De novo synthesis and functional analysis of the phosphatase-encoding gene acI-B of uncultured Actinobacteria from Lake Stechlin (NE Germany)

The National Center for Biotechnology Information [http://www.ncbi.nlm.nih.gov/guide/taxonomy/] database enlists more than 15,500 bacterial species. But this also includes a plethora of uncultured bacterial representations. Owing to their metabolism, they directly influence biogeochemical cycles, which underscores the the important status of bacteria on our planet. To study the function of a gene from an uncultured bacterium, we have undertaken a de novo gene synthesis approach. Actinobacteria of the acI-B subcluster are important but yet uncultured members of the bacterioplankton in temperate lakes of the northern hemisphere such as oligotrophic Lake Stechlin (NE Germany). This lake is relatively poor in phosphate (P) and harbors on average ~1.3 x 106 bacterial cells/ml, whereby Actinobacteria of the ac-I lineage can contribute to almost half of the entire bacterial community depending on seasonal variability. Single cell genome analysis of Actinobacterium SCGC AB141-P03, a member of the acI-B tribe in Lake Stechlin has revealed several phosphate-metabolizing genes. The genome of acI-B Actinobacteria indicates potential to degrade polyphosphate compound. To test for this genetic potential, we targeted the exoP-annotated gene potentially encoding polyphosphatase and synthesized it artificially to examine its biochemical role. Heterologous overexpression of the gene in Escherichia coli and protein purification revealed phosphatase activity. Comparative genome analysis suggested that homologs of this gene should be also present in other Actinobacteria of the acI lineages. This strategic retention of specialized genes in their genome provides a metabolic advantage over other members of the aquatic food web in a P-limited ecosystem. [Int Microbiol 2016; 19(1):39-47]Keywords: acI-B in Actinobacteria · phosphatases · single cell genomics · phosphate limitation · Lake Stechlin, NE German

De novo synthesis and functional analysis of the phosphatase-encoding gene acI-B of uncultured Actinobacteria from Lake Stechlin (NE Germany)

The National Center for Biotechnology Information [http://www.ncbi.nlm.nih.gov/guide/taxonomy/] database enlists more than 15,500 bacterial species. But this also includes a plethora of uncultured bacterial representations. Owing to their metabolism, they directly influence biogeochemical cycles, which underscores the the important status of bacteria on our planet. To study the function of a gene from an uncultured bacterium, we have undertaken a de novo gene synthesis approach. Actinobacteria of the acI-B subcluster are important but yet uncultured members of the bacterioplankton in temperate lakes of the northern hemisphere such as oligotrophic Lake Stechlin (NE Germany). This lake is relatively poor in phosphate (P) and harbors on average ~1.3 x 106 bacterial cells/ml, whereby Actinobacteria of the ac-I lineage can contribute to almost half of the entire bacterial community depending on seasonal variability. Single cell genome analysis of Actinobacterium SCGC AB141-P03, a member of the acI-B tribe in Lake Stechlin has revealed several phosphate-metabolizing genes. The genome of acI-B Actinobacteria indicates potential to degrade polyphosphate compound. To test for this genetic potential, we targeted the exoP-annotated gene potentially encoding polyphosphatase and synthesized it artificially to examine its biochemical role. Heterologous overexpression of the gene in Escherichia coli and protein purification revealed phosphatase activity. Comparative genome analysis suggested that homologs of this gene should be also present in other Actinobacteria of the acI lineages. This strategic retention of specialized genes in their genome provides a metabolic advantage over other members of the aquatic food web in a P-limited ecosystem. [Int Microbiol 2016; 19(1):39-47]Keywords: acI-B in Actinobacteria · phosphatases · single cell genomics · phosphate limitation · Lake Stechlin, NE German

Targeted Sorting of Single Virus-Infected Cells of the Coccolithophore Emiliania huxleyi

Discriminating infected from healthy cells is the first step to understanding the mechanisms and ecological implications of viral infection. We have developed a method for detecting, sorting, and performing molecular analysis of individual, infected cells of the important microalga Emiliania huxleyi, based on known physiological responses to viral infection. Of three fluorescent dyes tested, FM 1-43 (for detecting membrane blebbing) gave the most unequivocal and earliest separation of cells. Furthermore, we were able to amplify the genomes of single infected cells using Multiple Displacement Amplification. This novel method to reliably discriminate infected from healthy cells in cultures will allow researchers to answer numerous questions regarding the mechanisms and implications of viral infection of E. huxleyi. The method may be transferable to other virus-host systems

Phosphonate production by marine microbes: exploring new sources and potential function

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Acker, M., Hogle, S. L., Berube, P. M., Hackl, T., Coe, A., Stepanauskas, R., Chisholm, S. W., & Repeta, D. J. Phosphonate production by marine microbes: exploring new sources and potential function. Proceedings of the National Academy of Sciences of the United States of America, 119(11), (2022): e2113386119, https://doi.org/10.1073/pnas.2113386119.Phosphonates are organophosphorus metabolites with a characteristic C-P bond. They are ubiquitous in the marine environment, their degradation broadly supports ecosystem productivity, and they are key components of the marine phosphorus (P) cycle. However, the microbial producers that sustain the large oceanic inventory of phosphonates as well as the physiological and ecological roles of phosphonates are enigmatic. Here, we show that phosphonate synthesis genes are rare but widely distributed among diverse bacteria and archaea, including Prochlorococcus and SAR11, the two major groups of bacteria in the ocean. In addition, we show that Prochlorococcus can allocate over 40% of its total cellular P-quota toward phosphonate production. However, we find no evidence that Prochlorococcus uses phosphonates for surplus P storage, and nearly all producer genomes lack the genes necessary to degrade and assimilate phosphonates. Instead, we postulate that phosphonates are associated with cell-surface glycoproteins, suggesting that phosphonates mediate ecological interactions between the cell and its surrounding environment. Our findings indicate that the oligotrophic surface ocean phosphonate pool is sustained by a relatively small fraction of the bacterioplankton cells allocating a significant portion of their P quotas toward secondary metabolism and away from growth and reproduction.This work was supported in part by grants from the NSF (OCE-1153588 and DBI-0424599 to S.W.C.; OCE-1335810 and OIA-1826734 to R.S.; and OCE-1634080 to D.J.R.), the Gordon and Betty Moore Foundation (no. 6000 to D.J.R.), and the Simons Foundation (Life Sciences Project Award IDs 337262 and 647135 to S.W.C.; 510023 to R.S.; and Simons Collaboration on Ocean Processes and Ecology [SCOPE] Award ID 329108 to S.W.C. and D.J.R.)

Hiding in plain sight: the globally distributed bacterial candidate phylum PAUC34f

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Chen, M. L., Becraft, E. D., Pachiadaki, M., Brown, J. M., Jarett, J. K., Gasol, J. M., Ravin, N. V., Moser, D. P., Nunoura, T., Herndl, G. J., Woyke, T., & Stepanauskas, R. Hiding in plain sight: the globally distributed bacterial candidate phylum PAUC34f. Frontiers in Microbiology, 11, (2020): 376, doi: 10.3389/fmicb.2020.00376.Bacterial candidate phylum PAUC34f was originally discovered in marine sponges and is widely considered to be composed of sponge symbionts. Here, we report 21 single amplified genomes (SAGs) of PAUC34f from a variety of environments, including the dark ocean, lake sediments, and a terrestrial aquifer. The diverse origins of the SAGs and the results of metagenome fragment recruitment suggest that some PAUC34f lineages represent relatively abundant, free-living cells in environments other than sponge microbiomes, including the deep ocean. Both phylogenetic and biogeographic patterns, as well as genome content analyses suggest that PAUC34f associations with hosts evolved independently multiple times, while free-living lineages of PAUC34f are distinct and relatively abundant in a wide range of environments.This work was funded by the United States National Science Foundation grants 1460861 (REU site at Bigelow Laboratory for Ocean Sciences), 1441717, 1335810, and 1232982 to RS, and the Simons Foundation (Life Sciences Project Award ID 510023) to RS. NR was supported by the Ministry of Science and Higher Education of Russia. GH was supported by the Austrian Science Fund (FWF) project ARTEMIS (P28781-B21) and the European Research Council under the European Community’s Seventh Framework Program (FP7/2007-2013)/ERC (Grant Agreement No. 268595). JG was supported by Spanish project RTI2018-101025-B-I00. TW and JJ were funded by the U.S. Department of Energy, Joint Genome Institute, a DOE Office of Science User Facility supported under Contract No. DE-AC02-05CH11231

Single-cell genomics reveal low recombination frequencies in freshwater bacteria of the SAR11 clade

Zaremba-Niedzwiedzka K, Viklund J, Zhao W, et al. Single-cell genomics reveal low recombination frequencies in freshwater bacteria of the SAR11 clade. Genome biology. 2013;14(11): R130.BACKGROUND: The SAR11 group of Alphaproteobacteria is highly abundant in the oceans. It contains a recently diverged freshwater clade, which offers the opportunity to compare adaptations to salt- and freshwaters in a monophyletic bacterial group. However, there are no cultivated members of the freshwater SAR11 group and no genomes have been sequenced yet. RESULTS: We isolated ten single SAR11 cells from three freshwater lakes and sequenced and assembled their genomes. A phylogeny based on 57 proteins indicates that the cells are organized into distinct microclusters. We show that the freshwater genomes have evolved primarily by the accumulation of nucleotide substitutions and that they have among the lowest ratio of recombination to mutation estimated for bacteria. In contrast, members of the marine SAR11 clade have one of the highest ratios. Additional metagenome reads from six lakes confirm low recombination frequencies for the genome overall and reveal lake-specific variations in microcluster abundances. We identify hypervariable regions with gene contents broadly similar to those in the hypervariable regions of the marine isolates, containing genes putatively coding for cell surface molecules. CONCLUSIONS: We conclude that recombination rates differ dramatically in phylogenetic sister groups of the SAR11 clade adapted to freshwater and marine ecosystems. The results suggest that the transition from marine to freshwater systems has purged diversity and resulted in reduced opportunities for recombination with divergent members of the clade. The low recombination frequencies of the LD12 clade resemble the low genetic divergence of host-restricted pathogens that have recently shifted to a new host

Single cell genomics-based analysis of gene content and expression of prophages in a diffuse-flow deep-sea hydrothermal system

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Labonte, J. M., Pachiadaki, M., Fergusson, E., McNichol, J., Grosche, A., Gulmann, L. K., Vetriani, C., Sievert, S. M., & Stepanauskas, R. Single cell genomics-based analysis of gene content and expression of prophages in a diffuse-flow deep-sea hydrothermal system. Frontiers in Microbiology, 10, (2019): 1262, doi:10.3389/fmicb.2019.01262.Phage–host interactions likely play a major role in the composition and functioning of many microbiomes, yet remain poorly understood. Here, we employed single cell genomics to investigate phage–host interactions in a diffuse-flow, low-temperature hydrothermal vent that may be reflective of a broadly distributed biosphere in the subseafloor. We identified putative prophages in 13 of 126 sequenced single amplified genomes (SAGs), with no evidence for lytic infections, which is in stark contrast to findings in the surface ocean. Most were distantly related to known prophages, while their hosts included bacterial phyla Campylobacterota, Bacteroidetes, Chlorobi, Proteobacteria, Lentisphaerae, Spirochaetes, and Thermotogae. Our results suggest the predominance of lysogeny over lytic interaction in diffuse-flow, deep-sea hydrothermal vents, despite the high activity of the dominant Campylobacteria that would favor lytic infections. We show that some of the identified lysogens have co-evolved with their host over geological time scales and that their genes are transcribed in the environment. Functional annotations of lysogeny-related genes suggest involvement in horizontal gene transfer enabling host’s protection against toxic metals and antibacterial compounds.This work was supported by the U.S. National Science Foundation’s Dimensions of Biodiversity Program [OCE-1136488 (to RS), OCE-1136727 (to SMS) and OCE-1136451 (to CV)], as well as DEB-1441717 and OCE-1335810 (to RS), and the DOE JGI CSP project 1477