducing S. oneidensis cells. In vitro fumarate reductase FccA and c-type cytochrome MtrA were reduced by the cytoplasmic membrane-bound protein CymA. Electron transfer between CymA and MtrA was 1.4-fold faster than the CymA-catalyzed reduction of FccA. Further experiments showing a bidirectional electron transfer between FccA and MtrA provided evidence for an electron transfer network in the periplasmic space of S. oneidensis. Hence, FccA could function in both the electron transport to fumarate and via MtrA to mineral-phase Fe(III). Growth experiments with a DeltafccA deletion mutant suggest a role of FccA as a transient electron storage protein.

    View details for DOI 10.1128/AEM.01834-09

    View details for Web of Science ID 000272429100028

    View details for PubMedID 19837833

  • Localized Plasticity in the Streamlined Genomes of Vinyl Chloride Respiring Dehalococcoides PLOS GENETICS McMurdie, P. J., Behrens, S. F., Mueller, J. A., Goeke, J., Ritalahti, K. M., Wagner, R., Goltsman, E., Lapidus, A., Holmes, S., Loeffler, F. E., Spormann, A. M. 2009; 5 (11)


    Vinyl chloride (VC) is a human carcinogen and widespread priority pollutant. Here we report the first, to our knowledge, complete genome sequences of microorganisms able to respire VC, Dehalococcoides sp. strains VS and BAV1. Notably, the respective VC reductase encoding genes, vcrAB and bvcAB, were found embedded in distinct genomic islands (GEIs) with different predicted integration sites, suggesting that these genes were acquired horizontally and independently by distinct mechanisms. A comparative analysis that included two previously sequenced Dehalococcoides genomes revealed a contextually conserved core that is interrupted by two high plasticity regions (HPRs) near the Ori. These HPRs contain the majority of GEIs and strain-specific genes identified in the four Dehalococcoides genomes, an elevated number of repeated elements including insertion sequences (IS), as well as 91 of 96 rdhAB, genes that putatively encode terminal reductases in organohalide respiration. Only three core rdhA orthologous groups were identified, and only one of these groups is supported by synteny. The low number of core rdhAB, contrasted with the high rdhAB numbers per genome (up to 36 in strain VS), as well as their colocalization with GEIs and other signatures for horizontal transfer, suggests that niche adaptation via organohalide respiration is a fundamental ecological strategy in Dehalococccoides. This adaptation has been exacted through multiple mechanisms of recombination that are mainly confined within HPRs of an otherwise remarkably stable, syntenic, streamlined genome among the smallest of any free-living microorganism.

    View details for DOI 10.1371/journal.pgen.1000714

    View details for Web of Science ID 000272419500010

    View details for PubMedID 19893622

    View details for PubMedCentralID PMC2764846

  • Interaction of Zn(II)aq with mineral nano-and microparticles, bacterial surfaces, and biofilm-coated metal oxides Brown, G. E., Ha, J., Singer, D. M., Wang, Y., Gelabert, A., Farges, F., Trainor, T. P., Bargar, J. R., Eng, P., Spormann, A. M. AMER CHEMICAL SOC. 2009
  • Impact of Shewanella oneidensis MR-1 biofilm coatings on the reactivity of hematite Wang, Y., Gelabert, A., Choi, Y., Ha, J., Gescher, J., Bargar, J. R., Rogers, J., Eng, P., Cordova, C. D., Spormann, A. M., Brown, G. E. AMER CHEMICAL SOC. 2009
  • Monitoring abundance and expression of "Dehalococcoides" species chloroethene-reductive dehalogenases in a tetrachloroethene-dechlorinating flow column APPLIED AND ENVIRONMENTAL MICROBIOLOGY Behrens, S., Azizian, M. F., McMurdie, P. J., Sabalowsky, A., Dolan, M. E., Semprini, L., Spormann, A. M. 2008; 74 (18): 5695-5703


    We investigated the distribution and activity of chloroethene-degrading microorganisms and associated functional genes during reductive dehalogenation of tetrachloroethene to ethene in a laboratory continuous-flow column. Using real-time PCR, we quantified "Dehalococcoides" species 16S rRNA and chloroethene-reductive dehalogenase (RDase) genes (pceA, tceA, vcrA, and bvcA) in nucleic acid extracts from different sections of the column. Dehalococcoides 16S rRNA gene copies were highest at the inflow port [(3.6 +/- 0.6) x 10(6) (mean +/- standard deviation) per gram soil] where the electron donor and acceptor were introduced into the column. The highest transcript numbers for tceA, vcrA, and bvcA were detected 5 to 10 cm from the column inflow. bvcA was the most highly expressed of all RDase genes and the only vinyl chloride reductase-encoding transcript detectable close to the column outflow. Interestingly, no expression of pceA was detected in the column, despite the presence of the genes in the microbial community throughout the column. By comparing the 16S rRNA gene copy numbers to the sum of all four RDase genes, we found that 50% of the Dehalococcoides population in the first part of the column did not contain either one of the known chloroethene RDase genes. Analysis of 16S rRNA gene clone libraries from both ends of the flow column revealed a microbial community dominated by members of Firmicutes and Actinobacteria. Higher clone sequence diversity was observed near the column outflow. The results presented have implications for our understanding of the ecophysiology of reductively dehalogenating Dehalococcoides spp. and their role in bioremediation of chloroethenes.

    View details for DOI 10.1128/AEM.00926-08

    View details for Web of Science ID 000259017400012

    View details for PubMedID 18676701

    View details for PubMedCentralID PMC2547056

  • Continuous-flow column study of reductive dehalogenation of PCE upon bioaugmentation with the Evanite enrichment culture JOURNAL OF CONTAMINANT HYDROLOGY Azizian, M. F., Behrens, S., Sabalowsky, A., Dolan, M. E., Spormann, A. M., Semprini, L. 2008; 100 (1-2): 11-21


    A continuous-flow anaerobic column experiment was conducted to evaluate the reductive dechlorination of tetrachloroethene (PCE) in Hanford aquifer material after bioaugmentation with the Evanite (EV) culture. An influent PCE concentration of 0.09 mM was transformed to vinyl chloride (VC) and ethene (ETH) within a hydraulic residence time of 1.3 days. The experimental breakthrough curves were described by the one-dimensional two-site-nonequilibrium transport model. PCE dechlorination was observed after bioaugmentation and after the lactate concentration was increased from 0.35 to 0.67 mM. At the onset of reductive dehalogenation, cis-dichloroethene (c-DCE) concentrations in the column effluent exceeded the influent PCE concentration indicating enhanced PCE desorption and transformation. When the lactate concentration was increased to 1.34 mM, c-DCE reduction to vinyl chloride (VC) and ethene (ETH) occurred. Spatial rates of PCE and VC transformation were determined in batch-incubated microcosms constructed with aquifer samples obtained from the column. PCE transformation rates were highest in the first 5 cm from the column inlet and decreased towards the column effluent. Dehalococcoides cell numbers dropped from approximately 73.5% of the total Bacterial population in the original inocula, to about 0.5% to 4% throughout the column. The results were consistent with estimates of electron donor utilization, with 4% going towards dehalogenation reactions.

    View details for DOI 10.1016/j.jconhyd.2008.04.006

    View details for Web of Science ID 000259387600002

    View details for PubMedID 18550206

  • Towards environmental systems biology of Shewanella NATURE REVIEWS MICROBIOLOGY Fredrickson, J. K., Romine, M. F., Beliaev, A. S., Auchtung, J. M., Driscoll, M. E., Gardner, T. S., Nealson, K. H., Osterman, A. L., Pinchuk, G., Reed, J. L., Rodionov, D. A., Rodrigues, J. L., Saffarini, D. A., Serres, M. H., Spormann, A. M., Zhulin, I. B., Tiedje, J. M. 2008; 6 (8): 592-603


    Bacteria of the genus Shewanella are known for their versatile electron-accepting capacities, which allow them to couple the decomposition of organic matter to the reduction of the various terminal electron acceptors that they encounter in their stratified environments. Owing to their diverse metabolic capabilities, shewanellae are important for carbon cycling and have considerable potential for the remediation of contaminated environments and use in microbial fuel cells. Systems-level analysis of the model species Shewanella oneidensis MR-1 and other members of this genus has provided new insights into the signal-transduction proteins, regulators, and metabolic and respiratory subsystems that govern the remarkable versatility of the shewanellae.

    View details for DOI 10.1038/nrmicro1947

    View details for Web of Science ID 000257696600010

    View details for PubMedID 18604222

  • The NASA Astrobiology Roadmap ASTROBIOLOGY Marais, D. J., Nuth, J. A., Allamandola, L. J., Boss, A. P., Farmer, J. D., Hoehler, T. M., Jakosky, B. M., Meadows, V. S., Pohorille, A., Runnegar, B., Spormann, A. M. 2008; 8 (4): 715-730


    The NASA Astrobiology Roadmap provides guidance for research and technology development across the NASA enterprises that encompass the space, Earth, and biological sciences. The ongoing development of astrobiology roadmaps embodies the contributions of diverse scientists and technologists from government, universities, and private institutions. The Roadmap addresses three basic questions: how does life begin and evolve, does life exist elsewhere in the universe, and what is the future of life on Earth and beyond? Seven Science Goals outline the following key domains of investigation: understanding the nature and distribution of habitable environments in the universe, exploring for habitable environments and life in our own Solar System, understanding the emergence of life, determining how early life on Earth interacted and evolved with its changing environment, understanding the evolutionary mechanisms and environmental limits of life, determining the principles that will shape life in the future, and recognizing signatures of life on other worlds and on early Earth. For each of these goals, Science Objectives outline more specific high priority efforts for the next three to five years. These eighteen objectives are being integrated with NASA strategic planning.

    View details for DOI 10.1089/ast.2008.0819

    View details for Web of Science ID 000260369000002

    View details for PubMedID 18793098

  • Synchrotron X-ray studies of bacteria-mineral-metal ion interactions 18th Annual V M Goldschmidt Conference Brown, G. E., Gelabert, A., Wang, Y., Cismasu, C., Ha, J., Ona-Nguema, G., Benzerara, K., Morin, G., Yang, Y., Juillot, F., Guyot, F., CALAS, G., Yoon, T. H., Bargar, J. R., Eng, P. J., Templeton, A. S., Trainor, T. P., Spormann, A. M. PERGAMON-ELSEVIER SCIENCE LTD. 2008: A116–A116
  • Study of Proton, Pb+2 and Zn+2 adsorption onto Shewanella oneidensis MR-1 strain and a mutant Strain (Delta EPS): Spectroscopic observation and modeling approach 18th Annual V M Goldschmidt Conference Ha, J., Gelabert, A., Wang, Y., Spormann, A. M., Brown, G. E. PERGAMON-ELSEVIER SCIENCE LTD. 2008: A339–A339
  • Impact of S-oneidensis MR-1 biofilm coatings on trace element partitioning at metal-oxide/water interfaces: A long period XSW-FY study 18th Annual V M Goldschmidt Conference Wang, Y., Gelabert, A., Ha, J., Ona-Nguema, G., Gescher, J., Cordova-Ardy, C., Bargar, J. R., Rogers, J., Eng, P. J., Ghose, S. K., Spormann, A. M., Brown, G. E. PERGAMON-ELSEVIER SCIENCE LTD. 2008: A1003–A1003
  • Parameters controlling metal adsorption at the biofilm/mineral interface: Evidence for a diffusion limited process and comparison with thermodynamic modeling 18th Annual V M Goldschmidt Conference Gelabert, A., Wang, Y., Ha, J., Ona-Nguema, G., Spormann, A. M., Bargar, J. R., Rogers, J., Eng, P., Ghose, S., Brown, G. E. PERGAMON-ELSEVIER SCIENCE LTD. 2008: A301–A301
  • Dissimilatory iron reduction in Escherichia coli: identification of CymA of Shewanella oneidensis and NapC of E-coli as ferric reductases MOLECULAR MICROBIOLOGY Gescher, J. S., Cordova, C. D., Spormann, A. M. 2008; 68 (3): 706-719


    Over geological time scales, microbial reduction of chelated Fe(III) or Fe(III) minerals has profoundly affected today's composition of our bio- and geosphere. However, the electron transfer reactions that are specific and defining for dissimilatory iron(III)-reducing (DIR) bacteria are not well understood. Using a synthetic biology approach involving the reconstruction of the putative electron transport chain of the DIR bacterium Shewanella oneidensis MR-1 in Escherichia coli, we showed that expression of cymA was necessary and sufficient to convert E. coli into a DIR bacterium. In intact cells, the Fe(III)-reducing activity was limited to Fe(III) NTA as electron acceptor. In vitro biochemical analysis indicated that CymA, which is a cytoplasmic membrane-associated tetrahaem c-type cytochrome, carries reductase activity towards Fe(III) NTA, Fe(III) citrate, as well as to AQDS, a humic acid analogue. The in vitro specific activities of Fe(III) citrate reductase and AQDS reductase of E. coli spheroplasts were 10x and 30x higher, respectively, relative to the specific rates observed in intact cells, suggesting that access of chelated and insoluble forms of Fe(III) and AQDS is restricted in whole cells. Interestingly, the E. coli CymA orthologue NapC also carried ferric reductase activity. Our data support the argument that the biochemical mechanism of Fe(III) reduction per se was not the key innovation leading to environmental relevant DIR bacteria. Rather, the evolution of an extension of the electron transfer pathway from the Fe(III) reductase CymA to the cell surface via a system of periplasmic and outer membrane cytochrome proteins enabled access to diffusion-impaired electron acceptors.

    View details for DOI 10.1111/j.1365-2958.2008.06183.x

    View details for Web of Science ID 000254641600014

    View details for PubMedID 18394146

  • Linking microbial phylogeny to metabolic activity at the single-cell level by using enhanced element labeling-catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS APPLIED AND ENVIRONMENTAL MICROBIOLOGY Behrens, S., Loesekann, T., Pett-Ridge, J., Weber, P. K., Ng, W., Stevenson, B. S., Hutcheon, I. D., Relman, D. A., Spormann, A. M. 2008; 74 (10): 3143-3150


    To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with (13)C-carbon and (15)N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities.

    View details for DOI 10.1128/AEM.00191-08

    View details for Web of Science ID 000256074900028

    View details for PubMedID 18359832

    View details for PubMedCentralID PMC2394947

  • Synchrotron X-ray studies of heavy metal mineral-microbe interactions 8th International Symposium on the Geochemistry of the Earths Surface (GES-8) Brown, G. E., Wang, Y., Gelabert, A., Ha, J., Cismasu, C., Ona-Nguema, G., Benzerara, K., Miot, J., Menguy, N., Morin, G., Juillot, F., Guyot, F., CALAS, G., Farges, F., Trainor, T. P., Gescher, J., Cordova, C., Spormann, A. M. MINERALOGICAL SOC. 2008: 169–73
  • Physiology of microbes in biofilms BACTERIAL BIOFILMS Spormann, A. M. 2008; 322: 17-36


    Microbial biofilms are governed by an intricate interplay between physical-chemical factors and the physiological and genetic properties of the inhabiting microbes. Many of the physiological traits that are exhibited in a biofilm environment have been observed and studied previously in detail in planktonic cultures. However, their differential and combinatorial phenotypic expression in distinct subpopulations localized to different regions in a biofilm is the cause for the overall biofilm heterogeneity. In this chapter, the causes and consequences of this interplay are elaborated with a special focus on processes controlling biofilm stability and dispersal.

    View details for Web of Science ID 000258523900002

    View details for PubMedID 18453270

  • vpsA- and luxO-independent biofilms of Vibrio cholerae FEMS MICROBIOLOGY LETTERS Mueller, J., Miller, M. C., Nielsen, A. T., Schoolnik, G. K., Spormann, A. M. 2007; 275 (2): 199-206


    The natural life cycle of Vibrio cholerae involves the transitioning of cells between different environmental surfaces such as the chitinous shell of Crustaceae and the epithelial layer of the human intestine. Previous studies using static biofilm systems showed a strict dependence of biofilm formation on the vps and lux genes, which are essential for exopolysaccharide formation and cell-cell signaling, respectively. The authors' report here that in biofilms grown under hydrodynamic conditions, DeltavpsA and DeltaluxO mutants of V. cholerae do form pronounced, three-dimensional biofilms that resemble all aspects of wild-type biofilms. By genetic experiments, it was shown that in hydrodynamically grown biofilms this independence of vpsA is due to the expression of rpoS, which is a negative regulator of vpsA expression. Biofilms also underwent substantial dissolution after 96 h that could be induced by a simple stop of medium flow. The studies indicate that metabolic conditions control the reversible attachment of cells to the biofilm matrix and are key in regulating biofilm cell physiology via RpoS. Furthermore, the results redefine the roles of vps and quorum-sensing in V. cholerae biofilms.

    View details for DOI 10.1111/j.1574-6968.2007.00884.x

    View details for PubMedID 17697110

  • Designing a dissimilatory iron reducer. Reconstitution of the Fe(III)-reducing electron transport chain of Shewanella oneidensis MR-1 in Escherichia coli 17th Annual V M Goldschmidt Conference Gescher, J., Cordova-Ardy, C., Spormann, A. PERGAMON-ELSEVIER SCIENCE LTD. 2007: A318–A318
  • Unusual codon bias in vinyl chloride reductase genes of Dehalococcoides species APPLIED AND ENVIRONMENTAL MICROBIOLOGY McMurdie, P. J., Behrens, S. F., Holmes, S., Spormann, A. M. 2007; 73 (8): 2744-2747


    Vinyl chloride reductases (VC-RDase) are the key enzymes for complete microbial reductive dehalogenation of chloroethenes, including the groundwater pollutants tetrachloroethene and trichloroethene. Analysis of the codon usage of the VC-RDase genes vcrA and bvcA showed that these genes are highly unusual and are characterized by a low G+C fraction at the third position. The third position of codons in VC-RDase genes is biased toward the nucleotide T, even though available Dehalococcoides genome sequences indicate the absence of any tRNAs matching codons that end in T. The comparatively high level of abnormality in the codon usage of VC-RDase genes suggests an evolutionary history that is different from that of most other Dehalococcoides genes.

    View details for DOI 10.1128/AEM.02768-06

    View details for Web of Science ID 000246542400039

    View details for PubMedID 17308190

    View details for PubMedCentralID PMC1855607

  • Hydrogen metabolism in Shewanella oneidensis MR-1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY Meshulam-Simon, G., Behrens, S., Choo, A. D., Spormann, A. M. 2007; 73 (4): 1153-1165


    Shewanella oneidensis MR-1 is a facultative sediment microorganism which uses diverse compounds, such as oxygen and fumarate, as well as insoluble Fe(III) and Mn(IV) as electron acceptors. The electron donor spectrum is more limited and includes metabolic end products of primary fermenting bacteria, such as lactate, formate, and hydrogen. While the utilization of hydrogen as an electron donor has been described previously, we report here the formation of hydrogen from pyruvate under anaerobic, stationary-phase conditions in the absence of an external electron acceptor. Genes for the two S. oneidensis MR-1 hydrogenases, hydA, encoding a periplasmic [Fe-Fe] hydrogenase, and hyaB, encoding a periplasmic [Ni-Fe] hydrogenase, were found to be expressed only under anaerobic conditions during early exponential growth and into stationary-phase growth. Analyses of DeltahydA, DeltahyaB, and DeltahydA DeltahyaB in-frame-deletion mutants indicated that HydA functions primarily as a hydrogen-forming hydrogenase while HyaB has a bifunctional role and represents the dominant hydrogenase activity under the experimental conditions tested. Based on results from physiological and genetic experiments, we propose that hydrogen is formed from pyruvate by multiple parallel pathways, one pathway involving formate as an intermediate, pyruvate-formate lyase, and formate-hydrogen lyase, comprised of HydA hydrogenase and formate dehydrogenase, and a formate-independent pathway involving pyruvate dehydrogenase. A reverse electron transport chain is potentially involved in a formate-hydrogen lyase-independent pathway. While pyruvate does not support a fermentative mode of growth in this microorganism, pyruvate, in the absence of an electron acceptor, increased cell viability in anaerobic, stationary-phase cultures, suggesting a role in the survival of S. oneidensis MR-1 under stationary-phase conditions.

    View details for DOI 10.1128/AEM.01588-06

    View details for Web of Science ID 000244443700015

    View details for PubMedID 17189435

    View details for PubMedCentralID PMC1828657

  • COLL 82-Microbial reduction of hematite: Effects of particle size and exopolysaccharides Ha, J., Cordova, C. D., Yoon, T. H., Spormann, A. M., Brown, G. E. AMER CHEMICAL SOC. 2006
  • COLL 418-Soft X-ray spectromicroscopy studies of environmental interfaces Brown, G. E., Benzerara, K., Yoon, T. H., Ha, J., Cordova, C. D., Spormann, A. M., Morin, G., Calas, G., Tyliszczak, T. AMER CHEMICAL SOC. 2006
  • Applications of synchrotron radiation to processes at environmental interfaces 16th Annual V M Goldschmidt Conference Brown, G. E., Benzerara, K., Yoon, T. H., Ha, J., Cordova, C. D., Spormann, A. M., Tyliszcak, T., Tanwar, K. S., Trainor, T. P., Eng, P. J., Kendelewicz, T., Yamamoto, S., Bluhm, H., Ketteler, G., Salmeron, M., Nilsson, A. PERGAMON-ELSEVIER SCIENCE LTD. 2006: A69–A69
  • Microbial reduction of hematite: Effects of particle size and exopolysaccharides 16th Annual V M Goldschmidt Conference Ha, J., Cordova, C., Yoon, T., Spormann, A. M., Brown, G. E. PERGAMON-ELSEVIER SCIENCE LTD. 2006: A221–A221
  • Control of formation and cellular detachment from Shewanella oneidensis MR-1 biofilms by cyclic di-GMP JOURNAL OF BACTERIOLOGY Thormann, K. M., Duttler, S., Saville, R. M., Hyodo, M., Shukla, S., Hayakawa, Y., Spormann, A. M. 2006; 188 (7): 2681-2691


    Stability and resilience against environmental perturbations are critical properties of medical and environmental biofilms and pose important targets for their control. Biofilm stability is determined by two mutually exclusive processes: attachment of cells to and detachment from the biofilm matrix. Using Shewanella oneidensis MR-1, an environmentally versatile, Fe(III) and Mn(IV) mineral-reducing microorganism, we identified mxdABCD as a new set of genes essential for formation of a three-dimensional biofilm. Molecular analysis revealed that mxdA encodes a cyclic bis(3',5')guanylic acid (cyclic di-GMP)-forming enzyme with an unusual GGDEF motif, i.e., NVDEF, which is essential for its function. mxdB encodes a putative membrane-associated glycosyl transferase. Both genes are essential for matrix attachment. The attachment-deficient phenotype of a DeltamxdA mutant was rescued by ectopic expression of VCA0956, encoding another diguanylate cyclase. Interestingly, a rapid cellular detachment from the biofilm occurred upon induction of yhjH, a gene encoding an enzyme that has been shown to have phosphodiesterase activity. In this way, it was possible to bypass the previously identified sudden depletion of molecular oxygen as an environmental trigger to induce biofilm dissolution. We propose a model for c-di-GMP as a key intracellular regulator for controlling biofilm stability by shifting the state of a biofilm cell between attachment and detachment in a concentration-dependent manner.

    View details for DOI 10.1128/JB.188.7.2681-2691.2006

    View details for Web of Science ID 000236403300042

    View details for PubMedID 16547056

    View details for PubMedCentralID PMC1428383

  • Genomic comparisons among gamma-proteobacteria ENVIRONMENTAL MICROBIOLOGY Mrazek, J., Spormann, A. M., Karlin, S. 2006; 8 (2): 273-288


    Predicted highly expressed (PHX) genes are compared for 16 gamma-proteobacteria and their similarities and differences are interpreted with respect to known or predicted physiological characteristics of the organisms. Predicted highly expressed genes often reflect the organism's predominant lifestyle, habitat, nutrition sources and metabolic propensities. This technique allows to predict principal metabolic activities of the microorganisms operating in their natural habitats. Among our findings is an unusually high number of PHX enzymes acting in cell wall biosynthesis, amino acid biosynthesis and replication in the ant endosymbiont Blochmannia floridanus. We ascribe the abundance of these PHX genes to specific aspects of the relationship between the bacterium and its host. Xanthomonas campestris is unique with a very high number of PHX genes acting in flagellum biosynthesis, which may play a special role during its pathogenicity. Shewanella oneidensis possesses three protein complexes which all can function as complex I in the respiratory chain but only the Na(+)-transporting NADH:ubiquinone oxidoreductase nqr-2 operon is PHX. The PHX genes of Vibrio parahaemolyticus are consistent with the microorganism's adaptation to extremely fast growth rates. Comparative analysis of PHX genes from complex environmental genomic sequences as well as from uncultured pathogenic microbes can provide a novel, useful tool to predict global flux of matter and key intermediates.

    View details for DOI 10.1111/j.1462-2920.2005.00894.x

    View details for Web of Science ID 000234647600009

    View details for PubMedID 16423015

  • Evidence supporting predicted metabolic pathways for Vibrio cholerae: gene expression data and clinical tests NUCLEIC ACIDS RESEARCH Shi, J., Romero, P. R., Schoolnik, G. K., Spormann, A. M., Karp, P. D. 2006; 34 (8): 2438-2444


    Vibrio cholerae, the etiological agent of the diarrheal illness cholera, can kill an infected adult in 24 h. V.cholerae lives as an autochthonous microbe in estuaries, rivers and coastal waters. A better understanding of its metabolic pathways will assist the development of more effective treatments and will provide a deeper understanding of how this bacterium persists in natural aquatic habitats. Using the completed V.cholerae genome sequence and PathoLogic software, we created VchoCyc, a pathway-genome database that predicted 171 likely metabolic pathways in the bacterium. We report here experimental evidence supporting the computationally predicted pathways. The evidence comes from microarray gene expression studies of V.cholerae in the stools of three cholera patients [D. S. Merrell, S. M. Butler, F. Qadri, N. A. Dolganov, A. Alam, M. B. Cohen, S. B. Calderwood, G. K. Schoolnik and A. Camilli (2002) Nature, 417, 642-645.], from gene expression studies in minimal growth conditions and LB rich medium, and from clinical tests that identify V.cholerae. Expression data provide evidence supporting 92 (53%) of the 171 pathways. The clinical tests provide evidence supporting seven pathways, with six pathways supported by both methods. VchoCyc provides biologists with a useful tool for analyzing this organism's metabolic and genomic information, which could lead to potential insights into new anti-bacterial agents. VchoCyc is available in the BioCyc database collection (

    View details for DOI 10.1093/nar/gkl310

    View details for PubMedID 16682451

  • Dynamics of Shewanella oneidensis MR-1 biofilms developing on Fe(III) mineral surfaces 230th National Meeting of the American-Chemical-Society Cordova, C. D., Brown, G. E., Spormann, A. M. AMER CHEMICAL SOC. 2005: U1724–U1725
  • Electrical properties of bacterial outer membranes 230th National Meeting of the American-Chemical-Society Rosso, K. M., Yanina, S. V., Lower, B. H., Gorby, Y. A., Spormann, A. M., Brown, G. E. AMER CHEMICAL SOC. 2005: U1727–U1727
  • Year-one activities at the Stanford Environmental Molecular Science Institute 230th National Meeting of the American-Chemical-Society Brown, G. E., Bluhm, H., Brown, B. A., Chaka, A. M., Constantz, B., Fendorf, S., Foster, A. L., Myneni, S. C., Nilsson, A., Rosso, K. M., Salmeron, M., Saltzman, J., Spormann, A. M., Trainor, T. P. AMER CHEMICAL SOC. 2005: U1714–U1715
  • Regulation of Shewanella oneidensis MR-1 biofilms by c-di-GMP 230th National Meeting of the American-Chemical-Society Thormann, K. N., Saville, R. M., Duttler, S., Shukla, S., Spormann, A. M. AMER CHEMICAL SOC. 2005: U1728–U1728
  • Role of organic molecules and microbial organisms in metal ion sorption processes. 229th National Meeting of the American-Chemical-Society (ACS) Brown, G. E., Yoon, T. H., Johnson, S. B., Templeton, A. S., Trainor, T. P., Benzerara, K., Bostick, B. C., Kendelewicz, T., Doyle, C. S., Spormann, A. M. AMER CHEMICAL SOC. 2005: U784–U785
  • Induction of rapid detachment in Shewanella oneidensis MR-1 biofilms JOURNAL OF BACTERIOLOGY Thormann, K. M., Saville, R. M., Shukla, S., Spormann, A. M. 2005; 187 (3): 1014-1021


    Active detachment of cells from microbial biofilms is a critical yet poorly understood step in biofilm development. We discovered that detachment of cells from biofilms of Shewanella oneidensis MR-1 can be induced by arresting the medium flow in a hydrodynamic biofilm system. Induction of detachment was rapid, and substantial biofilm dispersal started as soon as 5 min after the stop of flow. We developed a confocal laser scanning microscopy-based assay to quantify detachment. The extent of biomass loss was found to be dependent on the time interval of flow stop and on the thickness of the biofilm. Up to 80% of the biomass of 16-h-old biofilms could be induced to detach. High-resolution microscopy studies revealed that detachment was associated with an overall loosening of the biofilm structure and a release of individual cells or small cell clusters. Swimming motility was not required for detachment. Although the loosening of cells from the biofilm structure was observed evenly throughout thin biofilms, the most pronounced detachment in thicker biofilms occurred in regions exposed to the flow of medium, suggesting a metabolic control of detachability. Deconvolution of the factors associated with the stop of medium flow revealed that a sudden decrease in oxygen tension is the predominant trigger for initiating detachment of individual cells. In contrast, carbon limitation did not trigger any substantial detachment, suggesting a physiological link between oxygen sensing or metabolism and detachment. In-frame deletions were introduced into genes encoding the known and putative global transcriptional regulators ArcA, CRP, and EtrA (FNR), which respond to changes in oxygen tension in S. oneidensis MR-1. Biofilms of null mutants in arcA and crp were severely impacted in the stop-of-flow-induced detachment response, suggesting a role for these genes in regulation of detachment. In contrast, an DeltaetrA mutant displayed a variable detachment phenotype. From this genetic evidence we conclude that detachment is a biologically controlled process and that a rapid change in oxygen concentration is a critical factor in detachment and, consequently, in dispersal of S. oneidensis cells from biofilms. Similar mechanisms might also operate in other bacteria.

    View details for DOI 10.1128/JB.187.3.1014-1021.2005

    View details for Web of Science ID 000226705200022

    View details for PubMedID 15659679

    View details for PubMedCentralID PMC545703

  • Environmental interfaces, heavy metals, microbes, and plants: Applications of XAFS spectroscopy and related synchrotron radiation methods to environmental science PHYSICA SCRIPTA Brown, G. E., Catalano, J. G., Templeton, A. S., Trainor, T. P., Farges, F., Bostick, B. C., Kendelewicz, T., Doyle, C. S., Spormann, A. M., Revill, K., Morin, G., Juillot, F., Calas, G. 2005; T115: 80-87
  • Dynamics and control of biofilms of the oligotrophic bacterium Caulobacter crescentus JOURNAL OF BACTERIOLOGY Entcheva-Dimitrov, P., Spormann, A. M. 2004; 186 (24): 8254-8266


    Caulobacter crescentus is an oligotrophic alpha-proteobacterium with a complex cell cycle involving sessile-stalked and piliated, flagellated swarmer cells. Because the natural lifestyle of C. crescentus intrinsically involves a surface-associated, sessile state, we investigated the dynamics and control of C. crescentus biofilms developing on glass surfaces in a hydrodynamic system. In contrast to biofilms of the well-studied Pseudomonas aeruginosa, Escherichia coli, and Vibrio cholerae, C. crescentus CB15 cells form biphasic biofilms, consisting predominantly of a cell monolayer biofilm and a biofilm containing densely packed, mushroom-shaped structures. Based on comparisons between the C. crescentus strain CB15 wild type and its holdfast (hfsA; DeltaCC0095), pili (DeltapilA-cpaF::Omegaaac3), motility (motA), flagellum (flgH) mutants, and a double mutant lacking holdfast and flagellum (hfsA; flgH), a model for biofilm formation in C. crescentus is proposed. For both biofilm forms, the holdfast structure at the tip of a stalked cell is crucial for mediating the initial attachment. Swimming motility by means of the single polar flagellum enhances initial attachment and enables progeny swarmer cells to escape from the monolayer biofilm. The flagellum structure also contributes to maintaining the mushroom structure. Type IV pili enhance but are not absolutely required for the initial adhesion phase. However, pili are essential for forming and maintaining the well-defined three-dimensional mushroom-shaped biofilm. The involvement of pili in mushroom architecture is a novel function for type IV pili in C. crescentus. These unique biofilm features demonstrate a spatial diversification of the C. crescentus population into a sessile, "stem cell"-like subpopulation (monolayer biofilm), which generates progeny cells capable of exploring the aqueous, oligotrophic environment by swimming motility and a subpopulation accumulating in large mushroom structures.

    View details for DOI 10.1128/JB.186.24.8254-8266.2004

    View details for Web of Science ID 000225670300012

    View details for PubMedID 15576774

    View details for PubMedCentralID PMC532430

  • Initial phases of biofilm formation in Shewanella oneidensis MR-1 JOURNAL OF BACTERIOLOGY Thormann, K. M., Saville, R. M., Shukla, S., Pelletier, D. A., Spormann, A. M. 2004; 186 (23): 8096-8104


    Shewanella oneidensis MR-1 is a facultative Fe(III)- and Mn(IV)-reducing microorganism and serves as a model for studying microbially induced dissolution of Fe or Mn oxide minerals as well as biogeochemical cycles. In soil and sediment environments, S. oneidensis biofilms form on mineral surfaces and are critical for mediating the metabolic interaction between this microbe and insoluble metal oxide phases. In order to develop an understanding of the molecular basis of biofilm formation, we investigated S. oneidensis biofilms developing on glass surfaces in a hydrodynamic flow chamber system. After initial attachment, growth of microcolonies and lateral spreading of biofilm cells on the surface occurred simultaneously within the first 24 h. Once surface coverage was almost complete, biofilm development proceeded with extensive vertical growth, resulting in formation of towering structures giving rise to pronounced three-dimensional architecture. Biofilm development was found to be dependent on the nutrient conditions, suggesting a metabolic control. In global transposon mutagenesis, 173 insertion mutants out of 15,000 mutants screened were identified carrying defects in initial attachment and/or early stages in biofilm formation. Seventy-one of those mutants exhibited a nonswimming phenotype, suggesting a role of swimming motility or motility elements in biofilm formation. Disruption mutations in motility genes (flhB, fliK, and pomA), however, did not affect initial attachment but affected progression of biofilm development into pronounced three-dimensional architecture. In contrast, mutants defective in mannose-sensitive hemagglutinin type IV pilus biosynthesis and in pilus retraction (pilT) showed severe defects in adhesion to abiotic surfaces and biofilm formation, respectively. The results provide a basis for understanding microbe-mineral interactions in natural environments.

    View details for DOI 10.1128/JB.186.23.8096-8104.2004

    View details for Web of Science ID 000225271700031

    View details for PubMedID 15547283

    View details for PubMedCentralID PMC529061

  • Scanning transmission X-ray microscopy study of microbial calcification GEOBIOLOGY Benzerara, K., Yoon, T. H., Tyliszczak, T., Constantz, B., Spormann, A. M., Brown, G. E. 2004; 2 (4): 249-259
  • Comparative evaluation of chloroethene dechlorination to ethene by Dehalococcoides-like microorganisms ENVIRONMENTAL SCIENCE & TECHNOLOGY Cupples, A. M., Spormann, A. M., McCarty, P. L. 2004; 38 (18): 4768-4774


    Reductive dehalogenation of tetrachloroethene (PCE), trichloroethene (TCE), cis-1,2-dichloroethene (DCE), and vinyl chloride (VC) was examined in four cultures containing Dehalococcoides-like microorganisms. Dechlorination and growth kinetics were compared using a Monod growth-rate model for multiple electron acceptor usage with competition. Included were the Victoria mixed culture containing Dehalococcoides species strain VS (from Victoria, TX), the mixed culture KB-1/VC (from southern Ontario), the Pinellas mixed culture (from Pinellas, FL), and D. ethenogenes strain 195. All cultures, with the exception of D. ethenogenes strain 195, grew with VC as catabolic electron acceptor. A dilution method was developed that allows a valid comparison to be made of dehalogenating kinetics between different mixed cultures. Using this procedure, maximum growth rates on VC were found to be similar for strain VS and KB-1/VC (0.42-0.49 +/- 0.02 d(-1)) but slower for the Pinellas culture (0.28 +/- 0.01 d(-1)). The 16S rRNA gene sequences were determined to ensure that no cross contamination between cultures had occurred. Following enrichment of the VC dechlorinating microorganisms on VC, the cultures were amended with DCE, TCE, or PCE. The three mixed cultures failed to dechlorinate PCE or did so very slowly. However, the dilution technique indicated that all experienced growth on TCE and DCE as well as on VC. Maximum growth rates on DCE alone were quite similar (0.43-0.46 d(-1)), while the Pinellas culture grew faster on TCE alone (0.49 d(-1)) than did the other two mixed cultures (0.33-0.35 d(-1)). Half-velocity and inhibition constants for growth on TCE were also determined for the three mixed cultures; both constants were found to be essentially equal and the same for the different cultures, varying between only 8.6 and 10.5 microM. The ability of the strain VS, KB-1/VC, and Pinellas cultures to utilize TCE rapidly with conversion to ethene is quite different from that of any other reported microorganism. It was separately confirmed with more traditional cell-counting techniques that strain VS coupled TCE, as well as DCE and VC, utilization with growth. This is the first report of an organism obtaining energy for growth through every step in the reduction of TCE to ethene. Also, as suggested by the dilution technique, the dehalogenating organisms in the KB-1/VC and Pinellas cultures appear to obtain growth from TCE utilization as well. Such ability to grow while dehalogenating TCE to ethene will be an important advantage for their use in bioaugmentation.

    View details for DOI 10.1021/es049965z

    View details for Web of Science ID 000223938500016

    View details for PubMedID 15487786

  • Molecular identification of the catabolic vinyl chloride reductase from Dehalococcoides sp strain VS and its environmental distribution APPLIED AND ENVIRONMENTAL MICROBIOLOGY Muller, J. A., Rosner, B. M., von Abendroth, G., Meshulam-Simon, G., McCarty, P. L., Spormann, A. M. 2004; 70 (8): 4880-4888


    Reductive dehalogenation of vinyl chloride (VC) to ethene is the key step in complete anaerobic degradation of chlorinated ethenes. VC-reductive dehalogenase was partially purified from a highly enriched culture of the VC-respiring Dehalococcoides sp. strain VS. The enzyme reduced VC and all dichloroethene (DCE) isomers, but not tetrachloroethene (PCE) or trichloroethene (TCE), at high rates. By using reversed genetics, the corresponding gene (vcrA) was isolated and characterized. Based on the predicted amino acid sequence, VC reductase is a novel member of the family of corrinoid/iron-sulfur cluster containing reductive dehalogenases. The vcrA gene was found to be cotranscribed with vcrB, encoding a small hydrophobic protein presumably acting as membrane anchor for VC reductase, and vcrC, encoding a protein with similarity to transcriptional regulators of the NosR/NirI family. The vcrAB genes were subsequently found to be present and expressed in other cultures containing VC-respiring Dehalococcoides organisms and could be detected in water samples from a field site contaminated with chlorinated ethenes. Therefore, the vcrA gene identified here may be a useful molecular target for evaluating, predicting, and monitoring in situ reductive VC dehalogenation.

    View details for DOI 10.1128/AEM.70.8.4880-4888.2004

    View details for Web of Science ID 000223290100061

    View details for PubMedID 15294827

    View details for PubMedCentralID PMC492378

  • The role of organic molecules and microbial organisms in metal ion sorption processes 14th Annual V M Goldschmidt Conference Brown, G. E., Yoon, T. H., Johnson, S. B., Templeton, A. S., Trainor, T. P., Bostick, B. C., Kendelewicz, T., Doyle, C. S., Spormann, A. M. PERGAMON-ELSEVIER SCIENCE LTD. 2004: A160–A160
  • A derivative of the menaquinone precursor 1,4-dihydroxy-2-naphthoate is involved in the reductive transformation of carbon tetrachloride by aerobically grown Shewanella oneidensis MR-1 APPLIED MICROBIOLOGY AND BIOTECHNOLOGY Ward, M. J., Fu, Q. S., Rhoads, K. R., Yeung, C. H., Spormann, A. M., Criddle, C. S. 2004; 63 (5): 571-577


    Transformation of carbon tetrachloride (CT) by Shewanella oneidensis MR-1 has been proposed to involve the anaerobic respiratory-chain component menaquinone. To investigate this hypothesis a series of menaquinone mutants were constructed. The menF mutant is blocked at the start of the menaquinone biosynthetic pathway. The menB, menA and menG mutants are all blocked towards the end of the pathway, being unable to produce 1,4-dihydroxy-2-naphthoic acid (DHNA), demethyl-menaquinone and menaquinone, respectively. Aerobically grown mutants unable to produce the menaquinone precursor DHNA (menF and menB mutants) showed a distinctly different CT transformation profile than mutants able to produce DHNA but unable to produce menaquinone (menA and menG mutants). While DHNA did not reduce CT in an abiotic assay, the addition of DHNA to the menF and menB mutants restored normal CT transformation activity. We conclude that a derivative of DHNA, that is distinct from menaquinone, is involved in the reduction of CT by aerobically grown S. oneidensis MR-1. When cells were grown anaerobically with trimethylamine-N-oxide as the terminal electron acceptor, all the menaquinone mutants showed wild-type levels of CT reduction. We conclude that S. oneidensis MR-1 produces two different factors capable of dehalogenating CT. The factor produced under anaerobic growth conditions is not a product of the menaquinone biosynthetic pathway.

    View details for DOI 10.1007/s00253-003-1407-3

    View details for Web of Science ID 000188456900013

    View details for PubMedID 12908086

  • In vitro system for simultaneous expression and maturation of iron-sulfur proteins. 227th National Meeting of the American-Chemical Society Boyer, M. E., Wang, C. W., Spormann, A. M., Swartz, J. AMER CHEMICAL SOC. 2004: U225–U225
  • Vinyl chloride and cis-dichloroethene dechlorination kinetics and microorganism growth under substrate limiting conditions ENVIRONMENTAL SCIENCE & TECHNOLOGY Cupples, A. M., Spormann, A. M., McCarty, P. L. 2004; 38 (4): 1102-1107


    The reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE) at contaminated sites often results in the accumulation of cis-1,2-dichloroethene (DCE) and vinyl chloride (VC), rather than the nonhazardous end product ethene. This accumulation may be caused by the absence of appropriate microorganisms, insufficient supply of donor substrate, or reaction kinetic limitations. Here, we address the issue of reaction kinetic limitations by investigating the effect of limiting substrate concentrations (electron donor and acceptor) on DCE and VC dechlorination kinetics and microorganism growth by bacterium VS. For this, a model based on Monod kinetics, but also accounting for competition between electron acceptors and the effect of low electron donor and acceptor concentrations (dual-substrate kinetics), was examined. Competitive coefficients for VC (7.8 +/- 1.5 microM) and DCE (3.6 +/- 1.1 microM) were obtained and included in the model. The half velocity coefficient for hydrogen, the electron donor, was experimentally determined (7 +/- 2 nM) through investigating dechlorination over different substrate concentrations. This complete model was then used, along with experimental data, to determine substrate concentrations at which the dechlorinating microorganisms would be in net decay. Notably, the model indicates net decay will result if the total electron acceptor concentration (DCE plus VC) is below 0.7 microM, regardless of electron donor levels. The ability to achieve sustainable bioremediation to acceptable levels can be greatly influenced by this threshold level.

    View details for DOI 10.1021/es0348647

    View details for Web of Science ID 000188996600025

    View details for PubMedID 14998024

  • Selenium speciation and partitioning within Burkholderia cepacia biofilms formed on alpha-Al2O3 surfaces GEOCHIMICA ET COSMOCHIMICA ACTA Templeton, A. S., Trainor, T. P., Spormann, A. M., Brown, G. E. 2003; 67 (19): 3547-3557
  • Rapid oxidation of pyrite surfaces by thiobacillus ferrooxidans and T. thiooxidans. 226th National Meeting of the American-Chemical-Society Bostick, B. C., Lester, K., Doyle, C., Kendelewicz, T., Brown, G. E., Fendorf, S., Spormann, A. M. AMER CHEMICAL SOC. 2003: U591–U591
  • Growth of a Dehalococcoides-like microorganism on vinyl chloride and cis-dichloroethene as electron acceptors as determined by competetive PCR (vol 69, pg 958, 2003) APPLIED AND ENVIRONMENTAL MICROBIOLOGY Cupples, A. M., Spormann, A. M., McCarty, P. L. 2003; 69 (7): 4342-4342
  • The NASA astrobiology roadmap ASTROBIOLOGY Marais, D. J., Allamandola, L. J., Benner, S. A., Boss, A. P., Deamer, D., Falkowski, P. G., Farmer, J. D., Hedges, S. B., Jakosky, B. M., Knoll, A. H., Liskowsky, D. R., Meadows, V. S., Meyer, M. A., Pilcher, C. B., Nealson, K. H., Spormann, A. M., Trent, J. D., Turner, W. W., Woolf, N. J., Yorke, H. W. 2003; 3 (2): 219-235


    The NASA Astrobiology Roadmap provides guidance for research and technology development across the NASA enterprises that encompass the space, Earth, and biological sciences. The ongoing development of astrobiology roadmaps embodies the contributions of diverse scientists and technologists from government, universities, and private institutions. The Roadmap addresses three basic questions: How does life begin and evolve, does life exist elsewhere in the universe, and what is the future of life on Earth and beyond? Seven Science Goals outline the following key domains of investigation: understanding the nature and distribution of habitable environments in the universe, exploring for habitable environments and life in our own solar system, understanding the emergence of life, determining how early life on Earth interacted and evolved with its changing environment, understanding the evolutionary mechanisms and environmental limits of life, determining the principles that will shape life in the future, and recognizing signatures of life on other worlds and on early Earth. For each of these goals, Science Objectives outline more specific high-priority efforts for the next 3-5 years. These 18 objectives are being integrated with NASA strategic planning.

    View details for Web of Science ID 000185588100002

    View details for PubMedID 14577870

  • Speciation of Pb(II) sorbed by Burkholderia cepacia/goethite composites ENVIRONMENTAL SCIENCE & TECHNOLOGY Templeton, A. S., Spormann, A. M., Brown, G. E. 2003; 37 (10): 2166-2172


    Bacterial-mineral composites are important in the retention of heavy metals such as Pb due to their large sorption capacity under a wide range of environmental conditions. However, the partitioning of heavy metals between components in such composites is not probed directly. Using Burkholderia cepacia biofilms coated with goethite (alpha-FeOOH) particles, the partitioning of Pb(II) between the biological and iron-(oxyhydr)oxide surfaces has been measured using an X-ray spectroscopic approach. EXAFS spectra were fit to quantitatively determine the fraction of Pb(II) associated with each component as a function of pH and [Pb]. At pH < 5.5, at least 50% of the total sorbed Pb(II) is associated with the biofilm component, whereas the total uptake within the composite is dominated by goethite (> 70% Pb/goethite) above pH 6. Direct comparison can be made between the amount of Pb(II) bound to each component in the composite vs separate binary systems (i.e., Pb/biofilm or Pb/goethite). At high pH, Pb(II) uptake on the biofilm is dramatically decreased due to competition with the goethite surface. In contrast, Pb uptake on goethite is significantly enhanced at low pH (2-fold increase at pH 5) compared to systems with no complexing ligands. The mode of Pb(II)-binding to the goethite component changes from low to high [Pb]. Structural fitting of the EXAFS spectra collected from 10(-5.6) to 10(-3.6) M [Pb]eq at pH 6 shows that the Pb-goethite surface complexes at low [Pb] are dominated by inner-sphere bidentate, binuclear complexes bridging two adjacent singly coordinated surface oxygens, giving rise to Pb-Fe distances of approximately 3.9 A. At high [Pb], the dominant Pb(II) inner-sphere complexes on the goethite surface shift to bidentate edge-sharing complexes with Pb-Fe distances of approximately 3.3 A.

    View details for DOI 10.1021/es026081b

    View details for Web of Science ID 000182866000020

    View details for PubMedID 12785522

  • Growth of a Dehalococcoides-like microorganism on vinyl chloride and cis-dichloroethene as electron acceptors as determined by competitive PCR APPLIED AND ENVIRONMENTAL MICROBIOLOGY Cupples, A. M., Spormann, A. M., McCarty, P. L. 2003; 69 (2): 953-959


    A competitive PCR (cPCR) assay targeting 16S ribosomal DNA was developed to enumerate growth of a Dehalococcoides-like microorganism, bacterium VS, from a mixed culture catalyzing the reductive dehalogenation of cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC), with hydrogen being used as an electron donor. The growth of bacterium VS was found to be coupled to the dehalogenation of VC and cDCE, suggesting unique metabolic capabilities. The average growth yield was (5.2 +/- 1.5) x 10(8) copies of the 16S rRNA gene/ micromol of Cl(-) (number of samples, 10), with VC being used as the electron acceptor and hydrogen as the electron donor. The maximum VC utilization rate (q) was determined to be 7.8 x 10(-10) micromol of Cl(-) (copy(-1) day(-1)), indicating a maximum growth rate of 0.4 day(-1). These average growth yield and q values agree well with values found previously for dechlorinating cultures. Decay coefficients were determined with growth (0.05 day(-1)) and no-growth (0.09 day(-1)) conditions. An important limitation of this cPCR assay was its inability to discriminate between active and inactive cells. This is an essential consideration for kinetic studies.

    View details for DOI 10.1128/AEM.69.2.953-959.2003

    View details for Web of Science ID 000180927100030

    View details for PubMedID 12571017

    View details for PubMedCentralID PMC143607

  • Sorption versus biomineralization of Pb(II) within Burkholderia cepacia biofilms ENVIRONMENTAL SCIENCE & TECHNOLOGY Templeton, A. S., Trainor, T. P., Spormann, A. M., Newville, M., Sutton, S. R., Dohnalkova, A., Gorby, Y., Brown, G. E. 2003; 37 (2): 300-307


    X-ray spectroscopy measurements have been combined with macroscopic uptake data and transmission electron microscopy (TEM) results to show that Pb(II) uptake by Burkholderia cepacia is due to simultaneous sorption and biomineralization processes. X-ray microprobe mapping of B. cepacia biofilms formed on alpha-Al2O3 surfaces shows that Pb(II) is distributed heterogeneously throughout the biofilms because of the formation of Pb "hot spots". EXAFS data and TEM observations show that the enhanced Pb accumulation is due to the formation of nanoscale crystals of pyromorphite (Pb5(PO4)3(OH)) adjacent to the outer-membrane of a fraction of the total population of B. cepacia cells. In contrast, B. cepacia cell suspensions or biofilms that were heat-killed or pretreated with X-rays do not form pyromorphite, which suggests that metabolic activity is required. Precipitation of pyromorphite occurs over several orders of magnitude in [H-] and [Pb] and accounts for approximately 90% of the total Pb uptake below pH 4.5 but only 45-60% at near-neutral pH because of the formation of additional Pb(II) adsorption complexes. Structural fits of Pb L(III) EXAFS data collected for heat-treated cells at near-neutral pH suggest that Pb(II) forms inner-sphere adsorption complexes with carboxyl functional groups in the biofilms.

    View details for DOI 10.1021/es025972g

    View details for Web of Science ID 000180501500014

    View details for PubMedID 12564901

  • Benzylsuccinate synthase of Azoarcus sp strain T: Cloning, sequencing, transcriptional organization, and its role in anaerobic toluene and m-xylene mineralization JOURNAL OF BACTERIOLOGY Achong, G. R., Rodriguez, A. M., Spormann, A. M. 2001; 183 (23): 6763-6770


    Biochemical studies in Azoarcus sp. strain T have demonstrated that anaerobic oxidation of both toluene and m-xylene is initiated by addition of the aromatic hydrocarbon to fumarate, forming benzylsuccinate and 3-methyl benzylsuccinate, respectively. Partially purified benzylsuccinate synthase was previously shown to catalyze both of these addition reactions. In this study, we identified and sequenced the genes encoding benzylsuccinate synthase from Azoarcus sp. strain T and examined the role of this enzyme in both anaerobic toluene and m-xylene mineralization. Based on reverse transcription-PCR experiments and transcriptional start site mapping, we found that the structural genes encoding benzylsuccinate synthase, bssCAB, together with two additional genes, bssD and bssE, were organized in an operon in the order bssDCABE. bssD is believed to encode an activating enzyme, similar in function to pyruvate formate-lyase activase. bssE shows homology to tutH from Thauera aromatica strain T1, whose function is currently unknown. A second operon that is upstream of bssDCABE and divergently transcribed contains two genes, tdiS and tdiR. The predicted amino acid sequences show similarity to sensor kinase and response regulator proteins of prokaryotic two-component regulatory systems. A chromosomal null bssA mutant was constructed (the bssA gene encodes the alpha-subunit of benzylsuccinate synthase). This bssA null mutant strain was unable to grow under denitrifying conditions on either toluene or m-xylene, while growth on benzoate was unaffected. The growth phenotype of the DeltabssA mutant could be rescued by reintroducing bssA in trans. These results demonstrate that benzylsuccinate synthase catalyzes the first step in anaerobic mineralization of both toluene and m-xylene.

    View details for Web of Science ID 000172158600006

    View details for PubMedID 11698363

  • Pb(II) distributions at biofilm-metal oxide interfaces PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Templeton, A. S., Trainor, T. P., Traina, S. J., Spormann, A. M., Brown, G. E. 2001; 98 (21): 11897-11902


    The distribution of aqueous Pb(II) sorbed at the interface between Burkholderia cepacia biofilms and hematite (alpha-Fe(2)O(3)) or corundum (alpha-Al(2)O(3)) surfaces has been probed by using an application of the long-period x-ray standing wave technique. Attached bacteria and adsorbed organic matter may interfere with sorption processes on metal oxide surfaces by changing the characteristics of the electrical double layer at the solid-solution interface, blocking surface sites, or providing a variety of new sites for metal binding. In this work, Pb L(alpha) fluorescence yield profiles for samples equilibrated with 10(-7) to 10(-3.8) M Pb(II) were measured and modeled to determine quantitatively the partitioning of Pb(II) at the biofilm-metal oxide interface. Our data show that the reactive sites on the metal oxide surfaces were not passivated by the formation of a monolayer biofilm. Instead, high-energy surface sites on the metal oxides form the dominant sink for Pb(II) at submicromolar concentrations, following the trend alpha-Fe(2)O(3) (0001) > alpha-Al(2)O(3) (1102) > alpha-Al(2)O(3) (0001), despite the greater site density within the overlying biofilms. At [Pb] > 10(-6) M, significant Pb uptake by the biofilms was observed.

    View details for Web of Science ID 000171558900016

    View details for PubMedID 11572932

  • Isolation and characterization of anaerobic ethylbenzene dehydrogenase, a novel Mo-Fe-S enzyme JOURNAL OF BACTERIOLOGY Johnson, H. A., Pelletier, D. A., Spormann, A. M. 2001; 183 (15): 4536-4542


    The first step in anaerobic ethylbenzene mineralization in denitrifying Azoarcus sp. strain EB1 is the oxidation of ethylbenzene to (S)-(-)-1-phenylethanol. Ethylbenzene dehydrogenase, which catalyzes this reaction, is a unique enzyme in that it mediates the stereoselective hydroxylation of an aromatic hydrocarbon in the absence of molecular oxygen. We purified ethylbenzene dehydrogenase to apparent homogeneity and showed that the enzyme is a heterotrimer (alphabetagamma) with subunit masses of 100 kDa (alpha), 35 kDa (beta), and 25 kDa (gamma). Purified ethylbenzene dehydrogenase contains approximately 0.5 mol of molybdenum, 16 mol of iron, and 15 mol of acid-labile sulfur per mol of holoenzyme, as well as a molydopterin cofactor. In addition to ethylbenzene, purified ethylbenzene dehydrogenase was found to oxidize 4-fluoro-ethylbenzene and the nonaromatic hydrocarbons 3-methyl-2-pentene and ethylidenecyclohexane. Sequencing of the encoding genes revealed that ebdA encodes the alpha subunit, a 974-amino-acid polypeptide containing a molybdopterin-binding domain. The ebdB gene encodes the beta subunit, a 352-amino-acid polypeptide with several 4Fe-4S binding domains. The ebdC gene encodes the gamma subunit, a 214-amino-acid polypeptide that is a potential membrane anchor subunit. Sequence analysis and biochemical data suggest that ethylbenzene dehydrogenase is a novel member of the dimethyl sulfoxide reductase family of molybdopterin-containing enzymes.

    View details for Web of Science ID 000169824000016

    View details for PubMedID 11443088

  • A stable organic free radical in anaerobic benzylsuccinate synthase of Azoarcus sp strain T JOURNAL OF BIOLOGICAL CHEMISTRY Krieger, C. J., Roseboom, W., Albracht, S. P., Spormann, A. M. 2001; 276 (16): 12924-12927


    The novel enzyme benzylsuccinate synthase initiates anaerobic toluene metabolism by catalyzing the addition of toluene to fumarate, forming benzylsuccinate. Based primarily on its sequence similarity to the glycyl radical enzymes, pyruvate formate-lyase and anaerobic ribonucleotide reductase, benzylsuccinate synthase was speculated to be a glycyl radical enzyme. In this report we use EPR spectroscopy to demonstrate for the first time that active benzylsuccinate synthase from the denitrifying bacterium Azoarcus sp. strain T harbors an oxygen-sensitive stable organic free radical. The EPR signal of the radical was centered at g = 2.0021 and was characterized by a major 2-fold splitting of about 1.5 millitesla. The strong similarities between the EPR signal of the benzylsuccinate synthase radical and that of the glycyl radicals of pyruvate formate-lyase and anaerobic ribonucleotide reductase provide evidence that the benzylsuccinate synthase radical is located on a glycine residue, presumably glycine 828 in Azoarcus sp. strain T benzylsuccinate synthase.

    View details for Web of Science ID 000168198600066

    View details for PubMedID 11278506

  • Solution interactions of uranyl (UO22+) with Pseuodomonas fluorescens: Bioavailability of complexed ligand, toxicity, and fate of uranyl. Bencheikh-Latmani, R., Leckie, J. O., Spormann, A. M. AMER CHEMICAL SOC. 2000: U370–U370
  • Metabolism of alkylbenzenes, alkanes, and other hydrocarbons in anaerobic bacteria BIODEGRADATION Spormann, A. M., Widdel, F. 2000; 11 (2-3): 85-105


    Aromatic and aliphatic hydrocarbons are the main constituents of petroleum and its refined products. Whereas degradation of hydrocarbons by oxygen-respiring microorganisms has been known for about a century, utilization of hydrocarbons under anoxic conditions has been investigated only during the past decade. Diverse strains of anaerobic bacteria have been isolated that degrade toluene anaerobically, using nitrate, iron(III), or sulfate as electron acceptors. Also, other alkylbenzenes such as m-xylene or ethylbenzene are utilized by a number of strains. The capacity for anaerobic utilization of alkylbenzenes has been observed in members of the alpha-, beta-, gamma- and delta-subclasses of the Proteobacteria. Furthermore, denitrifying bacteria and sulfate-reducing bacteria with the capacity for anaerobic alkane degradation have been isolated, which are members of the beta- and delta-subclass, respectively. The mechanism of the activation of hydrocarbons as apolar molecules in the absence of oxygen is of particular interest. The biochemistry of anaerobic toluene degradation has been studied in detail. Toluene is activated by addition to fumarate to yield benzylsuccinate, which is then further metabolized via benzoyl-CoA. The toluene-activating enzyme presents a novel type of glycine radical protein. Another principle of anaerobic alkylbenzene activation has been observed in the anaerobic degradation of ethylbenzene. Ethylbenzene in denitrifying bacteria is dehydrogenated to 1-phenylethanol and further to acetophenone; the latter is also metabolized to benzoyl-CoA. Naphthalene is presumably activated under anoxic conditions by a carboxylation reaction. Investigations into the pathway of anaerobic alkane degradation are only at the beginning. The saturated hydrocarbons are most likely activated by addition of a carbon compound rather than by desaturation and hydration, as speculated about in some early studies. An anaerobic oxidation of methane with sulfate as electron acceptor has been documented in aquatic sediments. The process is assumed to involve a reversal of methanogenesis catalyzed by Archaea, and scavenge of an electron-carrying metabolite by sulfate-reducing bacteria. Among unsaturated non-aromatic hydrocarbons, anaerobic bacterial degradation has been demonstrated and investigated with n-alkenes, alkenoic terpenes and the alkyne, acetylene.

    View details for Web of Science ID 000168530600002

    View details for PubMedID 11440245

  • Initial reactions in anaerobic oxidation of m-xylene by the denitrifying bacterium Azoarcus sp strain T JOURNAL OF BACTERIOLOGY Krieger, C. J., Beller, H. R., Reinhard, M., Spormann, A. M. 1999; 181 (20): 6403-6410


    The initial enzymatic steps in anaerobic m-xylene oxidation were studied in Azoarcus sp. strain T, a denitrifying bacterium capable of mineralizing m-xylene via 3-methylbenzoate. Permeabilized cells of m-xylene-grown Azoarcus sp. strain T catalyzed the addition of m-xylene to fumarate to form (3-methylbenzyl)succinate. In the presence of succinyl coenzyme A (CoA) and nitrate, (3-methylbenzyl)succinate was oxidized to E-(3-methylphenyl)itaconate (or a closely related isomer) and 3-methylbenzoate. Kinetic studies conducted with permeabilized cells and whole-cell suspensions of m-xylene-grown Azoarcus sp. strain T demonstrated that the specific rate of in vitro (3-methylbenzyl)succinate formation accounts for at least 15% of the specific rate of in vivo m-xylene consumption. Based on these findings, we propose that Azoarcus sp. strain T anaerobically oxidizes m-xylene to 3-methylbenzoate (or its CoA thioester) via (3-methylbenzyl)succinate and E-(3-methylphenyl)itaconate (or its CoA thioester) in a series of reactions that are analogous to those recently proposed for anaerobic toluene oxidation to benzoyl-CoA. A deuterium kinetic isotope effect was observed in the (3-methylbenzyl)succinate synthase reaction (and the benzylsuccinate synthase reaction), suggesting that a rate-determining step in this novel fumarate addition reaction involves breaking a C-H bond.

    View details for Web of Science ID 000083006500024

    View details for PubMedID 10515931

  • Gliding motility in bacteria: Insights from studies of Myxococcus xanthus MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS Spormann, A. M. 1999; 63 (3): 621-?


    Gliding motility is observed in a large variety of phylogenetically unrelated bacteria. Gliding provides a means for microbes to travel in environments with a low water content, such as might be found in biofilms, microbial mats, and soil. Gliding is defined as the movement of a cell on a surface in the direction of the long axis of the cell. Because this definition is operational and not mechanistic, the underlying molecular motor(s) may be quite different in diverse microbes. In fact, studies on the gliding bacterium Myxococcus xanthus suggest that two independent gliding machineries, encoded by two multigene systems, operate in this microorganism. One machinery, which allows individual cells to glide on a surface, independent of whether the cells are moving alone or in groups, requires the function of the genes of the A-motility system. More than 37 A-motility genes are known to be required for this form of movement. Depending on an additional phenotype, these genes are divided into two subclasses, the agl and cgl genes. Videomicroscopic studies on gliding movement, as well as ultrastructural observations of two myxobacteria, suggest that the A-system motor may consist of multiple single motor elements that are arrayed along the entire cell body. Each motor element is proposed to be localized to the periplasmic space and to be anchored to the peptidoglycan layer. The force to glide which may be generated here is coupled to adhesion sites that move freely in the outer membrane. These adhesion sites provide a specific contact with the substratum. Based on single-cell observations, similar models have been proposed to operate in the unrelated gliding bacteria Flavobacterium johnsoniae (formerly Cytophaga johnsonae), Cytophaga strain U67, and Flexibacter polymorphus (a filamentous glider). Although this model has not been verified experimentally, M. xanthus seems to be the ideal organism with which to test it, given the genetic tools available. The second gliding motor in M. xanthus controls cell movement in groups (S-motility system). It is dependent on functional type IV pili and is operative only when cells are in close proximity to each other. Type IV pili are known to be involved in another mode of bacterial surface translocation, called twitching motility. S-motility may well represent a variation of twitching motility in M. xanthus. However, twitching differs from gliding since it involves cell movements that are jerky and abrupt and that lack the organization and smoothness observed in gliding. Components of this motor are encoded by genes of the S-system, which appear to be homologs of genes involved in the biosynthesis, assembly, and function of type IV pili in Pseudomonas aeruginosa and Neisseria gonorrhoeae. How type IV pili generate force in S-motility is currently unknown, but it is to be expected that ongoing physiological, genetic, and biochemical studies in M. xanthus, in conjunction with studies on twitching in P. aeruginosa and N. gonorrhoeae, will provide important insights into this microbial motor. The two motility systems of M. xanthus are affected to different degrees by the MglA protein, which shows similarity to a small GTPase. Bacterial chemotaxis-like sensory transduction systems control gliding motility in M. xanthus. The frz genes appear to regulate gliding movement of individual cells and movement by the S-motility system, suggesting that the two motors found in this bacterium can be regulated to result in coordinated multicellular movements. In contrast, the dif genes affect only S-system-dependent swarming.

    View details for Web of Science ID 000082409400005

    View details for PubMedID 10477310

  • Substrate range of benzylsuccinate synthase from Azoarcus sp strain T FEMS MICROBIOLOGY LETTERS Beller, H. R., Spormann, A. M. 1999; 178 (1): 147-153


    Benzylsuccinate synthase, which catalyzes the anaerobic addition of the methyl carbon of toluene to fumarate, has recently been reported in several denitrifying and sulfate-reducing, toluene-degrading bacteria. In substrate range studies with partially purified benzylsuccinate synthase from denitrifying Azoarcus sp. strain T, benzylsuccinate analogs were observed as a result of fumarate addition to the following toluene surrogates: xylenes, monofluorotoluenes, benzaldehyde, and 1-methyl-1-cyclohexene (but not 4-methyl-l-cyclohexene or methylcyclohexane). Benzylsuccinate was also observed as a result of toluene addition to maleate, but no products were observed from assays with toluene and either crotonate or trans-glutaconate. Toluene-maleate addition, like toluene-fumarate addition, resulted in highly stereospecific formation of the (+)-benzylsuccinic acid enantiomer [(R)-2-benzyl-3-carboxypropionic acid]. The previously reported finding that the methyl H atom abstracted from toluene is retained in the succinyl moiety of benzylsuccinate was found to apply to several toluene surrogates. The implications of these observations for the mechanism of benzylsuccinate synthase will be discussed.

    View details for Web of Science ID 000082299800021

    View details for PubMedID 10483734

  • In vitro studies on the initial reactions of anaerobic ethylbenzene mineralization JOURNAL OF BACTERIOLOGY Johnson, H. A., Spormann, A. M. 1999; 181 (18): 5662-5668


    Anaerobic mineralization of ethylbenzene by the denitrifying bacterium Azoarcus sp. strain EB1 was recently shown to be initiated by dehydrogenation of ethylbenzene to 1-phenylethanol. 1-Phenylethanol is converted to benzoate (benzoyl coenzyme A) via acetophenone as transient intermediate. We developed in vitro assays to examine ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities in cell extracts of this strain. With p-benzoquinone as the electron acceptor, cell extracts of Azoarcus sp. strain EB1 catalyzed ethylbenzene oxidation at a specific rate of 10 nmol min(-1) [mg of protein](-1) and an apparent K(m) for ethylbenzene of approximately 60 microM. The membrane-associated ethylbenzene dehydrogenase activity was found to oxidize 4-fluoroethylbenzene and propylbenzene but was unable to transform 4-chloro-ethylbenzene, the ethyltoluenes, and styrene. Enzymatic ethylbenzene oxidation was stereospecific, with (S)-(-)-1-phenylethanol being the only enantiomer detected by chiral high-pressure liquid chromatography analysis. Moreover, cell extracts catalyzed the oxidation of (S)-(-)-1-phenylethanol but not of (R)-(+)-1-phenylethanol to acetophenone. When cell extracts were dialyzed, (S)-(-)-1-phenylethanol oxidation occurred only in the presence of NAD(+), suggesting that NAD(+) is the physiological electron acceptor of 1-phenylethanol dehydrogenase. Both ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities were present in Azoarcus sp. strain EB1 cells that were grown anaerobically on ethylbenzene, 1-phenylethanol, and acetophenone, but these activities were absent in benzoate-grown cells.

    View details for Web of Science ID 000082534700015

    View details for PubMedID 10482506

  • A chimeric prokaryotic ancestry of mitochondria and primitive eukaryotes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Karlin, S., Brocchieri, L., Mrazek, J., Campbell, A. M., Spormann, A. M. 1999; 96 (16): 9190-9195


    We provide data and analysis to support the hypothesis that the ancestor of animal mitochondria (Mt) and many primitive amitochondrial (a-Mt) eukaryotes was a fusion microbe composed of a Clostridium-like eubacterium and a Sulfolobus-like archaebacterium. The analysis is based on several observations: (i) The genome signatures (dinucleotide relative abundance values) of Clostridium and Sulfolobus are compatible (sufficiently similar) and each has significantly more similarity in genome signatures with animal Mt sequences than do all other available prokaryotes. That stable fusions may require compatibility in genome signatures is suggested by the compatibility of plasmids and hosts. (ii) The expanded energy metabolism of the fusion organism was strongly selective for cementing such a fusion. (iii) The molecular apparatus of endospore formation in Clostridium serves as raw material for the development of the nucleus and cytoplasm of the eukaryotic cell.

    View details for Web of Science ID 000081835500076

    View details for PubMedID 10430918

  • Genetic and molecular analysis of cglB, a gene essential for single-cell gliding in Myxococcus xanthus JOURNAL OF BACTERIOLOGY Rodriguez, A. M., Spormann, A. M. 1999; 181 (14): 4381-4390


    Gliding movements of individual isolated Myxococcus xanthus cells depend on the genes of the A-motility system (agl and cgl genes). Mutants carrying defects in those genes are unable to translocate as isolated cells on solid surfaces. The motility defect of cgl mutants can be transiently restored to wild type by extracellular complementation upon mixing mutant cells with wild-type or other motility mutant cells. To develop a molecular understanding of the function of a Cgl protein in gliding motility, we cloned the cglB wild-type allele by genetic complementation of the mutant phenotype. The nucleotide sequence of a 2.85-kb fragment was determined and shown to encode two complete open reading frames. The CglB protein was determined to be a 416-amino-acid putative lipoprotein with an unusually high cysteine content. The CglB antigen localized to the membrane fraction. The swarming and gliding defects of a constructed DeltacglB mutant were fully restored upon complementation with the cglB wild-type allele. Experiments with a cglB allele encoding a CglB protein with a polyhistidine tag at the C terminus showed that this allele also promoted wild-type levels of swarming and single-cell gliding, but was unable to stimulate DeltacglB cells to move. Possible functions of CglB as a mechanical component or as a signal protein in single cell gliding are discussed.

    View details for Web of Science ID 000081360100030

    View details for PubMedID 10400597

  • XAFS and XSW study of the distribution of Pb(II) sorbed to biofilms on alpha-Al2O3 and alpha-FeOOH surfaces 10th International Conference on XAFS (XAFS X) Templeton, A. S., Ostergren, J. D., Trainor, T. P., Foster, A. L., Traina, S. J., Spormann, A., Brown, G. E. WILEY-BLACKWELL. 1999: 642–644

    View details for Web of Science ID 000081221700186

    View details for PubMedID 15263408

  • Gliding mutants of Myxococcus xanthus with high reversal frequencies and small displacements JOURNAL OF BACTERIOLOGY Spormann, A. M., Kaiser, D. 1999; 181 (8): 2593-2601


    Myxococcus xanthus cells move on a solid surface by gliding motility. Several genes required for gliding motility have been identified, including those of the A- and S-motility systems as well as the mgl and frz genes. However, the cellular defects in gliding movement in many of these mutants were unknown. We conducted quantitative, high-resolution single-cell motility assays and found that mutants defective in mglAB or in cglB, an A-motility gene, reversed the direction of gliding at frequencies which were more than 1 order of magnitude higher than that of wild type cells (2.9 min-1 for DeltamglAB mutants and 2.7 min-1 for cglB mutants, compared to 0.17 min-1 for wild-type cells). The average gliding speed of DeltamglAB mutant cells was 40% of that of wild-type cells (on average 1.9 micrometers/min for DeltamglAB mutants, compared to 4.4 micrometers/min for wild-type cells). The mglA-dependent reversals and gliding speeds were dependent on the level of intracellular MglA protein: mglB mutant cells, which contain only 15 to 20% of the wild-type level of MglA protein, glided with an average reversal frequency of about 1.8 min-1 and an average speed of 2.6 micrometers/min. These values range between those exhibited by wild-type cells and by DeltamglAB mutant cells. Epistasis analysis of frz mutants, which are defective in aggregation and in single-cell reversals, showed that a frzD mutation, but not a frzE mutation, partially suppressed the mglA phenotype. In contrast to mgl mutants, cglB mutant cells were able to move with wild-type speeds only when in close proximity to each other. However, under those conditions, these mutant cells were found to glide less often with those speeds. By analyzing double mutants, the high reversing movements and gliding speeds of cglB cells were found to be strictly dependent on type IV pili, encoded by S-motility genes, whereas the high-reversal pattern of mglAB cells was only partially reduced by a pilR mutation. These results suggest that the MglA protein is required for both control of reversal frequency and gliding speed and that in the absence of A motility, type IV pilus-dependent cell movement includes reversals at high frequency. Furthermore, mglAB mutants behave as if they were severely defective in A motility but only partially defective in S motility.

    View details for Web of Science ID 000079706600037

    View details for PubMedID 10198026

  • Predicting microbial biodegradation pathways ASM NEWS Wackett, L. P., Ellis, L. B., Speedie, S. M., Hershberger, C. D., Knackmuss, H. J., Spormann, A. M., Walsh, C. T., Forney, L. J., Punch, W. F., Kazic, T., Kanehisa, M., Berndt, D. J. 1999; 65 (2): 87-93
  • Anaerobic bacterial metabolism of hydrocarbons FEMS MICROBIOLOGY REVIEWS Heider, J., Spormann, A. M., Beller, H. R., Widdel, F. 1998; 22 (5): 459-473
  • Analysis of the novel benzylsuccinate synthase reaction for anaerobic toluene activation based on structural studies of the product JOURNAL OF BACTERIOLOGY Beller, H. R., Spormann, A. M. 1998; 180 (20): 5454-5457


    Recent studies of anaerobic toluene catabolism have demonstrated a novel reaction for anaerobic hydrocarbon activation: the addition of the methyl carbon of toluene to fumarate to form benzylsuccinate. In vitro studies of the anaerobic benzylsuccinate synthase reaction indicate that the H atom abstracted from the toluene methyl group during addition to fumarate is retained in the succinyl moiety of benzylsuccinate. Based on structural studies of benzylsuccinate formed during anaerobic, in vitro assays with denitrifying, toluene-mineralizing strain T, we now report the following characteristics of the benzylsuccinate synthase reaction: (i) it is highly stereospecific, resulting in >95% formation of the (+)-benzylsuccinic acid enantiomer [(R)-2-benzyl-3-carboxypropionic acid], and (ii) active benzylsuccinate synthase does not contain an abstracted methyl H atom from toluene at the beginning or at the end of a catalytic cycle.

    View details for Web of Science ID 000076346600024

    View details for PubMedID 9765580

  • In vitro studies on reductive vinyl chloride dehalogenation by an anaerobic mixed culture APPLIED AND ENVIRONMENTAL MICROBIOLOGY Rosner, B. M., McCarty, P. L., Spormann, A. M. 1997; 63 (11): 4139-4144


    Reductive dehalogenation of vinyl chloride (VC) was studied in an anaerobic mixed bacterial culture. In growth experiments, ethene formation from VC increased exponentially at a rate of about 0.019 h(sup-1). Reductive VC dehalogenation was measured in vitro by using cell extracts of the mixed culture. The apparent K(infm) for VC was determined to be about 76 (mu)M; the V(infmax) was about 28 nmol (middot) min(sup-1) (middot) mg of protein(sup-1). The VC-dehalogenating activity was membrane associated. Propyl iodide had an inhibitory effect on the VC-dehalogenating activity in the in vitro assay. However, this inhibition could not be reversed by illumination. Cell extracts also catalyzed the reductive dehalogenation of cis-1,2-dichloroethene (cis-DCE) and, at a lower rate, of trichloroethene (TCE). Tetrachloroethene (PCE) was not transformed. The results indicate that the reductive dehalogenation of VC and cis-DCE described here is different from previously reported reductive dehalogenation of PCE and TCE.

    View details for Web of Science ID A1997YE28100001

    View details for PubMedID 16535722

  • Benzylsuccinate formation as a means of anaerobic toluene activation by sulfate-reducing strain PRTOL1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY Beller, H. R., Spormann, A. M. 1997; 63 (9): 3729-3731


    Permeabilized cells of toluene-mineralizing, sulfate-reducing strain PRTOL1 catalyzed the addition of toluene to fumarate to form benzylsuccinate under anaerobic conditions. Recent in vitro studies with two toluene-mineralizing, denitrifying bacteria demonstrated the same fumarate addition reaction and indicated that it may be the first step of anaerobic toluene degradation. This study with strain PRTOL1 shows that anaerobic toluene activation by fumarate addition occurs in bacteria as disparate as sulfate-reducing and denitrifying species (members of the delta and beta subclasses of the Proteobacteria, respectively).

    View details for Web of Science ID A1997XV51000063

    View details for PubMedID 16535701

  • Anaerobic activation of toluene and o-xylene by addition to fumarate in denitrifying strain T JOURNAL OF BACTERIOLOGY Beller, H. R., Spormann, A. M. 1997; 179 (3): 670-676


    Anaerobic assays conducted with strain T, a denitrifying bacterium capable of mineralizing toluene to carbon dioxide, demonstrated that toluene-grown, permeabilized cells catalyzed the addition of toluene to fumarate to form benzylsuccinate. This reaction was not dependent on the presence of coenzyme A (CoA) or ATP. In the presence of CoA, formation of E-phenylitaconate from benzylsuccinate was also observed. Kinetic studies demonstrated that the specific rate of benzylsuccinate formation from toluene and fumarate in assays with permeabilized cells was >30% of the specific rate of toluene consumption in whole-cell suspensions with nitrate; this observation suggests that benzylsuccinate formation may be the first reaction in anaerobic toluene degradation by strain T. Use of deuterium-labeled toluene and gas chromatography-mass spectrometry indicated that the H atom abstracted from the toluene methyl group during addition to fumarate was retained in the succinyl moiety of benzylsuccinate. In this study, no evidence was found to support previously proposed reactions of toluene with acetyl-CoA or succinyl-CoA. Toluene-grown, permeabilized cells of strain T also catalyzed the addition of o-xylene to fumarate to form (2-methylbenzyl)succinate. o-Xylene is not a growth substrate for strain T, and its transformation was probably cometabolic. With the exception of specific reaction rates, the observed characteristics of the toluene-fumarate addition reaction (i.e., retention of a methyl H atom and independence from CoA and ATP) also apply to the o-xylene-fumarate addition reaction. Thus, addition to fumarate may be a biochemical strategy to anaerobically activate a range of methylbenzenes.

    View details for Web of Science ID A1997WE44000014

    View details for PubMedID 9006019

  • Initial reactions in anaerobic ethylbenzene oxidation by a denitrifying bacterium, strain EB1 JOURNAL OF BACTERIOLOGY Ball, H. A., Johnson, H. A., Reinhard, M., Spormann, A. M. 1996; 178 (19): 5755-5761


    Initial reactions in anaerobic oxidation of ethylbenzene were investigated in a denitrifying bacterium, strain EB1. Cells of strain EB1 mineralized ethylbenzene to CO2 under denitrifying conditions, as demonstrated by conversion of 69% of [14C]ethylbenzene to 14CO2. In anaerobic suspensions of strain EB1 cells metabolizing ethylbenzene, the transient formation and consumption of 1-phenylethanol, acetophenone, and an as yet unidentified compound were observed. On the basis of growth experiments and spectroscopic data, the unknown compound is proposed to be benzoyl acetate. Cell suspension experiments using H2(18)O demonstrated that the hydroxyl group of the first product of anoxic ethylbenzene oxidation, 1-phenylethanol, is derived from water. A tentative pathway for anaerobic ethylbenzene mineralization by strain EB1 is proposed.

    View details for Web of Science ID A1996VK78900027

    View details for PubMedID 8824622

  • Isolation and characterization of a novel toluene-degrading, sulfate-reducing bacterium APPLIED AND ENVIRONMENTAL MICROBIOLOGY Beller, H. R., Spormann, A. M., Sharma, P. K., Cole, J. R., Reinhard, M. 1996; 62 (4): 1188-1196


    A novel sulfate-reducing bacterium isolated from fuel-contaminated subsurface soil, strain PRTOL1, mineralizes toluene as the sole electron donor and carbon source under strictly anaerobic conditions. The mineralization of 80% of toluene carbon to CO2 was demonstrated in experiments with [ring-U-14C]toluene; 15% of toluene carbon was converted to biomass and nonvolatile metabolic by-products, primarily the former. The observed stoichiometric ratio of moles of sulfate consumed per mole of toluene consumed was consistent with the theoretical ratio for mineralization of toluene coupled with the reduction of sulfate to hydrogen sulfide. Strain PRTOL1 also transforms o- and p-xylene to metabolic products when grown with toluene. However, xylene transformation by PRTOL1 is slow relative to toluene degradation and cannot be sustained over time. Stable isotope-labeled substrates were used in conjunction with gas chromatography-mass spectrometry to investigate the by-products of toluene and xylene metabolism. The predominant by-products from toluene, o-xylene, and p-xylene were benzylsuccinic acid, (2-methylbenzyl)succinic acid, and 4-methylbenzoic acid (or p-toluic acid), respectively. Metabolic by-products accounted for nearly all of the o-xylene consumed. Enzyme assays indicated that acetyl coenzyme A oxidation proceeded via the carbon monoxide dehydrogenase pathway. Compared with the only other reported toluene-degrading, sulfate-reducing bacterium, strain PRTOL1 is distinct in that it has a novel 16S rRNA gene sequence and was derived from a freshwater rather than marine environment.

    View details for Web of Science ID A1996UD95000010

    View details for PubMedID 8919780

  • GLIDING MOVEMENTS IN MYXOCOCCUS-XANTHUS JOURNAL OF BACTERIOLOGY Spormann, A. M., Kaiser, A. D. 1995; 177 (20): 5846-5852


    Prokaryotic gliding motility is described as the movement of a cell on a solid surface in the direction of the cell's long axis, but its mechanics are unknown. To investigate the basis of gliding, movements of individual Myxococcus xanthus cells were monitored by employing a video microscopy method by which displacements as small as 0.03 micron could be detected and speeds as low as 1 micron/min could be resolved. Single cells were observed to glide with speeds varying between 1 and 20 microns/min. We found that speed variation was due to differences in distance between the moving cell and the nearest cell. Cells separated by less than one cell diameter (0.5 micron) moved with an average speed of 5.0 micron/min, whereas cells separated by more than 0.5 micron glided with an average speed of 3.8 microns/min. The power to glide was found to be carried separately at both ends of a cell.

    View details for Web of Science ID A1995RZ77000019

    View details for PubMedID 7592333