Members of the acI lineage of Actinobacteria are the most abundant microorganisms in most freshwater lakes; however, our understanding of the keys to their success and their role in carbon and nutrient cycling in freshwater systems has been hampered by the lack of pure cultures and genomes. We obtained draft genome assemblies from 11 single cells representing three acI tribes (acI-A1, acI-A7, acI-B1) from four temperate lakes in the United States and Europe. Comparative analysis of acI SAGs and other available freshwater bacterial genomes showed that acI has more gene content directed toward carbohydrate acquisition as compared to Polynucleobacter and LD12 Alphaproteobacteria, which seem to specialize more on carboxylic acids. The acI genomes contain actinorhodopsin as well as some genes involved in anaplerotic carbon fixation indicating the capacity to supplement their known heterotrophic lifestyle. Genome-level differences between the acI-A and acI-B clades suggest specialization at the clade level for carbon substrate acquisition. Overall, the acI genomes appear to be highly streamlined versions of Actinobacteria that include some genes allowing it to take advantage of sunlight and N-rich organic compounds such as polyamines, di-and oligopeptides, branched-chain amino acids and cyanophycin. This work significantly expands the known metabolic potential of the cosmopolitan freshwater acI lineage and its ecological and genetic traits.
Sulfide:quinone oxidoreductase (SQR) catalyzes sulfide oxidation during sulfide-dependent chemo-and phototrophic growth in bacteria. The green sulfur bacterium Chlorobaculum tepidum (formerly Chlorobium tepidum) can grow on sulfide as the sole electron donor and sulfur source. C. tepidum contains genes encoding three SQR homologs: CT0117, CT0876, and CT1087. This study examined which, if any, of the SQR homologs possess sulfide-dependent ubiquinone reduction activity and are required for growth on sulfide. In contrast to CT0117 and CT0876, transcripts of CT1087 were detected only when cells actively oxidized sulfide. Mutation of CT0117 or CT1087 in C. tepidum decreased SQR activity in membrane fractions, and the CT1087 mutant could not grow with >6 mM sulfide. Mutation of both CT0117 and CT1087 in C. tepidum completely abolished SQR activity, and the double mutant failed to grow with >4 mM sulfide. A C-terminal His 6 -tagged CT1087 protein was membrane localized, as was SQR activity. Epitope-tagged CT1087 was detected only when sulfide was actively consumed by cells. Recombinantly produced CT1087 and CT0117 proteins had SQR activity, while CT0876 did not. In summary, we conclude that, under the conditions tested, both CT0117 and CT1087 function as SQR proteins in C. tepidum. CT0876 may support the growth of C. tepidum at low sulfide concentrations, but no evidence was found for SQR activity associated with this protein.
Freshwater Verrucomicrobia spp. are cosmopolitan in lakes and rivers, and yet their roles and ecophysiology are not well understood, as cultured freshwater Verrucomicrobia spp. are restricted to one subdivision of this phylum. Here, we greatly expanded the known genomic diversity of this freshwater lineage by recovering 19 Verrucomicrobia draft genomes from 184 metagenomes collected from a eutrophic lake and a humic bog across multiple years. Most of these genomes represent the first freshwater representatives of several Verrucomicrobia subdivisions. Genomic analysis revealed Verrucomicrobia to be potential (poly)saccharide degraders and suggested their adaptation to carbon sources of different origins in the two contrasting ecosystems. We identified putative extracellular electron transfer genes and so-called “Planctomycete-specific” cytochrome c-encoding genes and identified their distinct distribution patterns between the lakes/layers. Overall, our analysis greatly advances the understanding of the function, ecophysiology, and distribution of freshwater Verrucomicrobia, while highlighting their potential role in freshwater carbon cycling.
The specific enzymes employed by Chlorobium tepidum for the anaerobic oxidation of thiosulfate, sulfide and elemental sulfur during anoxygenic photosynthesis are not well defined. In particular, it is unclear how C. tepidum completely oxidizes thiosulfate. A C. tepidum genomic region, encoding a putative quinone-interacting membrane-bound oxidoreductase (Qmo) complex (CT0866-0868), hypothetical proteins (CT0869-0875) and a sulfide : quinone oxidoreductase (SQR) homologue (CT0876), was analysed for its role in anaerobic sulfur oxidation. Transcripts of genes encoding the Qmo complex, which is similar to archaeal heterodisulfide reductases, were detected by RT-PCR only while sulfide or elemental sulfur were being oxidized, whereas the SQR homologue and CT0872 were expressed during thiosulfate oxidation and into early stationary phase. A mutant of C. tepidum was obtained in which the region between CT0868 and CT0876 was replaced by a transposon insertion resulting in the truncation or deletion of nine genes. This strain, C5, was completely defective for growth on thiosulfate as the sole electron donor in C. tepidum, but only slightly defective for growth on sulfide or thiosulfate plus sulfide. Strain C5 did not oxidize thiosulfate and also displayed a defect in acetate assimilation under all growth conditions. A gene of unknown function, CT0872, deleted in strain C5 that is conserved in chemolithotrophic sulfur-oxidizing bacteria and archaea is the most likely candidate for the thiosulfate oxidation phenotype observed in this strain. The defect in acetate assimilation may be explained by deletion of CT0874, which encodes a homologue of 3-oxoacyl acyl carrier protein synthase.
Microbes are critical in carbon and nutrient cycling in freshwater ecosystems. Members of the Verrucomicrobia are ubiquitous in such systems, yet their roles and ecophysiology are not well understood. In this study, we recovered 19 Verrucomicrobia draft genomes by sequencing 184 time-series metagenomes from a eutrophic lake and a humic bog that differ in carbon source and nutrient availabilities. These genomes span four of the seven previously defined Verrucomicrobia subdivisions, and greatly expand the known genomic diversity of freshwater Verrucomicrobia. Genome analysis revealed their potential role as (poly)saccharide-degraders in freshwater, uncovered interesting genomic features for this life style, and suggested their adaptation to nutrient availabilities in their environments. Between the two lakes, Verrucomicrobia populations differ significantly in glycoside hydrolase gene abundance and functional profiles, reflecting the autochthonous and terrestrially-derived allochthonous carbon sources of the two ecosystems respectively. Interestingly, a number of genomes recovered from the bog contained gene clusters that potentially encode a novel porin-multiheme cytochrome c complex and might be involved in extracellular electron transfer in the anoxic humic-rich environment. Notably, most epilimnion genomes have large numbers of so-called “Planctomycete-specific” cytochrome c-containing genes, which exhibited nearly opposite distribution patterns with glycoside hydrolase genes, probably associated with the different environmental oxygen availability and carbohydrate complexity between lakes/layers. Overall, the recovered genomes are a major step towards understanding the role, ecophysiology and distribution of Verrucomicrobia in freshwater.IMPORTANCEFreshwater Verrucomicrobia are cosmopolitan in lakes and rivers, yet their roles and ecophysiology are not well understood, as cultured freshwater Verrucomicrobia are restricted to one subdivision of this phylum. Here, we greatly expand the known genomic diversity of this freshwater lineage by recovering 19 Verrucomicrobia draft genomes from 184 metagenomes collected from a eutrophic lake and a humic bog across multiple years. Most of these genomes represent first freshwater representatives of several Verrucomicrobia subdivisions. Genomic analysis revealed Verrucomicrobia as potential (poly)saccharide-degraders, and suggested their adaptation to carbon source of different origins in the two contrasting ecosystems. We identified putative extracellular electron transfer genes and so-called “Planctomycete-specific” cytochrome c-containing genes, and found their distinct distribution patterns between the lakes/layers. Overall, our analysis greatly advances the understanding of the function, ecophysiology and distribution of freshwater Verrucomicrobia, while highlighting their potential role in freshwater carbon cycling.
Marine bacteria drive the biogeochemical processing of oceanic dissolved organic carbon (DOC), a 750-Tg C reservoir that is a critical component of the global C cycle. Catabolism of DOC is thought to be regulated by the biomass composition of heterotrophic bacteria, as cells maintain a C:N:P ratio of ∼50:10:1 during DOC processing. Yet a complicating factor in stoichiometry-based analyses is that bacteria can change the C:N:P ratio of their biomass in response to resource composition. We investigated the physiological mechanisms of resource-driven shifts in biomass stoichiometry in continuous cultures of the marine heterotrophic bacterium Ruegeria pomeroyi (a member of the Roseobacter clade) under four element limitation regimes (C, N, P, and S). Microarray analysis indicated that the bacterium scavenged for alternate sources of the scarce element when cells were C-, N-, or P-limited; reworked the ratios of biomolecules when C- and P- limited; and exerted tighter control over import/export and cytoplasmic pools when N-limited. Under S limitation, a scenario not existing naturally for surface ocean microbes, stress responses dominated transcriptional changes. Resource-driven changes in C:N ratios of up to 2.5-fold and in C:P ratios of up to sixfold were measured in R. pomeroyi biomass. These changes were best explained if the C and P content of the cells was flexible in the face of shifting resources but N content was not, achieved through the net balance of different transcriptional strategies. The cellular-level metabolic trade-offs that govern biomass stoichiometry in R. pomeroyi may have implications for global carbon cycling if extendable to other heterotrophic bacteria. Strong homeostatic responses to N limitation by marine bacteria would intensify competition with autotrophs. Modification of cellular inventories in C- and P-limited heterotrophs would vary the elemental ratio of particulate organic matter sequestered in the deep ocean.
Polyamines (PAs) are a group of nitrogen-rich dissolved organic nitrogen (DON) compounds that are ubiquitously distributed in marine environments. To identify bacteria that are involved in PA transformations, coastal bacterioplankton microcosms were amended with a single PA model compound, i.e. putrescine (PUT) or spermidine (SPD), or with no addition as controls (CTRs). Bromodeoxyuridine (BrdU) was added to all the microcosms to label newly synthesized DNAs. Fluorescence-activated cell sorting (FACS) analysis indicated significant increases in numbers of total cells and cells with both high and low levels of BrdU incorporation in the PUT and SPD microcosms, but not in the CTRs. 16S rDNA pyrotag sequencing of FACS-sorted cells indicated that PUT- and SPD-transforming bacteria were composed similarly of a diverse group of taxa affiliated with Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria (especially Roseobacter of its alpha lineage). Broad taxonomic distribution of PA-transforming bacteria was also indicated by the abundance and distribution of PA transporter gene homologues in a survey of sequenced marine bacterial genomes. Our results suggest that PAs may be common DON substrates for marine bacterioplankton, in line with the hypothesis that bacterially mediated PA transformation accounts for an important proportion of marine DON flux.
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