Lev Tsypin

All Publications

• Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation. bioRxiv : the preprint server for biology Tsypin, L. M., Saunders, S. H., Chen, A. W., Newman, D. K. 2023

  Abstract

  The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa.

  View details for DOI 10.1101/2023.11.14.567096

  View details for PubMedID 38014283

  View details for PubMedCentralID PMC10680695

• An apical membrane complex for triggering rhoptry exocytosis and invasion in Toxoplasma EMBO JOURNAL Sparvoli, D., Delabre, J., Penarete-Vargas, D., Mageswaran, S., Tsypin, L. M., Heckendorn, J., Theveny, L., Maynadier, M., Cova, M., Berry-Sterkers, L., Guerin, A., Dubremetz, J., Urbach, S., Striepen, B., Turkewitz, A. P., Chang, Y., Lebrun, M. 2022; 41 (22): e111158

  Abstract

  Apicomplexan parasites possess secretory organelles called rhoptries that undergo regulated exocytosis upon contact with the host. This process is essential for the parasitic lifestyle of these pathogens and relies on an exocytic machinery sharing structural features and molecular components with free-living ciliates. However, how the parasites coordinate exocytosis with host interaction is unknown. Here, we performed a Tetrahymena-based transcriptomic screen to uncover novel exocytic factors in Ciliata and conserved in Apicomplexa. We identified membrane-bound proteins, named CRMPs, forming part of a large complex essential for rhoptry secretion and invasion in Toxoplasma. Using cutting-edge imaging tools, including expansion microscopy and cryo-electron tomography, we show that, unlike previously described rhoptry exocytic factors, TgCRMPs are not required for the assembly of the rhoptry secretion machinery and only transiently associate with the exocytic site-prior to the invasion. CRMPs and their partners contain putative host cell-binding domains, and CRMPa shares similarities with GPCR proteins. Collectively our data imply that the CRMP complex acts as a host-molecular sensor to ensure that rhoptry exocytosis occurs when the parasite contacts the host cell.

  View details for DOI 10.15252/embj.2022111158

  View details for Web of Science ID 000868492700001

  View details for PubMedID 36245278

  View details for PubMedCentralID PMC9670195

• Nitrate Reduction Stimulates and Is Stimulated by Phenazine-1-Carboxylic Acid Oxidation by Citrobacter portucalensis MBL MBIO Tsypin, L. M., Newman, D. K. 2021; 12 (4): e0226521

  Abstract

  Phenazines are secreted metabolites that microbes use in diverse ways, from quorum sensing to antimicrobial warfare to energy conservation. Phenazines are able to contribute to these activities due to their redox activity. The physiological consequences of cellular phenazine reduction have been extensively studied, but the counterpart phenazine oxidation has been largely overlooked. Phenazine-1-carboxylic acid (PCA) is common in the environment and readily reduced by its producers. Here, we describe its anaerobic oxidation by Citrobacter portucalensis strain MBL, which was isolated from topsoil in Falmouth, MA, and which does not produce phenazines itself. This activity depends on the availability of a suitable terminal electron acceptor, specifically nitrate. When C. portucalensis MBL is provided reduced PCA and nitrate, it oxidizes the PCA at a rate that is environmentally relevant. We compared this terminal electron acceptor-dependent PCA-oxidizing activity of C. portucalensis MBL to that of several other gammaproteobacteria with various capacities to respire nitrate. We found that PCA oxidation by these strains in a nitrate-dependent manner is decoupled from growth and strain dependent. We infer that bacterial PCA oxidation is widespread and genetically determined. Notably, oxidizing PCA enhances the rate of nitrate reduction to nitrite by C. portucalensis MBL beyond the stoichiometric exchange of electrons from PCA to nitrate, which we attribute to C. portucalensis MBL's ability to also reduce oxidized PCA, thereby catalyzing a complete PCA redox cycle. This bidirectionality highlights the versatility of PCA as a biological redox agent. IMPORTANCE Phenazines are increasingly appreciated for their roles in structuring microbial communities. These tricyclic aromatic molecules have been found to regulate gene expression, be toxic, promote antibiotic tolerance, and promote survival under oxygen starvation. In all of these contexts, however, phenazines are studied as electron acceptors. Even if their utility arises primarily from being readily reduced, they need to be oxidized in order to be recycled. While oxygen and ferric iron can oxidize phenazines abiotically, biotic oxidation of phenazines has not been studied previously. We observed bacteria that readily oxidize phenazine-1-carboxylic acid (PCA) in a nitrate-dependent fashion, concomitantly increasing the rate of nitrate reduction to nitrite. Because nitrate is a prevalent terminal electron acceptor in diverse anoxic environments, including soils, and phenazine producers are widespread, this observation of linked phenazine and nitrogen redox cycling suggests an underappreciated role for redox-active secreted metabolites in the environment.

  View details for DOI 10.1128/mBio.02265-21

  View details for Web of Science ID 000757946900041

  View details for PubMedID 34465028

  View details for PubMedCentralID PMC8437036

• Draft Genome Sequence of the Redox-Active Enteric Bacterium Citrobacter portucalensis Strain MBL MICROBIOLOGY RESOURCE ANNOUNCEMENTS Tsypin, L. M., Saunders, S. H., Bar-On, Y., Leadbetter, J. R., Newman, D. K. 2020; 9 (32)

  Abstract

  We grew a soil enrichment culture to identify organisms that anaerobically oxidize phenazine-1-carboxylic acid. A strain of Citrobacter portucalensis was isolated from this enrichment and sequenced by both Illumina and PacBio technologies. It has a genome with a length of 5.3 Mb, a G+C content of 51.8%, and at least one plasmid.

  View details for DOI 10.1128/MRA.00695-20

  View details for Web of Science ID 000560673400014

  View details for PubMedID 32763937

  View details for PubMedCentralID PMC7409854

• Genetic tool development in marine protists: emerging model organisms for experimental cell biology NATURE METHODS Faktorova, D., Nisbet, R. R., Robledo, J., Casacuberta, E., Sudek, L., Allen, A. E., Ares, M., Areste, C., Balestreri, C., Barbrook, A. C., Beardslee, P., Bender, S., Booth, D. S., Bouget, F., Bowler, C., Breglia, S. A., Brownlee, C., Burger, G., Cerutti, H., Cesaroni, R., Chiurillo, M. A., Clemente, T., Coles, D. B., Collier, J. L., Cooney, E. C., Coyne, K., Docampo, R., Dupont, C. L., Edgcomb, V., Einarsson, E., Elustondo, P. A., Federici, F., Freire-Beneitez, V., Freyria, N. J., Fukuda, K., Garcia, P. A., Girguis, P. R., Gomaa, F., Gornik, S. G., Guo, J., Hampl, V., Hanawa, Y., Haro-Contreras, E. R., Hehenberger, E., Highfield, A., Hirakawa, Y., Hopes, A., Howe, C. J., Hu, I., Ibanez, J., Irwin, N. T., Ishii, Y., Janowicz, N., Jones, A. C., Kachale, A., Fujimura-Kamada, K., Kaur, B., Kaye, J. Z., Kazana, E., Keeling, P. J., King, N., Klobutcher, L. A., Lander, N., Lassadi, I., Li, Z., Lin, S., Lozano, J., Luan, F., Maruyama, S., Matute, T., Miceli, C., Minagawa, J., Moosburner, M., Najle, S. R., Nanjappa, D., Nimmo, I. C., Noble, L., Vanclova, A., Nowacki, M., Nunez, I., Pain, A., Piersanti, A., Pucciarelli, S., Pyrih, J., Rest, J. S., Rius, M., Robertson, D., Ruaud, A., Ruiz-Trillo, I., Sigg, M. A., Silver, P. A., Slamovits, C. H., Smith, G., Sprecher, B. N., Stern, R., Swart, E. C., Tsaousis, A. D., Tsypin, L., Turkewitz, A., Turnsek, J., Valach, M., Verge, V., von Dassow, P., von der Haar, T., Waller, R. F., Wang, L., Wen, X., Wheeler, G., Woods, A., Zhang, H., Mock, T., Worden, A. Z., Lukes, J. 2020; 17 (5): 481-+

  Abstract

  Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

  View details for DOI 10.1038/s41592-020-0796-x

  View details for Web of Science ID 000523954200003

  View details for PubMedID 32251396

  View details for PubMedCentralID PMC7200600

• The Co-regulation Data Harvester: automating gene annotation starting from a transcriptome database. SoftwareX Tsypin, L. M., Turkewitz, A. P. 2017; 6: 165-171

  Abstract

  Identifying co-regulated genes provides a useful approach for defining pathway-specific machinery in an organism. To be efficient, this approach relies on thorough genome annotation, a process much slower than genome sequencing per se. Tetrahymena thermophila, a unicellular eukaryote, has been a useful model organism and has a fully sequenced but sparsely annotated genome. One important resource for studying this organism has been an online transcriptomic database. We have developed an automated approach to gene annotation in the context of transcriptome data in T. thermophila, called the Co-regulation Data Harvester (CDH). Beginning with a gene of interest, the CDH identifies co-regulated genes by accessing the Tetrahymena transcriptome database. It then identifies their closely related genes (orthologs) in other organisms by using reciprocal BLAST searches. Finally, it collates the annotations of those orthologs' functions, which provides the user with information to help predict the cellular role of the initial query. The CDH, which is freely available, represents a powerful new tool for analyzing cell biological pathways in Tetrahymena. Moreover, to the extent that genes and pathways are conserved between organisms, the inferences obtained via the CDH should be relevant, and can be explored, in many other systems.

  View details for DOI 10.1016/j.softx.2017.06.006

  View details for PubMedID 29104906

  View details for PubMedCentralID PMC5663188