Professor (by Courtesy), Biology
Deciphering proteolysis pathways for the error-prone DNA polymerase in Cyanobacteria.
Protein quality control pathways require AAA+ proteases, such as Clp and Lon. Lon protease maintains UmuD, an important component of the error-prone DNA repair polymerase (Pol V), at very low levels in E. coli. Most members of the phylum Cyanobacteria lack Lon (including the model cyanobacterium, Synechocystis sp. PCC6803), so maintenance of UmuD at low levels must employ different proteases. We demonstrate that the first nineteen residues from the N-terminus of UmuD (Sug1-19 ) fused to a reporter protein are adequate to trigger complete proteolysis and that mutation of a single leucine residue (L6) to aspartic acid inhibits proteolysis. This process appears to follow the N-end rule and is mediated by ClpA/P protease and the ClpS adaptor. Additionally, mutations of arginine residues in the Sug1-19 tag suggest that the ClpX/P pathway also plays a role in proteolysis. We propose that there is a dual degron at the N-terminus of the UmuD protein in Synechocystis sp. PCC6803, which is distinct from the degron required for degradation of UmuD in E. coli. The use of two proteolysis pathways to tune levels of UmuD might reflect how a photosynthetic organism responds to multiple environmental stressors. This article is protected by copyright. All rights reserved.
View details for DOI 10.1111/1462-2920.14911
View details for PubMedID 31908125
Building an Inducible T7 RNA Polymerase/T7 Promoter Circuit in Synechocystis sp. PCC6803.
ACS synthetic biology
To develop tightly regulated orthogonal gene expression circuits in the photoautotrophic cyanobacterium Synechocystis sp. PCC6803 (Syn6803), we designed a circuit in which a native inducible promoter drives the expression of phage T7 RNA polymerase (T7RNAP). T7RNAP, in turn, specifically recognizes the T7 promoter that is designed to drive GFP expression. In Syn6803, this T7RNAP/T7promoter-GFP circuit produces high GFP fluorescence, which was further enhanced by using mutant T7 promoters. We also tested two orthogonal inducible promoters, Trc1O and L03, but these promoters drive T7RNAP to levels that are toxic in E. coli. Introduction of a protein degradation tag alleviated this problem. However, in Syn6803, these circuits did not function successfully. This highlights the underappreciated fact that similar circuits work with varying efficiencies in different chassis organisms. This lays the groundwork for developing new orthogonally controlled phage RNA polymerase-dependent expression systems in Syn6803.
View details for DOI 10.1021/acssynbio.8b00515
View details for PubMedID 30935196
- Construction of a Shuttle Vector Using an Endogenous Plasmid From the Cyanobacterium Synechocystis sp PCC6803 FRONTIERS IN MICROBIOLOGY 2018; 9
Construction of a Shuttle Vector Using an Endogenous Plasmid From the Cyanobacterium Synechocystis sp. PCC6803.
Frontiers in microbiology
2018; 9: 1662
To advance synthetic biology in the photosynthetic cyanobacterium Synechocystis sp. PCC6803 (Syn6803), we constructed a shuttle vector with some versatile features. This shuttle vector, pSCB-YFP, consists of a putative replicon identified on the plasmid pCC5.2, the origin of replication of pMB1 from E. coli, as well as the YFP reporter gene and a spectinomycin/streptomycin resistance cassette. pSCB-YFP is stably maintained in Syn6803M (a motile strain that lacks the endogenous pCC5.2) and expresses YFP. In addition, we engineered a fragment into pSCB-YFP that has multiple cloning sites and other features such that this plasmid can also be used as an expression vector (pSCBe). The shuttle vector pSCB-YFP can be stably maintained for at least 50 generations without antibiotic selection. It is a high copy number plasmid and can stably co-exist with the RSF1010-based pPMQAK1-GFP.
View details for PubMedID 30087668
View details for PubMedCentralID PMC6066503
Probing the ecological and evolutionary history of a thermophilic cyanobacterial population via statistical properties of its microdiversity.
2018; 13 (11): e0205396
Despite extensive DNA sequencing data derived from natural microbial communities, it remains a major challenge to identify the key evolutionary and ecological forces that shape microbial populations. We have focused on the extensive microdiversity of the cyanobacterium Synechococcus sp., which is a dominant member of the dense phototrophic biofilms in the hot springs of Yellowstone National Park. From deep amplicon sequencing of many loci and statistical analyses of these data, we showed previously that the population has undergone an unexpectedly high degree of homologous recombination, unlinking synonymous SNP-pair correlations even on intragenic length scales. Here, we analyze the genic amino acid diversity, which provides new evidence of selection and insights into the evolutionary history of the population. Surprisingly, some features of the data, including the spectrum of distances between genic-alleles, appear consistent with primarily asexual neutral drift. Yet the non-synonymous site frequency spectrum has too large an excess of low-frequency polymorphisms to result from negative selection on deleterious mutations given the distribution of coalescent times that we infer. And our previous analyses showed that the population is not asexual. Taken together, these apparently contradictory data suggest that selection, epistasis, and hitchhiking all play essential roles in generating and stabilizing the diversity. We discuss these as well as potential roles of ecological niches at genomic and genic levels. From quantitative properties of the diversity and comparative genomic data, we infer aspects of the history and inter-spring dispersal of the meta-population since it was established in the Yellowstone Caldera. Our investigations illustrate the need for combining multiple types of sequencing data and quantitative statistical analyses to develop an understanding of microdiversity in natural microbial populations.
View details for PubMedID 30427861
Emergent Phototactic Responses of Cyanobacteria under Complex Light Regimes
2017; 8 (2)
Environmental cues can stimulate a variety of single-cell responses, as well as collective behaviors that emerge within a bacterial community. These responses require signal integration and transduction, which can occur on a variety of time scales and often involve feedback between processes, for example, between growth and motility. Here, we investigate the dynamics of responses of the phototactic, unicellular cyanobacterium Synechocystis sp. PCC6803 to complex light inputs that simulate the natural environments that cells typically encounter. We quantified single-cell motility characteristics in response to light of different wavelengths and intensities. We found that red and green light primarily affected motility bias rather than speed, while blue light inhibited motility altogether. When light signals were simultaneously presented from different directions, cells exhibited phototaxis along the vector sum of the light directions, indicating that cells can sense and combine multiple signals into an integrated motility response. Under a combination of antagonistic light signal regimes (phototaxis-promoting green light and phototaxis-inhibiting blue light), the ensuing bias was continuously tuned by competition between the wavelengths, and the community response was dependent on both bias and cell growth. The phototactic dynamics upon a rapid light shift revealed a wavelength dependence on the time scales of photoreceptor activation/deactivation. Thus, Synechocystis cells achieve exquisite integration of light inputs at the cellular scale through continuous tuning of motility, and the pattern of collective behavior depends on single-cell motility and population growth.IMPORTANCE The photosynthetic cyanobacterium Synechocystis sp. exhibits phototaxis that is dependent on the incident light wavelength through the action of various photoreceptors. In natural environments, cells experience a set of highly dynamic and complex light inputs, yet how cells transduce multiple or dynamic inputs into motion is unknown. In this study, we measured the phototactic behaviors of single cells and communities as a function of light intensity or when illuminated by combinations of lights of different wavelengths or incidence directions. Responses to a spectrum of light regimes revealed that Synechocystis sp. integrates information about the light environment to tune its phototactic response, which is likely generated by competition among photoreceptors and the degree of wavelength-regulated growth to sensitively control the direction and degree of movement.
View details for DOI 10.1128/mBio.02330-16
View details for Web of Science ID 000400575700011
View details for PubMedID 28270586
Long-term microfluidic tracking of coccoid cyanobacterial cells reveals robust control of division timing
Cyanobacteria are important agents in global carbon and nitrogen cycling and hold great promise for biotechnological applications. Model organisms such as Synechocystis sp. and Synechococcus sp. have advanced our understanding of photosynthetic capacity and circadian behavior, mostly using population-level measurements in which the behavior of individuals cannot be monitored. Synechocystis sp. cells are small and divide slowly, requiring long-term experiments to track single cells. Thus, the cumulative effects of drift over long periods can cause difficulties in monitoring and quantifying cell growth and division dynamics.To overcome this challenge, we enhanced a microfluidic cell-culture device and developed an image analysis pipeline for robust lineage reconstruction. This allowed simultaneous tracking of many cells over multiple generations, and revealed that cells expand exponentially throughout their cell cycle. Generation times were highly correlated for sister cells, but not between mother and daughter cells. Relationships between birth size, division size, and generation time indicated that cell-size control was inconsistent with the "sizer" rule, where division timing is based on cell size, or the "timer" rule, where division occurs after a fixed time interval. Instead, single cell growth statistics were most consistent with the "adder" rule, in which division occurs after a constant increment in cell volume. Cells exposed to light-dark cycles exhibited growth and division only during the light period; dark phases pause but do not disrupt cell-cycle control.Our analyses revealed that the "adder" model can explain both the growth-related statistics of single Synechocystis cells and the correlation between sister cell generation times. We also observed rapid phenotypic response to light-dark transitions at the single cell level, highlighting the critical role of light in cyanobacterial cell-cycle control. Our findings suggest that by monitoring the growth kinetics of individual cells we can build testable models of circadian control of the cell cycle in cyanobacteria.
View details for DOI 10.1186/s12915-016-0344-4
View details for Web of Science ID 000394057800001
View details for PubMedID 28196492
On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires.
2017; 8 (4)
Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium-Arthrospira platensis Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown.IMPORTANCE While the majority of CRISPR-Cas immune systems adapt to foreign genetic elements by capturing segments of invasive DNA, some systems carry reverse transcriptases (RTs) that enable adaptation to RNA molecules. From analysis of available bacterial sequence data, we find evidence that RT-based RNA adaptation machinery has been able to join with CRISPR-Cas immune systems in many, diverse bacterial species. To investigate whether the abilities to adapt to DNA and RNA molecules are utilized for defense against distinct classes of invaders in nature, we sequenced CRISPR arrays from samples of commercial-scale open-air cultures of Arthrospira platensis, a cyanobacterium that contains both RT-lacking and RT-containing CRISPR-Cas systems. We uncovered a diverse pool of naturally occurring immune memories, with the RT-lacking locus acquiring a number of segments matching known viral or bacterial genes, while the RT-containing locus has acquired spacers from a distinct sequence pool for which the source remains enigmatic.
View details for PubMedID 28698278
View details for PubMedCentralID PMC5513706
Diversity in a Polymicrobial Community Revealed by Analysis of Viromes, Endolysins and CRISPR Spacers
2016; 11 (9)
The polymicrobial biofilm communities in Mushroom and Octopus Spring in Yellowstone National Park (YNP) are well characterized, yet little is known about the phage populations. Dominant species, Synechococcus sp. JA-2-3B'a(2-13), Synechococcus sp. JA-3-3Ab, Chloroflexus sp. Y-400-fl, and Roseiflexus sp. RS-1, contain multiple CRISPR-Cas arrays, suggesting complex interactions with phage predators. To analyze phage populations from Octopus Spring biofilms, we sequenced a viral enriched fraction. To assemble and analyze phage metagenomic data, we developed a custom module, VIRITAS, implemented within the MetAMOS framework. This module bins contigs into groups based on tetranucleotide frequencies and CRISPR spacer-protospacer matching and ORF calling. Using this pipeline we were able to assemble phage sequences into contigs and bin them into three clusters that corroborated with their potential host range. The virome contained 52,348 predicted ORFs; some were clearly phage-like; 9319 ORFs had a recognizable Pfam domain while the rest were hypothetical. Of the recognized domains with CRISPR spacer matches, was the phage endolysin used by lytic phage to disrupt cells. Analysis of the endolysins present in the thermophilic cyanophage contigs revealed a subset of characterized endolysins as well as a Glyco_hydro_108 (PF05838) domain not previously associated with sequenced cyanophages. A search for CRISPR spacer matches to all identified phage endolysins demonstrated that a majority of endolysin domains were targets. This strategy provides a general way to link host and phage as endolysins are known to be widely distributed in bacteriophage. Endolysins can also provide information about host cell wall composition and have the additional potential to be used as targets for novel therapeutics.
View details for DOI 10.1371/journal.pone.0160574
View details for Web of Science ID 000383255900008
View details for PubMedID 27611571
Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase-Cas1 fusion protein
2016; 351 (6276): 932-?
CRISPR systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in type I and II CRISPR systems by the acquisition of short segments of DNA (spacers) from invasive elements. In several type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we showed that a RT-Cas1 fusion protein enables the acquisition of RNA spacers in vivo in a RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze the ligation of RNA segments into the CRISPR array, which is followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA.
View details for DOI 10.1126/science.aad4234
View details for Web of Science ID 000370821400031
View details for PubMedID 26917774
View details for PubMedCentralID PMC4898656
- Challenges of metagenomics and single-cell genomics approaches for exploring cyanobacterial diversity. Photosynthesis research 2015; 126 (1): 135-146
- Fine-scale diversity and extensive recombination in a quasisexual bacterial population occupying a broad niche SCIENCE 2015; 348 (6238): 1019-1023
Microbial diversity. Fine-scale diversity and extensive recombination in a quasisexual bacterial population occupying a broad niche.
2015; 348 (6238): 1019-1023
Extensive fine-scale genetic diversity is found in many microbial species across varied environments, but for most, the evolutionary scenarios that generate the observed variation remain unclear. Deep sequencing of a thermophilic cyanobacterial population and analysis of the statistics of synonymous single-nucleotide polymorphisms revealed a high rate of homologous recombination and departures from neutral drift consistent with the effects of genetic hitchhiking. A sequenced isolate genome resembled an unlinked random mixture of the allelic diversity at the sampled loci. These observations suggested a quasisexual microbial population that occupies a broad ecological niche, with selection driving frequencies of alleles rather than whole genomes.
View details for DOI 10.1126/science.aaa4456
View details for PubMedID 26023139
Maintenance of Motility Bias during Cyanobacterial Phototaxis
2015; 108 (7): 1623-1632
Signal transduction in bacteria is complex, ranging across scales from molecular signal detectors and effectors to cellular and community responses to stimuli. The unicellular, photosynthetic cyanobacterium Synechocystis sp. PCC6803 transduces a light stimulus into directional movement known as phototaxis. This response occurs via a biased random walk toward or away from a directional light source, which is sensed by intracellular photoreceptors and mediated by Type IV pili. It is unknown how quickly cells can respond to changes in the presence or directionality of light, or how photoreceptors affect single-cell motility behavior. In this study, we use time-lapse microscopy coupled with quantitative single-cell tracking to investigate the timescale of the cellular response to various light conditions and to characterize the contribution of the photoreceptor TaxD1 (PixJ1) to phototaxis. We first demonstrate that a community of cells exhibits both spatial and population heterogeneity in its phototactic response. We then show that individual cells respond within minutes to changes in light conditions, and that movement directionality is conferred only by the current light directionality, rather than by a long-term memory of previous conditions. Our measurements indicate that motility bias likely results from the polarization of pilus activity, yielding variable levels of movement in different directions. Experiments with a photoreceptor (taxD1) mutant suggest a supplementary role of TaxD1 in enhancing movement directionality, in addition to its previously identified role in promoting positive phototaxis. Motivated by the behavior of the taxD1 mutant, we demonstrate using a reaction-diffusion model that diffusion anisotropy is sufficient to produce the observed changes in the pattern of collective motility. Taken together, our results establish that single-cell tracking can be used to determine the factors that affect motility bias, which can then be coupled with biophysical simulations to connect changes in motility behaviors at the cellular scale with group dynamics.
View details for DOI 10.1016/j.bpj.2015.01.042
View details for Web of Science ID 000352498100010
View details for PubMedID 25863054
View details for PubMedCentralID PMC4390813
Creation and Analysis of a Virome: Using CRISPR Spacers.
Methods in molecular biology (Clifton, N.J.)
2015; 1311: 307-316
Advances in sequencing technology have allowed for the study of complex and previously unexplored microbial and viral populations; however, linking host-phage partners using in silico techniques has been challenging. Here, we describe the flow-through for creation of a virome, and its subsequent analysis with the viral assembly and analysis module "Viritas," which we have recently developed. This module allows for binning of contigs based on tetranucleotide frequencies, putative phage-host partner identification by CRISPR spacer matching, and identification of ORFs.
View details for DOI 10.1007/978-1-4939-2687-9_20
View details for PubMedID 25981482
- Motility Enhancement through Surface Modification Is Sufficient for Cyanobacterial Community Organization during Phototaxis. PLoS computational biology 2013; 9 (9)
Role of Polyphosphate in Thermophilic Synechococcus sp from Microbial Mats
JOURNAL OF BACTERIOLOGY
2013; 195 (15): 3309-3319
Synechococcus OS-B' , a thermophilic unicellular cyanobacterium, recently isolated from the microbial mats in Octopus Spring (Yellowstone National Park), induces a suite of genes, including phosphatases and transporters, in response to phosphorus (P) starvation. Here we describe two different approaches to examine the ability of Synechococcus OS-B' to synthesize and breakdown polyphosphate (poly P), a key storage compound in many prokaryotes. First, we developed a transformation protocol to create mutants in the polyphosphate kinase (ppk), the major enzyme responsible for the synthesis of poly P. The ppk mutant exhibited a pleiotropic phenotype with defects in poly P accumulation, aberrant levels of pho regulon transcripts, growth defects and changes in cell size and exopolysaccharide levels, among others. Second, we measured transcripts of ppk and ppx (encoding the polyphosphatase) directly from mat samples and found that the levels varied dramatically over a diel cycle. We also used Western blot analysis to quantify levels of PPK and PPX and found that these enzymes differentially accumulated during the diel cycle. Levels of polyphosphate kinase peaked at night, while polyphosphatase levels were highest during the early morning hours. We hypothesize that the opposing activities of these two enzymes allow cells to store and utilize poly P to optimize growth over a diel cycle.
View details for DOI 10.1128/JB.00207-13
View details for Web of Science ID 000321559400002
View details for PubMedID 23687278
Motility enhancement through surface modification is sufficient for cyanobacterial community organization during phototaxis.
PLoS computational biology
2013; 9 (9)
The emergent behaviors of communities of genotypically identical cells cannot be easily predicted from the behaviors of individual cells. In many cases, it is thought that direct cell-cell communication plays a critical role in the transition from individual to community behaviors. In the unicellular photosynthetic cyanobacterium Synechocystis sp. PCC 6803, individual cells exhibit light-directed motility ("phototaxis") over surfaces, resulting in the emergence of dynamic spatial organization of multicellular communities. To probe this striking community behavior, we carried out time-lapse video microscopy coupled with quantitative analysis of single-cell dynamics under varying light conditions. These analyses suggest that cells secrete an extracellular substance that modifies the physical properties of the substrate, leading to enhanced motility and the ability for groups of cells to passively guide one another. We developed a biophysical model that demonstrates that this form of indirect, surface-based communication is sufficient to create distinct motile groups whose shape, velocity, and dynamics qualitatively match our experimental observations, even in the absence of direct cellular interactions or changes in single-cell behavior. Our computational analysis of the predicted community behavior, across a matrix of cellular concentrations and light biases, demonstrates that spatial patterning follows robust scaling laws and provides a useful resource for the generation of testable hypotheses regarding phototactic behavior. In addition, we predict that degradation of the surface modification may account for the secondary patterns occasionally observed after the initial formation of a community structure. Taken together, our modeling and experiments provide a framework to show that the emergent spatial organization of phototactic communities requires modification of the substrate, and this form of surface-based communication could provide insight into the behavior of a wide array of biological communities.
View details for DOI 10.1371/journal.pcbi.1003205
View details for PubMedID 24039562
View details for PubMedCentralID PMC3763999
Modeling local interactions during the motion of cyanobacteria
JOURNAL OF THEORETICAL BIOLOGY
2012; 309: 147-158
Synechocystis sp., a common unicellular freshwater cyanobacterium, has been used as a model organism to study phototaxis, an ability to move in the direction of a light source. This microorganism displays a number of additional characteristics such as delayed motion, surface dependence, and a quasi-random motion, where cells move in a seemingly disordered fashion instead of in the direction of the light source, a global force on the system. These unexplained motions are thought to be modulated by local interactions between cells such as intercellular communication. In this paper, we consider only local interactions of these phototactic cells in order to mathematically model this quasi-random motion. We analyze an experimental data set to illustrate the presence of quasi-random motion and then derive a stochastic dynamic particle system modeling interacting phototactic cells. The simulations of our model are consistent with experimentally observed phototactic motion.
View details for DOI 10.1016/j.jtbi.2012.06.013
View details for Web of Science ID 000307526200016
View details for PubMedID 22713858
Novel Miniature Transposable Elements in Thermophilic Synechococcus Strains and Their Impact on an Environmental Population
JOURNAL OF BACTERIOLOGY
2012; 194 (14): 3636-3642
The genomes of the two closely related freshwater thermophilic cyanobacteria Synechococcus sp. strain JA-3-3Ab and Synechococcus sp. strain JA-2-3B'a(2-13) each host several families of insertion sequences (ISSoc families) at various copy numbers, resulting in an overall high abundance of insertion sequences in the genomes. In addition to full-length copies, a large number of internal deletion variants have been identified. ISSoc2 has two variants (ISSoc2∂-1 and ISSoc2∂-2) that are observed to have multiple near-exact copies. Comparison of environmental metagenomic sequences to the Synechococcus genomes reveals novel placement of copies of ISSoc2, ISSoc2∂-1, and ISSoc2∂-2. Thus, ISSoc2∂-1 and ISSoc2∂-2 appear to be active nonautonomous mobile elements derived by internal deletion from ISSoc2. Insertion sites interrupting genes that are likely critical for cell viability were detected; however, most insertions either were intergenic or were within genes of unknown function. Most novel insertions detected in the metagenome were rare, suggesting a stringent selective environment. Evidence for mobility of internal deletion variants of other insertion sequences in these isolates suggests that this is a general mechanism for the formation of miniature insertion sequences.
View details for DOI 10.1128/JB.00333-12
View details for Web of Science ID 000306099700008
View details for PubMedID 22563047
Community ecology of hot spring cyanobacterial mats: predominant populations and their functional potential
2011; 5 (8): 1262-1278
Phototrophic microbial mat communities from 60°C and 65°C regions in the effluent channels of Mushroom and Octopus Springs (Yellowstone National Park, WY, USA) were investigated by shotgun metagenomic sequencing. Analyses of assembled metagenomic sequences resolved six dominant chlorophototrophic populations and permitted the discovery and characterization of undescribed but predominant community members and their physiological potential. Linkage of phylogenetic marker genes and functional genes showed novel chlorophototrophic bacteria belonging to uncharacterized lineages within the order Chlorobiales and within the Kingdom Chloroflexi. The latter is the first chlorophototrophic member of Kingdom Chloroflexi that lies outside the monophyletic group of chlorophototrophs of the Order Chloroflexales. Direct comparison of unassembled metagenomic sequences to genomes of representative isolates showed extensive genetic diversity, genomic rearrangements and novel physiological potential in native populations as compared with genomic references. Synechococcus spp. metagenomic sequences showed a high degree of synteny with the reference genomes of Synechococcus spp. strains A and B', but synteny declined with decreasing sequence relatedness to these references. There was evidence of horizontal gene transfer among native populations, but the frequency of these events was inversely proportional to phylogenetic relatedness.
View details for DOI 10.1038/ismej.2011.73
View details for Web of Science ID 000295782200004
View details for PubMedID 21697961
View details for PubMedCentralID PMC3146275
Analysis of Insertion Sequences in Thermophilic Cyanobacteria: Exploring the Mechanisms of Establishing, Maintaining, and Withstanding High Insertion Sequence Abundance
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
2011; 77 (15): 5458-5466
Insertion sequences (ISs) are simple mobile genetic elements capable of relocating within a genome. Through this transposition activity, they are known to create mutations which are mostly deleterious to the cell, although occasionally they are beneficial. Two closely related isolates of thermophilic Synechococcus species from hot spring microbial mats are known to harbor a large number of diverse ISs. To explore the mechanism of IS acquisition within natural populations and survival in the face of high IS abundance, we examined IS content and location in natural populations of Synechococcus by comparing metagenomic data to the genomes of fully sequenced cultured isolates. The observed IS distribution in the metagenome was equivalent to the distribution in the isolates, indicating that the cultured isolates are appropriate models for the environmental population. High sequence conservation between IS families shared between the two isolates suggests that ISs are able to move between individuals within populations and between species via lateral gene transfer, consistent with models for IS family accumulation. Most IS families show evidence of recent activity, and interruption of critical genes in some individuals was observed, demonstrating that transposition is an ongoing mutational force in the populations.
View details for DOI 10.1128/AEM.05090-11
View details for Web of Science ID 000293224500044
View details for PubMedID 21666019
Whole gene amplification and protein separation from a few cells
2011; 411 (1): 64-70
Despite the growing interest to explore untapped microbial gene and protein diversity, no single platform has been able to acquire both gene and protein information from just a few cells. We present a microfluidic system that simultaneously performs on-chip capillary electrophoresis for protein analysis and whole genome amplification (WGA), and we demonstrate this by doing both for the same cohort of cyanobacterial cells. This technology opens avenues for studying protein profiles of precious environmental microbial samples and simultaneously accessing genomic information based on WGA.
View details for DOI 10.1016/j.ab.2010.12.028
View details for Web of Science ID 000287618900009
View details for PubMedID 21185803
In situ dynamics of O-2, pH and cyanobacterial transcripts associated with CCM, photosynthesis and detoxification of ROS
2011; 5 (2): 317-328
The relative abundance of transcripts encoding proteins involved in inorganic carbon concentrating mechanisms (CCM), detoxification of reactive oxygen species (ROS) and photosynthesis in the thermophilic cyanobacterium Synechococcus OS-B' was measured in hot spring microbial mats over two diel cycles, and was coupled with in situ determinations of incoming irradiance and microenvironmental dynamics of O(2) and pH. Fluctuations in pH and O(2) in the mats were largely driven by the diel cycle of solar irradiance, with a pH variation from ∼7.0 to ∼9.5, and O(2) levels ranging from anoxia to supersaturation during night and day, respectively. Levels of various transcripts from mat cyanobacteria revealed several patterns that correlated with incident irradiance, O(2) and pH within the mat matrix. Transcript abundances for most genes increased during the morning dark-light transition. Some transcripts remained at a near constant level throughout the light period, whereas others showed an additional increase in abundance as the mat underwent transition from low-to-high light (potentially reflecting changes in O(2) concentration and pH), followed by either a decreased abundance in the early afternoon, or a gradual decline during the early afternoon and into the evening. One specific transcipt, psbA1, was the lowest during mid-day under high irradiance and increased when the light levels declined. We discuss these complex in situ transcriptional patterns with respect to environmental and endogenous cues that might impact and regulate transcription over the diel cycle.
View details for DOI 10.1038/ismej.2010.131
View details for Web of Science ID 000290020000015
View details for PubMedID 20740024
View details for PubMedCentralID PMC3105686
Alternative pathways for phosphonate metabolism in thermophilic cyanobacteria from microbial mats
2011; 5 (1): 141-149
Synechococcus sp. represents an ecologically diverse group of cyanobacteria found in numerous environments, including hot-spring microbial mats, where they are spatially distributed along thermal, light and oxygen gradients. These thermophiles engage in photosynthesis and aerobic respiration during the day, but switch to fermentative metabolism and nitrogen fixation at night. The genome of Synechococcus OS-B', isolated from Octopus Spring (Yellowstone National Park) contains a phn gene cluster encoding a phosphonate (Phn) transporter and a C-P lyase. A closely related isolate, Synechococcus OS-A, lacks this cluster, but contains genes encoding putative phosphonatases (Phnases) that appear to be active only in the presence of the Phn substrate. Both isolates grow well on several different Phns as a sole phosphorus (P) source. Interestingly, Synechococcus OS-B' can use the organic carbon backbones of Phns for heterotrophic growth in the dark, whereas in the light this strain releases organic carbon from Phn as ethane or methane (depending on the specific Phn available); Synechococcus OS-A has neither of these capabilities. These differences in metabolic strategies for assimilating the P and C of Phn by two closely related Synechococcus spp. are suggestive of niche-specific constraints in the evolution of nutrient assimilation pathways and syntrophic relationships among the microbial populations of the hot-spring mats. Thus, it is critical to evaluate levels of various P sources, including Phn, in thermally active habitats and the potential importance of these compounds in the biogeochemical cycling of P and C (some Phn compounds also contain N) in diverse terrestrial environments.
View details for DOI 10.1038/ismej.2010.96
View details for Web of Science ID 000285845200014
View details for PubMedID 20631809
View details for PubMedCentralID PMC3105666
CRISPR-Cas Systems in Bacteria and Archaea: Versatile Small RNAs for Adaptive Defense and Regulation
ANNUAL REVIEW GENETICS, VOL 45
2011; 45: 273-297
Bacteria and archaea have evolved defense and regulatory mechanisms to cope with various environmental stressors, including virus attack. This arsenal has been expanded by the recent discovery of the versatile CRISPR-Cas system, which has two novel features. First, the host can specifically incorporate short sequences from invading genetic elements (virus or plasmid) into a region of its genome that is distinguished by clustered regularly interspaced short palindromic repeats (CRISPRs). Second, when these sequences are transcribed and precisely processed into small RNAs, they guide a multifunctional protein complex (Cas proteins) to recognize and cleave incoming foreign genetic material. This adaptive immunity system, which uses a library of small noncoding RNAs as a potent weapon against fast-evolving viruses, is also used as a regulatory system by the host. Exciting breakthroughs in understanding the mechanisms of the CRISPR-Cas system and its potential for biotechnological applications and understanding evolutionary dynamics are discussed.
View details for DOI 10.1146/annurev-genet-110410-132430
View details for Web of Science ID 000299299600013
View details for PubMedID 22060043
Germ Warfare in a Microbial Mat Community: CRISPRs Provide Insights into the Co-Evolution of Host and Viral Genomes
2009; 4 (1)
CRISPR arrays and associated cas genes are widespread in bacteria and archaea and confer acquired resistance to viruses. To examine viral immunity in the context of naturally evolving microbial populations we analyzed genomic data from two thermophilic Synechococcus isolates (Syn OS-A and Syn OS-B') as well as a prokaryotic metagenome and viral metagenome derived from microbial mats in hotsprings at Yellowstone National Park. Two distinct CRISPR types, distinguished by the repeat sequence, are found in both the Syn OS-A and Syn OS-B' genomes. The genome of Syn OS-A contains a third CRISPR type with a distinct repeat sequence, which is not found in Syn OS-B', but appears to be shared with other microorganisms that inhabit the mat. The CRISPR repeats identified in the microbial metagenome are highly conserved, while the spacer sequences (hereafter referred to as "viritopes" to emphasize their critical role in viral immunity) were mostly unique and had no high identity matches when searched against GenBank. Searching the viritopes against the viral metagenome, however, yielded several matches with high similarity some of which were within a gene identified as a likely viral lysozyme/lysin protein. Analysis of viral metagenome sequences corresponding to this lysozyme/lysin protein revealed several mutations all of which translate into silent or conservative mutations which are unlikely to affect protein function, but may help the virus evade the host CRISPR resistance mechanism. These results demonstrate the varied challenges presented by a natural virus population, and support the notion that the CRISPR/viritope system must be able to adapt quickly to provide host immunity. The ability of metagenomics to track population-level variation in viritope sequences allows for a culture-independent method for evaluating the fast co-evolution of host and viral genomes and its consequence on the structuring of complex microbial communities.
View details for DOI 10.1371/journal.pone.0004169
View details for Web of Science ID 000265473700008
View details for PubMedID 19132092
Phosphorus Deprivation Responses and Phosphonate Utilization in a Thermophilic Synechococcus sp from Microbial Mats
JOURNAL OF BACTERIOLOGY
2008; 190 (24): 8171-8184
The genomes of two closely related thermophilic cyanobacterial isolates, designated Synechococcus isolate OS-A and Synechococcus isolate OS-B', from the microbial mats of Octopus Spring (Yellowstone National Park) have been sequenced. An extensive suite of genes that are controlled by phosphate levels constitute the putative Pho regulon in these cyanobacteria. We examined physiological responses of an axenic OS-B' isolate as well as transcript abundances of Pho regulon genes as the cells acclimated to phosphorus-limiting conditions. Upon imposition of phosphorus deprivation, OS-B' stopped dividing after three to four doublings, and absorbance spectra measurements indicated that the cells had lost most of their phycobiliproteins and chlorophyll a. Alkaline phosphatase activity peaked and remained high after 48 h of phosphorus starvation, and there was an accumulation of transcripts from putative Pho regulon genes. Interestingly, the genome of Synechococcus isolate OS-B' harbors a cluster of phn genes that are not present in OS-A isolates. The proteins encoded by the phn genes function in the transport and metabolism of phosphonates, which could serve as an alternative phosphorus source when exogenous phosphate is low. The phn genes were upregulated within a day of eliminating the source of phosphate from the medium. However, the ability of OS-B' to utilize methylphosphonate as a sole phosphorus source occurred only after an extensive period of exposure to the substrate. Once acclimated, the cells grew rapidly in fresh medium with methylphosphonate as the only source of phosphorus. The possible implications of these results are discussed with respect to the ecophysiology of the microbial mats.
View details for DOI 10.1128/JB.01011-08
View details for Web of Science ID 000261217900036
View details for PubMedID 18931115
View details for PubMedCentralID PMC2593230
Characterization of two thermostable cyanobacterial phytochromes reveals global movements in the chromophore-binding domain during photoconversion
JOURNAL OF BIOLOGICAL CHEMISTRY
2008; 283 (30): 21251-21266
Photointerconversion between the red light-absorbing (Pr) form and the far-red light-absorbing (Pfr) form is the central feature that allows members of the phytochrome (Phy) superfamily to act as reversible switches in light perception. Whereas the chromophore structure and surrounding binding pocket of Pr have been described, those for Pfr have remained enigmatic for various technical reasons. Here we describe a novel pair of Phys from two thermophilic cyanobacteria, Synechococcus sp. OS-A and OS-B', that overcome several of these limitations. Like other cyanobacterial Phys, SyA-Cph1 and SyB-Cph1 covalently bind the bilin phycocyanobilin via their cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) domains and then assume the photointerconvertible Pr and Pfr states with absorption maxima at 630 and 704 nm, respectively. However, they are naturally missing the N-terminal Per/Arndt/Sim domain common to others in the Phy superfamily. Importantly, truncations containing only the GAF domain are monomeric, photochromic, and remarkably thermostable. Resonance Raman and NMR spectroscopy show that all four pyrrole ring nitrogens of phycocyanobilin are protonated both as Pr and following red light irradiation, indicating that the GAF domain by itself can complete the Pr to Pfr photocycle. (1)H-(15)N two-dimensional NMR spectra of isotopically labeled preparations of the SyB-Cph1 GAF domain revealed that a number of amino acids change their environment during photoconversion of Pr to Pfr, which can be reversed by subsequent photoconversion back to Pr. Through three-dimensional NMR spectroscopy before and after light photoexcitation, it should now be possible to define the movements of the chromophore and binding pocket during photoconversion. We also generated a series of strongly red fluorescent derivatives of SyB-Cph1, which based on their small size and thermostability may be useful as cell biological reporters.
View details for DOI 10.1074/jbc.M801592200
View details for Web of Science ID 000257746100067
View details for PubMedID 18480055
Tracking phototactic responses and modeling motility of Synechocystis sp strain PCC6803
JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY
2008; 91 (2-3): 77-86
The unicellular cyanobacterium Synechocystis sp. Strain PCC 6,803 exhibits phototaxis by moving along a surface towards a light source. This process requires Type IV pili and a phytochrome-like photoreceptor coupled to a complex signal transduction pathway. Cells progress through different phases of interaction before the development of finger-like projections moving in the direction of the light that are characteristic of phototaxis. To probe the interaction between individual cells during the initial phase of phototaxis we tracked and analyzed a large number of cells. We observed that individual cells have limited motility, but when cells divide and/or aggregate to attain a certain minimal group size, enhanced motility and phototaxis is observed. At the later stages of motility, there is noticeable phototactic behavior which results in the appearance of the finger-like projections. Our results indicate that cells prefer to move over areas previously traversed by other cells and confine themselves to these areas and that cells alter local surface characteristics allowing for enhanced motility. Based on cell tracking data we present a preliminary random walk model showing the forces that might interact to create the typical phases of phototaxis and motility. In this model, we can simulate the formation of finger-like projections that are characteristic of phototaxis.
View details for DOI 10.1016/j.jphotobiol.2008.01.012
View details for Web of Science ID 000256725500002
View details for PubMedID 18343151
Regulation of nif gene expression and the energetics of N-2 fixation over the diel cycle in a hot spring microbial mat
2008; 2 (4): 364-378
Nitrogen fixation, a prokaryotic, O2-inhibited process that reduces N2 gas to biomass, is of paramount importance in biogeochemical cycling of nitrogen. We analyzed the levels of nif transcripts of Synechococcus ecotypes, NifH subunit and nitrogenase activity over the diel cycle in the microbial mat of an alkaline hot spring in Yellowstone National Park. The results showed a rise in nif transcripts in the evening, with a subsequent decline over the course of the night. In contrast, immunological data demonstrated that the level of the NifH polypeptide remained stable during the night, and only declined when the mat became oxic in the morning. Nitrogenase activity was low throughout the night; however, it exhibited two peaks, a small one in the evening and a large one in the early morning, when light began to stimulate cyanobacterial photosynthetic activity, but O2 consumption by respiration still exceeded the rate of O2 evolution. Once the irradiance increased to the point at which the mat became oxic, the nitrogenase activity was strongly inhibited. Transcripts for proteins associated with energy-producing metabolisms in the cell also followed diel patterns, with fermentation-related transcripts accumulating at night, photosynthesis- and respiration-related transcripts accumulating during the day and late afternoon, respectively. These results are discussed with respect to the energetics and regulation of N2 fixation in hot spring mats and factors that can markedly influence the extent of N2 fixation over the diel cycle.
View details for DOI 10.1038/ismej.2007.117
View details for Web of Science ID 000255288700003
View details for PubMedID 18323780
Genomics, environmental genomics and the issue of microbial species
2008; 100 (2): 207-219
A microbial species concept is crucial for interpreting the variation detected by genomics and environmental genomics among cultivated microorganisms and within natural microbial populations. Comparative genomic analyses of prokaryotic species as they are presently described and named have led to the provocative idea that prokaryotes may not form species as we think about them for plants and animals. There are good reasons to doubt whether presently recognized prokaryotic species are truly species. To achieve a better understanding of microbial species, we believe it is necessary to (i) re-evaluate traditional approaches in light of evolutionary and ecological theory, (ii) consider that different microbial species may have evolved in different ways and (iii) integrate genomic, metagenomic and genome-wide expression approaches with ecological and evolutionary theory. Here, we outline how we are using genomic methods to (i) identify ecologically distinct populations (ecotypes) predicted by theory to be species-like fundamental units of microbial communities, and (ii) test their species-like character through in situ distribution and gene expression studies. By comparing metagenomic sequences obtained from well-studied hot spring cyanobacterial mats with genomic sequences of two cultivated cyanobacterial ecotypes, closely related to predominant native populations, we can conduct in situ population genetics studies that identify putative ecotypes and functional genes that determine the ecotypes' ecological distinctness. If individuals within microbial communities are found to be grouped into ecologically distinct, species-like populations, knowing about such populations should guide us to a better understanding of how genomic variation is linked to community function.
View details for DOI 10.1038/sj.hdy.6801011
View details for Web of Science ID 000252585800014
View details for PubMedID 17551524
A novel two domain-fusion protein in cyanobacteria with similarity to the CAB/ELIP/HLIP superfamily: Evolutionary implications and regulation
2008; 1 (1): 155-166
Vascular plants contain abundant, light-harvesting complexes in the thylakoid membrane that are non-covalently associated with chlorophylls and carotenoids. These light-harvesting chlorophyll a/b binding (LHC) proteins are members of an extended CAB/ELIP/HLIP superfamily of distantly related polypeptides, which have between one and four transmembrane helices (TMH). This superfamily includes the single TMH, high-light-inducible proteins (Hlips), found in cyanobacteria that are induced by various stress conditions, including high light, and are considered ancestral to the LHC proteins. The roles of, and evolutionary relationships between, these superfamily members are of particular interest, since they function in both light harvesting and photoprotection and may have evolved through tandem gene duplication and fusion events. We have investigated the Hlips (hli gene family) in the thermophilic unicellular cyanobacterium Synechococcus OS-B'. The five hli genes present on the genome of Synechococcus OS-B' are relatively similar, but transcript analyses indicate that there are different patterns of transcript accumulation when the cells are exposed to various growth conditions, suggesting that different Hlips may have specific functions. Hlip5 has an additional TMH at the N-terminus as a result of a novel fusion event. This additional TMH is very similar to a conserved hypothetical, single membrane-spanning polypeptide present in most cyanobacteria. The evolutionary significance of these results is discussed.
View details for DOI 10.1093/mp/ssm019
View details for Web of Science ID 000259068900015
View details for PubMedID 20031922
Population level functional diversity in a microbial community revealed by comparative genomic and metagenomic analyses
2007; 1 (8): 703-713
In microbial mat communities of Yellowstone hot springs, ribosomal RNA (rRNA) sequence diversity patterns indicate the presence of closely related bacterial populations along environmental gradients of temperature and light. To identify the functional bases for adaptation, we sequenced the genomes of two cyanobacterial (Synechococcus OS-A and OS-B') isolates representing ecologically distinct populations that dominate at different temperatures and are major primary producers in the mat. There was a marked lack of conserved large-scale gene order between the two Synechococcus genomes, indicative of extensive genomic rearrangements. Comparative genomic analyses showed that the isolates shared a large fraction of their gene content at high identity, yet, differences in phosphate and nitrogen utilization pathways indicated that they have adapted differentially to nutrient fluxes, possibly by the acquisition of genes by lateral gene transfer or their loss in certain populations. Comparisons of the Synechococcus genomes to metagenomic sequences derived from mats where these Synechococcus stains were originally isolated, revealed new facets of microbial diversity. First, Synechococcus populations at the lower temperature regions of the mat showed greater sequence diversity than those at high temperatures, consistent with a greater number of ecologically distinct populations at the lower temperature. Second, we found evidence of a specialized population that is apparently very closely related to Synechococcus OS-B', but contains genes that function in the uptake of reduced ferrous iron. In situ expression studies demonstrated that these genes are differentially expressed over the diel cycle, with highest expression when the mats are anoxic and iron may be in the reduced state. Genomic information from these mat-specific isolates and metagenomic information can be coupled to detect naturally occurring populations that are associated with different functionalities, not always represented by isolates, but which may nevertheless be important for niche partitioning and the establishment of microbial community structure.
View details for DOI 10.1038/ismej.2007.46
View details for Web of Science ID 000251946500004
View details for PubMedID 18059494
Responses of a thermophilic Synechococcus isolate from the microbial mat of octopus spring to light
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
2007; 73 (13): 4268-4278
Thermophilic cyanobacteria of the genus Synechococcus are major contributors to photosynthetic carbon fixation in the photic zone of microbial mats in Octopus Spring, Yellowstone National Park. Synechococcus OS-B' was characterized with regard to the ability to acclimate to a range of different light irradiances; it grows well at 25 to 200 micromol photons m(-2) s(-1) but dies when the irradiance is increased to 400 micromol photons m(-2) s(-1). At 200 micromol photons m(-2) s(-1) (high light [HL]), we noted several responses that had previously been associated with HL acclimation of cyanobacteria, including cell bleaching, reduced levels of phycobilisomes and chlorophyll, and elevated levels of a specific carotenoid. Synechococcus OS-B' synthesizes the carotenoids zeaxanthin and beta,beta-carotene and a novel myxol-anhydrohexoside. Interestingly, 77-K fluorescence emission spectra suggest that Synechococcus OS-B' accumulates very small amounts of photosystem II relative to that of photosystem I. This ratio further decreased at higher growth irradiances, which may reflect potential photodamage following exposure to HL. We also noted that HL caused reduced levels of transcripts encoding phycobilisome components, particularly that for CpcH, a 20.5-kDa rod linker polypeptide. There was enhanced transcript abundance of genes encoding terminal oxidases, superoxide dismutase, tocopherol cyclase, and phytoene desaturase. Genes encoding the photosystem II D1:1 and D1:2 isoforms (psbAI and psbAII/psbAIII, respectively) were also regulated according to the light regimen. The results are discussed in the context of how Synechococcus OS-B' may cope with high light irradiances in the high-temperature environment of the microbial mat.
View details for DOI 10.1128/AEM.00201-07
View details for Web of Science ID 000248070000023
View details for PubMedID 17483258
View details for PubMedCentralID PMC1932787
Counting low-copy number proteins in a single cell
2007; 315 (5808): 81-84
We have designed a microfluidic device in which we can manipulate, lyse, label, separate, and quantify the protein contents of a single cell using single-molecule fluorescence counting. Generic labeling of proteins is achieved through fluorescent-antibody binding. The use of cylindrical optics enables high-efficiency (approximately 60%) counting of molecules in micrometer-sized channels. We used this microfluidic device to quantify beta2 adrenergic receptors expressed in insect cells (SF9). We also analyzed phycobiliprotein contents in individual cyanobacterial cells (Synechococcus sp. PCC 7942) and observed marked differences in the levels of specific complexes in cell populations that were grown under nitrogen-depleted conditions.
View details for DOI 10.1126/science.1133992
View details for Web of Science ID 000243259100039
View details for PubMedID 17204646
Negative control of the high light-inducible hliA gene and implications for the activities of the NblS sensor kinase in the cyanobacterium Synechococcus elongatus strain PCC 7942
ARCHIVES OF MICROBIOLOGY
2006; 186 (5): 403-413
The hliA gene of the cyanobacterium Synechococcus elongatus PCC 7942 is known to be upregulated by high-intensity light through the activity of the NblS sensor kinase. In this work it was found that, within the hliA upstream region, changes to the sequence around -30 to -25 (relative to the transcriptional start site) resulted in elevated hliA expression, implicating this region in negative regulation of the gene. Electrophoretic mobility shift assays performed were consistent with a protein binding this region that acts to keep the gene off in lower light. A reduction in gene dosage of nblS in vivo resulted in enhanced hliA expression, suggesting that negative control of hliA is mediated through NblS. An extended version of the high light regulatory 1 (HLR1) motif (previously described in Synechocystis PCC 6803) was identified within the sequence surrounding -30 to -25 of hliA. The extended HLR1 sequence was found upstream of other NblS-controlled genes from S. elongatus and Synechocystis PCC 6803 and upstream of hli genes from a variety of cyanobacterial and related genomes. These results point to the evolutionary conservation of the HLR1 element and its importance in NblS-mediated signaling and yield new insight into NblS-mediated control of gene expression.
View details for DOI 10.1007/s00203-006-0154-0
View details for Web of Science ID 000241795400007
View details for PubMedID 16897032
Phototaxis and impaired motility in adenylyl cyclase and cyclase receptor protein mutants of Synechocystis sp strain PCC 6803
JOURNAL OF BACTERIOLOGY
2006; 188 (20): 7306-7310
We have carefully characterized and reexamined the motility and phototactic responses of Synechocystis sp. adenylyl cyclase (Cya1) and catabolite activator protein (SYCRP1) mutants to different light regimens, glucose, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and cyclic AMP. We find that contrary to earlier reports, cya1 and sycrp1 mutants are motile and phototactic but are impaired in one particular phase of phototaxis in comparison with wild-type Synechocystis sp.
View details for DOI 10.1128/JB.00573-06
View details for Web of Science ID 000241239600030
View details for PubMedID 17015670
View details for PubMedCentralID PMC1636242
- Examination of diel changes in global transcript accumulation in Synechocystis (cyanobacteria) JOURNAL OF PHYCOLOGY 2006; 42 (3): 622-636
In situ analysis of nitrogen fixation and metabolic switching in unicellular thermophilic cyanobacteria inhabiting hot spring microbial mats
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2006; 103 (7): 2398-2403
Genome sequences of two Synechococcus ecotypes inhabiting the Octopus Spring microbial mat in Yellowstone National Park revealed the presence of all genes required for nitrogenase biosynthesis. We demonstrate that nif genes of the Synechococcus ecotypes are expressed in situ in a region of the mat that varies in temperature from 53.5 degrees C to 63.4 degrees C (average 60 degrees C); transcripts are only detected at the end of the day when the mat becomes anoxic. Nitrogenase activity in mat samples was also detected in the evening. Hitherto, N2 fixation in hot spring mats was attributed either to filamentous cyanobacteria (not present at >50 degrees C in these mats) or to heterotrophic bacteria. To explore how energy-generating processes of the Synechococcus ecotypes track natural light and O2 conditions, we evaluated accumulation of transcripts encoding proteins involved in photosynthesis, respiration, and fermentation. Transcripts from photosynthesis (cpcF, cpcE, psaB, and psbB) and respiration (coxA and cydA) genes declined in the evening. In contrast, transcripts encoding enzymes that may participate in fermentation fell into two categories; some (ldh, pdhB, ald, and ackA) decreased in the evening, whereas others (pflB, pflA, adhE, and acs) increased at the end of the day and remained high into the night. Energy required for N2 fixation during the night may be derived from fermentation pathways that become prominent as the mat becomes anoxic. In a broader context, our data suggest that there are critical regulatory switches in situ that are linked to the diel cycle and that these switches alter many metabolic processes within the microbial mat.
View details for DOI 10.1073/pnas.0507513103
View details for Web of Science ID 000235411600071
View details for PubMedID 16467157
View details for PubMedCentralID PMC1413695
Light matters: phototaxis and signal transduction in unicellular cyanobacteria
2004; 53 (3): 745-754
Many photosynthetic microorganisms have evolved the ability to sense light quality and/or quantity and can steer themselves into optimal conditions within the environment. Phototaxis and gliding motility in unicellular cyanobacteria require type IV pili, which are multifunctional cell surface appendages. Screens for cells exhibiting aberrant motility uncovered several non-motile mutants as well as some that had lost positive phototaxis (consequently, they were negatively phototactic). Several negatively phototactic mutants mapped to the tax1 locus, which contains five chemotaxis-like genes. This locus includes a gene that encodes a putative photoreceptor (TaxD1) for positive phototaxis. A second chemotaxis-like cluster (tax3 locus) appears to be involved in pilus biogenesis. The biosynthesis and regulation of type IV pilus-based motility as well as the communication between the pilus motor and photosensory molecules appear to be complex and tightly regulated. Furthermore, the discovery that cyclic AMP and novel gene products are necessary for phototaxis/motility suggests that there might be additional levels of communication and signal processing.
View details for DOI 10.1111/j.1365-2958.2004.04160.x
View details for Web of Science ID 000222722000004
View details for PubMedID 15255889
Multiple light inputs control phototaxis in Synechocystis sp strain PCC6803
JOURNAL OF BACTERIOLOGY
2003; 185 (5): 1599-1607
The phototactic behavior of individual cells of the cyanobacterium Synechocystis sp. strain PCC6803 was studied with a glass slide-based phototaxis assay. Data from fluence rate-response curves and action spectra suggested that there were at least two light input pathways regulating phototaxis. We observed that positive phototaxis in wild-type cells was a low fluence response, with peak spectral sensitivity at 645 and 704 nm. This red-light-induced phototaxis was inhibited or photoreversible by infrared light (760 nm). Previous work demonstrated that a taxD1 mutant (Cyanobase accession no. sll0041; also called pisJ1) lacked positive but maintained negative phototaxis. Therefore, the TaxD1 protein, which has domains that are similar to sequences found in both bacteriophytochrome and the methyl-accepting chemoreceptor protein, is likely to be the photoreceptor that mediates positive phototaxis. Wild-type cells exhibited negative phototaxis under high-intensity broad-spectrum light. This phenomenon is predominantly blue light responsive, with a maximum sensitivity at approximately 470 nm. A weakly negative phototactic response was also observed in the spectral region between 600 and 700 nm. A deltataxD1 mutant, which exhibits negative phototaxis even under low-fluence light, has a similar action maximum in the blue region of the spectrum, with minor peaks from green to infrared (500 to 740 nm). These results suggest that while positive phototaxis is controlled by the red light photoreceptor TaxD1, negative phototaxis in Synechocystis sp. strain PCC6803 is mediated by one or more (as yet) unidentified blue light photoreceptors.
View details for DOI 10.1128/JB.185.5.1599-1607.2003
View details for Web of Science ID 000181151200015
View details for PubMedID 12591877
View details for PubMedCentralID PMC148062
Analysis of the hli gene family in marine and freshwater cyanobacteria
FEMS MICROBIOLOGY LETTERS
2002; 215 (2): 209-219
Certain cyanobacteria thrive in natural habitats in which light intensities can reach 2000 micromol photon m(-2) s(-1) and nutrient levels are extremely low. Recently, a family of genes designated hli was demonstrated to be important for survival of cyanobacteria during exposure to high light. In this study we have identified members of the hli gene family in seven cyanobacterial genomes, including those of a marine cyanobacterium adapted to high-light growth in surface waters of the open ocean (Prochlorococcus sp. strain Med4), three marine cyanobacteria adapted to growth in moderate- or low-light (Prochlorococcus sp. strain MIT9313, Prochlorococcus marinus SS120, and Synechococcus WH8102), and three freshwater strains (the unicellular Synechocystis sp. strain PCC6803 and the filamentous species Nostoc punctiforme strain ATCC29133 and Anabaena sp. [Nostoc] strain PCC7120). The high-light-adapted Prochlorococcus Med4 has the smallest genome (1.7 Mb), yet it has more than twice as many hli genes as any of the other six cyanobacterial species, some of which appear to have arisen from recent duplication events. Based on cluster analysis, some groups of hli genes appear to be specific to either marine or freshwater cyanobacteria. This information is discussed with respect to the role of hli genes in the acclimation of cyanobacteria to high light, and the possible relationships among members of this diverse gene family.
View details for Web of Science ID 000178923300007
View details for PubMedID 12399037
Novel motility mutants of Synechocystis strain PCC 6803 generated by in vitro transposon mutagenesis
JOURNAL OF BACTERIOLOGY
2001; 183 (20): 6140-6143
We screened for transposon-generated mutants of Synechocystis sp. strain PCC 6803 that exhibited aberrant phototactic movement. Of the 300 mutants generated, about 50 have been partially characterized; several contained transposons in genes encoding chemotaxis-related proteins, while others mapped to novel genes. These novel genes and their possible roles in motility are discussed.
View details for Web of Science ID 000171267100040
View details for PubMedID 11567015
View details for PubMedCentralID PMC99694
Light regulation of type IV pilus-dependent motility by chemosensor-like elements in Synechocystis PCC6803
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2001; 98 (13): 7540-7545
To optimize photosynthesis, cyanobacteria move toward or away from a light source by a process known as phototaxis. Phototactic movement of the cyanobacterium Synechocystis PCC6803 is a surface-dependent phenomenon that requires type IV pili, cellular appendages implicated in twitching and social motility in a range of bacteria. To elucidate regulation of cyanobacterial motility, we generated transposon-tagged mutants with aberrant phototaxis; mutants were either nonmotile or exhibited an "inverted motility response" (negative phototaxis) relative to wild-type cells. Several mutants contained transposons in genes similar to those involved in bacterial chemotaxis. Synechocystis PCC6803 has three loci with chemotaxis-like genes, of which two, Tax1 and Tax3, are involved in phototaxis. Transposons interrupting the Tax1 locus yielded mutants that exhibited an inverted motility response, suggesting that this locus is involved in controlling positive phototaxis. However, a strain null for taxAY1 was nonmotile and hyperpiliated. Interestingly, whereas the C-terminal region of the TaxD1 polypeptide is similar to the signaling domain of enteric methyl-accepting chemoreceptor proteins, the N terminus has two domains resembling chromophore-binding domains of phytochrome, a photoreceptor in plants. Hence, TaxD1 may play a role in perceiving the light stimulus. Mutants in the Tax3 locus are nonmotile and do not make type IV pili. These findings establish links between chemotaxis-like regulatory elements and type IV pilus-mediated phototaxis.
View details for Web of Science ID 000169456600098
View details for PubMedID 11404477
View details for PubMedCentralID PMC34704
- Tracking the light environment by cyanobacteria and the dynamic nature of light harvesting JOURNAL OF BIOLOGICAL CHEMISTRY 2001; 276 (15): 11449-11452
Highly expressed and alien genes of the Synechocystis genome
NUCLEIC ACIDS RESEARCH
2001; 29 (7): 1590-1601
Comparisons of codon frequencies of genes to several gene classes are used to characterize highly expressed and alien genes on the SYNECHOCYSTIS: PCC6803 genome. The primary gene classes include the ensemble of all genes (average gene), ribosomal protein (RP) genes, translation processing factors (TF) and genes encoding chaperone/degradation proteins (CH). A gene is predicted highly expressed (PHX) if its codon usage is close to that of the RP/TF/CH standards but strongly deviant from the average gene. Putative alien (PA) genes are those for which codon usage is significantly different from all four classes of gene standards. In SYNECHOCYSTIS:, 380 genes were identified as PHX. The genes with the highest predicted expression levels include many that encode proteins vital for photosynthesis. Nearly all of the genes of the RP/TF/CH gene classes are PHX. The principal glycolysis enzymes, which may also function in CO(2) fixation, are PHX, while none of the genes encoding TCA cycle enzymes are PHX. The PA genes are mostly of unknown function or encode transposases. Several PA genes encode polypeptides that function in lipopolysaccharide biosynthesis. Both PHX and PA genes often form significant clusters (operons). The proteins encoded by PHX and PA genes are described with respect to functional classifications, their organization in the genome and their stoichiometry in multi-subunit complexes.
View details for Web of Science ID 000167970300024
View details for PubMedID 11266562
View details for PubMedCentralID PMC31270
The Arabidopsis Information Resource (TAIR): a comprehensive database and web-based information retrieval, analysis, and visualization system for a model plant
NUCLEIC ACIDS RESEARCH
2001; 29 (1): 102-105
Arabidopsis thaliana, a small annual plant belonging to the mustard family, is the subject of study by an estimated 7000 researchers around the world. In addition to the large body of genetic, physiological and biochemical data gathered for this plant, it will be the first higher plant genome to be completely sequenced, with completion expected at the end of the year 2000. The sequencing effort has been coordinated by an international collaboration, the Arabidopsis Genome Initiative (AGI). The rationale for intensive investigation of Arabidopsis is that it is an excellent model for higher plants. In order to maximize use of the knowledge gained about this plant, there is a need for a comprehensive database and information retrieval and analysis system that will provide user-friendly access to Arabidopsis information. This paper describes the initial steps we have taken toward realizing these goals in a project called The Arabidopsis Information Resource (TAIR) (www.arabidopsis.org).
View details for Web of Science ID 000166360300025
View details for PubMedID 11125061
View details for PubMedCentralID PMC29827
Isolation of regulated genes of the cyanobacterium Synechocystis sp strain PCC 6803 by differential display
JOURNAL OF BACTERIOLOGY
2000; 182 (20): 5692-5699
Global identification of differentially regulated genes in prokaryotes is constrained because the mRNA does not have a 3' polyadenylation extension; this precludes specific separation of mRNA from rRNA and tRNA and synthesis of cDNAs from the entire mRNA population. Knowledge of the entire genome sequence of Synechocystis sp. strain PCC 6803 has enabled us to develop a differential display procedure that takes advantage of a short palindromic sequence that is dispersed throughout the Synechocystis sp. strain PCC 6803 genome. This sequence, designated the HIP (highly iterated palindrome) element, occurs in approximately half of the Synechocystis sp. strain PCC 6803 genes but is absent in rRNA and tRNA genes. To determine the feasibility of exploiting the HIP element, alone or in combination with specific primer subsets, for analyzing differential gene expression, we used HIP-based primers to identify light intensity-regulated genes. Several gene fragments, including those encoding ribosomal proteins and phycobiliprotein subunits, were differentially amplified from RNA templates derived from cells grown in low light or exposed to high light for 3 h. One novel finding was that expression of certain genes of the pho regulon, which are under the control of environmental phosphate levels, were markedly elevated in high light. High-light activation of pho regulon genes correlated with elevated growth rates that occur when the cells are transferred from low to high light. These results suggest that in high light, the rate of growth of Synechocystis sp. strain PCC 6803 exceeds its capacity to assimilate phosphate, which, in turn, may trigger a phosphate starvation response and activation of the pho regulon.
View details for Web of Science ID 000089576300007
View details for PubMedID 11004166
View details for PubMedCentralID PMC94689
Type IV pilus biogenesis and motility in the cyanobacterium Synechocystis sp PCC6803
2000; 37 (4): 941-951
We have recently shown that phototactic movement in the unicellular cyanobacterium Synechocystis sp. PCC6803 requires type IV pilins. To elucidate further type IV pilus-dependent motility, we inactivated key genes implicated in pilus biogenesis and function. Wild-type Synechocystis sp. PCC6803 cells have two morphologically distinct pilus types (thick and thin pili). Of these, the thick pilus morphotype, absent in a mutant disrupted for the pilin-encoding pilA1 gene, appears to be required for motility. The thin pilus morphotype does not appear to be altered in the pilA1 mutant, raising the possibility that thin pili have a function distinct from that of motility. Mutants disrupted for pilA2, which encodes a second pilin-like protein, are still motile and exhibit no difference in morphology or density of cell-surface pili. In contrast, inactivation of pilD (encoding the leader peptidase) or pilC (encoding a protein required for pilus assembly) abolishes cell motility, and both pilus morphotypes are absent. Thus, the PilA1 polypeptide is required for the biogenesis of the thick pilus morphotype which, in turn, is necessary for motility (hence we refer to them as type IV pili). Furthermore, PilA2 is critical neither for motility nor for pilus biogenesis. Two genes encoding proteins with similarity to PilT, which is considered to be a component of the motor essential for type IV pilus-dependent movement, were also inactivated. A pilT1 mutant is (i) non-motile, (ii) hyperpiliated and (iii) accumulates higher than normal levels of the pilA1 transcript. In contrast, pilT2 mutants are motile, but are negatively phototactic under conditions in which wild-type cells are positively phototactic.
View details for Web of Science ID 000089054100022
View details for PubMedID 10972813
The role of an alternative sigma factor in motility and pilus formation in the cyanobacterium Synechocystis sp. strain PCC6803
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1999; 96 (6): 3188-3193
Disruption of a gene for an alternative sigma factor, designated sigF, in the freshwater, unicellular cyanobacterium Synechocystis sp. strain PCC6803 resulted in a pleiotropic phenotype. Most notably, this mutant lost phototactic movement with a concomitant loss of pili, which are abundant on the surface of wild-type cells. The sigF mutant also secreted both high levels of yellow-brown and UV-absorbing pigments and a polypeptide that is similar to a large family of extracellular proteins that includes the hemolysins. Furthermore, the sigF mutant had a dramatically reduced level of the transcript from two tandemly arranged pilA genes (sll1694 and sll1695), which encode major structural components of type IV pili. Inactivation of these pilA genes eliminated phototactic movement, though some pili were still present in this strain. Together, these results demonstrate that SigF plays a critical role in motility via the control of pili formation and is also likely to regulate other features of the cell surface. Furthermore, the data provide evidence that type IV pili are required for phototactic movement in certain cyanobacteria and suggest that different populations of pili present on the Synechocystis cell surface may perform different functions.
View details for Web of Science ID 000079224500113
View details for PubMedID 10077659
View details for PubMedCentralID PMC15917
Molecular responses of cyanobacteria to macronutrient limitation
JOURNAL OF SCIENTIFIC & INDUSTRIAL RESEARCH
1996; 55 (8-9): 630-637
View details for Web of Science ID A1996VB44000007
CYANOBACTERIAL PROTEIN WITH SIMILARITY TO THE CHLOROPHYLL A/B BINDING-PROTEINS OF HIGHER-PLANTS - EVOLUTION AND REGULATION
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1995; 92 (2): 636-640
We have isolated, from the prokaryotic cyanobacterium Synechococcus sp. strain PCC 7942, a gene encoding a protein of 72 amino acids [designated high light inducible protein (HLIP)] with similarity to the extended family of eukaryotic chlorophyll a/b binding proteins (CABs). HLIP has a single membrane-spanning alpha-helix, whereas both the CABs and the related early light inducible proteins have three membrane-spanning helices. Hence, HLIP may represent an evolutionary progenitor of the eukaryotic members of the CAB extended family. We also show that the gene encoding HLIP is induced by high light and blue/UV-A radiation. The evolution, regulation, and potential function of HLIP are discussed.
View details for Web of Science ID A1995QC87400063
View details for PubMedID 7831342
View details for PubMedCentralID PMC42797
Light-harvesting complexes in oxygenic photosynthesis: Diversity, control, and evolution
ANNUAL REVIEW OF GENETICS
1995; 29: 231-288
This article focuses on light-harvesting complexes (LHCs) in oxygen evolving photosynthetic organisms. These organisms include cyanobacteria, red algae, plants, green algae, brown algae, diatoms, chrysophytes, and dinoflagellates. We highlight the diversity of pigment-protein complexes that fuel the conversion of radiant energy to chemical bond energy in land plants and the diverse groups of the algae, detail the ways in which environmental parameters (i.e. light quantity and quality, nutrients) modulate the synthesis of these complexes, and discuss the evolutionary relationships among the LHC structural polypeptides.
View details for Web of Science ID A1995TL71900010
View details for PubMedID 8825475
CHARACTERIZATION OF GENES ENCODING THE LIGHT-HARVESTING PROTEINS IN DIATOMS - BIOGENESIS OF THE FUCOXANTHIN CHLOROPHYLL-A/C PROTEIN COMPLEX
Colloquium on Microalgal Biotechnology and Commercial Applications, at the 47th Annual Meeting of the Phycological-Society-of-America
KLUWER ACADEMIC PUBL. 1994: 225–30
View details for Web of Science ID A1994NJ47300015
SPECIFIC AND GENERAL RESPONSES OF CYANOBACTERIA TO MACRONUTRIENT DEPRIVATION
International Symposium on Cellular and Molecular Biology of Phosphate and Phosphorylated Compounds in Microorganisms
AMER SOC MICROBIOLOGY. 1994: 112–118
View details for Web of Science ID A1994BB56S00019
CHARACTERIZATION OF GENE CLUSTERS ENCODING THE FUCOXANTHIN CHLOROPHYLL PROTEINS OF THE DIATOM PHAEODACTYLUM-TRICORNUTUM
NUCLEIC ACIDS RESEARCH
1993; 21 (19): 4458-4466
We are studying the multigene family encoding the fucoxanthin-chlorophyll binding proteins (fcp genes) that constitute the major component of the photosystem II-associated light harvesting complex in diatoms and brown algae. The characteristics of clusters of fcp genes on the genome of the diatom Phaeodactylum tricornutum are described. Sequence analysis of two genomic clones, PT5 and PT4, has demonstrated the presence of four fcp genes (fcpA, fcpB, fcpC, fcpD) on the former and two fcp genes (fcpE, fcpF) on the latter. The proteins encoded by the six characterized fcp genes range in similarity from 86% to 99%. The genes within each cluster are separated by short intergenic sequences (between 0.5 to 1.1 kb). None of these genes contain introns and all appear to be transcribed with short 5' transcribed, untranslated leader sequences; the transcription initiation sites were mapped 26 to 48 bases upstream of the ATG translation start site. Small conserved motifs are found among all of the genes just upstream of both the translation and the transcription start sites. The codon bias is similar in all of the fcp genes, with a predominance of pyrimidines in the third positions of codons of the four codon families. The two fcp genes that are most similar are fcpC and fcpD, and might represent a recent gene duplication. Southern analyses using fcp cDNAs as hybridization probes suggest that there may be additional sequences on the P. tricornutum genome that resemble the characterized fcp sequences.
View details for Web of Science ID A1993MB07000004
View details for PubMedID 8233779
View details for PubMedCentralID PMC311176
TARGETING PROTEINS TO DIATOM PLASTIDS INVOLVES TRANSPORT THROUGH AN ENDOPLASMIC-RETICULUM
MOLECULAR & GENERAL GENETICS
1991; 229 (3): 400-404
Diatoms and related algae, in contrast to higher plants, have a xanthophyll-dominated light harvesting complex and an endoplasmic reticulum (ER) network surrounding the plastid. We have previously demonstrated that polypeptide constituents of the light harvesting complex from the diatom Phaeodactylum tricornutum are nuclear encoded and synthesized as higher molecular weight precursors in the cytoplasm. The amino-termini of the precursor proteins, as deduced from their gene sequences, have features of a signal peptide. Here, we show that the precursor polypeptides can be cotranslationally imported and processed by an in vitro microsomal membrane system, suggesting that cytoplasmically synthesized proteins require a signal peptide to traverse an ER before entering the plastid. These results are discussed in the context of plastid evolution.
View details for Web of Science ID A1991GM74400011
View details for PubMedID 1944228
ISOLATION, TRANSCRIPTION, AND INACTIVATION OF THE GENE FOR AN ATYPICAL ALKALINE-PHOSPHATASE OF SYNECHOCOCCUS-SP STRAIN PCC 7942
JOURNAL OF BACTERIOLOGY
1991; 173 (14): 4297-4309
The alkaline phosphatase of Synechococcus sp. strain PCC 7942 is 145 kDa, which is larger than any alkaline phosphatase previously characterized and approximately three times the size of the analogous enzyme in Escherichia coli. The gene for the alkaline phosphatase, phoA, was cloned and sequenced, and the protein that it encodes was found to have little similarity to other phosphatases. Some sequence similarities were observed between the Synechococcus sp. strain PCC 7942 alkaline phosphatase, the alpha subunit of the ATPase from bacteria and chloroplasts, and the UshA sugar hydrolase of E. coli. Also, limited sequence similarity was observed between a region of the phosphatase and a motif implicated in nucleotide binding. Interestingly, although the alkaline phosphatase is transported across the inner cytoplasmic membrane and into the periplasmic space, it does not appear to have a cleavable signal sequence at its amino terminus. The half-life of the mRNA encoding the alkaline phosphatase, measured after inhibition of RNA synthesis, is approximately 5 min. Similar kinetics for the loss of alkaline phosphatase mRNA occur upon the addition of phosphate to phosphate-depleted cultures, suggesting that high levels of this nutrient inhibit transcription from phoA almost immediately. The phoA gene also appears to be the first gene of an operon; the largest detectable transcript that hybridizes to a phoA gene-specific probe is 11 kb, over twice the size needed to encode the mature protein. Other phosphate-regulated mRNAs are also transcribed upstream of the phoA gene. Insertional inactivation of phoA results in the loss of extracellular, phosphate-regulated phosphatase activity but does not alter the capacity of the cell for phosphate uptake.
View details for Web of Science ID A1991FX32700007
View details for PubMedID 1712356
View details for PubMedCentralID PMC208089