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.