Insights into the phylogeny and coding potential of microbial dark matter.

Genome sequencing enhances our understanding of the biological world by providing blueprints for the evolutionary and functional diversity that shapes the biosphere. However, microbial genomes that are currently available are of limited phylogenetic breadth, owing to our historical inability to cultivate most microorganisms in the laboratory. We apply single-cell genomics to target and sequence 201 uncultivated archaeal and bacterial cells from nine diverse habitats belonging to 29 major mostly uncharted branches of the tree of life, so-called 'microbial dark matter'. With this additional genomic information, we are able to resolve many intra- and inter-phylum-level relationships and to propose two new superphyla. We uncover unexpected metabolic features that extend our understanding of biology and challenge established boundaries between the three domains of life. These include a novel amino acid use for the opal stop codon, an archaeal-type purine synthesis in Bacteria and complete sigma factors in Archaea similar to those in Bacteria. The single-cell genomes also served to phylogenetically anchor up to 20% of metagenomic reads in some habitats, facilitating organism-level interpretation of ecosystem function. This study greatly expands the genomic representation of the tree of life and provides a systematic step towards a better understanding of biological evolution on our planet.

UGA is an additional glycine codon in uncultured SR1 bacteria from the human microbiota.

The composition of the human microbiota is recognized as an important factor in human health and disease. Many of our cohabitating microbes belong to phylum-level divisions for which there are no cultivated representatives and are only represented by small subunit rRNA sequences. For one such taxon (SR1), which includes bacteria with elevated abundance in periodontitis, we provide a single-cell genome sequence from a healthy oral sample. SR1 bacteria use a unique genetic code. In-frame TGA (opal) codons are found in most genes (85%), often at loci normally encoding conserved glycine residues. UGA appears not to function as a stop codon and is in equilibrium with the canonical GGN glycine codons, displaying strain-specific variation across the human population. SR1 encodes a divergent tRNA(Gly)UCA with an opal-decoding anticodon. SR1 glycyl-tRNA synthetase acylates tRNA(Gly)UCA with glycine in vitro with similar activity compared with normal tRNA(Gly)UCC. Coexpression of SR1 glycyl-tRNA synthetase and tRNA(Gly)UCA in Escherichia coli yields significant ß-galactosidase activity in vivo from a lacZ gene containing an in-frame TGA codon. Comparative genomic analysis with Human Microbiome Project data revealed that the human body harbors a striking diversity of SR1 bacteria. This is a surprising finding because SR1 is most closely related to bacteria that live in anoxic and thermal environments. Some of these bacteria share common genetic and metabolic features with SR1, including UGA to glycine reassignment and an archaeal-type ribulose-1,5-bisphosphate carboxylase (RubisCO) involved in AMP recycling. UGA codon reassignment renders SR1 genes untranslatable by other bacteria, which impacts horizontal gene transfer within the human microbiota.

Prevalent genome streamlining and latitudinal divergence of planktonic bacteria in the surface ocean.

Planktonic bacteria dominate surface ocean biomass and influence global biogeochemical processes, but remain poorly characterized owing to difficulties in cultivation. Using large-scale single cell genomics, we obtained insight into the genome content and biogeography of many bacterial lineages inhabiting the surface ocean. We found that, compared with existing cultures, natural bacterioplankton have smaller genomes, fewer gene duplications, and are depleted in guanine and cytosine, noncoding nucleotides, and genes encoding transcription, signal transduction, and noncytoplasmic proteins. These findings provide strong evidence that genome streamlining and oligotrophy are prevalent features among diverse, free-living bacterioplankton, whereas existing laboratory cultures consist primarily of copiotrophs. The apparent ubiquity of metabolic specialization and mixotrophy, as predicted from single cell genomes, also may contribute to the difficulty in bacterioplankton cultivation. Using metagenome fragment recruitment against single cell genomes, we show that the global distribution of surface ocean bacterioplankton correlates with temperature and latitude and is not limited by dispersal at the time scales required for nucleotide substitution to exceed the current operational definition of bacterial species. Single cell genomes with highly similar small subunit rRNA gene sequences exhibited significant genomic and biogeographic variability, highlighting challenges in the interpretation of individual gene surveys and metagenome assemblies in environmental microbiology. Our study demonstrates the utility of single cell genomics for gaining an improved understanding of the composition and dynamics of natural microbial assemblages.

Diversity and genomic insights into the uncultured Chloroflexi from the human microbiota.

Many microbial phyla that are widely distributed in open environments have few or no representatives within animal-associated microbiota. Among them, the Chloroflexi comprises taxonomically and physiologically diverse lineages adapted to a wide range of aquatic and terrestrial habitats. A distinct group of uncultured chloroflexi related to free-living anaerobic Anaerolineae inhabits the mammalian gastrointestinal tract and includes low-abundance human oral bacteria that appear to proliferate in periodontitis. Using a single-cell genomics approach, we obtained the first draft genomic reconstruction for these organisms and compared their inferred metabolic potential with free-living chloroflexi. Genomic data suggest that oral chloroflexi are anaerobic heterotrophs, encoding abundant carbohydrate transport and metabolism functionalities, similar to those seen in environmental Anaerolineae isolates. The presence of genes for a unique phosphotransferase system and N-acetylglucosamine metabolism suggests an important ecological niche for oral chloroflexi in scavenging material from lysed bacterial cells and the human tissue. The inferred ability to produce sialic acid for cell membrane decoration may enable them to evade the host defence system and colonize the subgingival space. As with other low abundance but persistent members of the microbiota, discerning community and host factors that influence the proliferation of oral chloroflexi may help understand the emergence of oral pathogens and the microbiota dynamics in health and disease states.

Myocardial transcriptome analysis of human arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy primarily of the right ventricle characterized through fibrofatty replacement of cardiomyocytes. The genetic etiology in ARVC patients is most commonly caused by dominant inheritance and high genetic heterogeneity. Though histological examinations of ARVC-affected human myocardium reveals fibrolipomatous replacement, the molecular mechanisms leading to loss of cardiomyocytes are largely unknown. We therefore analyzed the transcriptomes of six ARVC hearts and compared our findings to six nonfailing donor hearts (NF). To characterize the ARVC-specific transcriptome, we compared our findings to samples from seven patients with idiopathic dilated cardiomyopathy (DCM). The myocardial DCM and ARVC samples were prepared from hearts explanted during an orthotopic heart transplantation representing myocardium from end-stage heart failure patients (NYHA IV). From each heart, left (LV) and right ventricular (RV) myocardial samples were analyzed by Affymetrix HG-U133 Plus 2.0 arrays, adding up to six sample groups. Unsupervised cluster analyses of the groups revealed a clear separation of NF and cardiomyopathy samples. However, in contrast to the other samples, the analyses revealed no distinct expression pattern in LV and RV of myocardial ARVC samples. We further identified differentially expressed transcripts using t-tests and found transcripts separating diseased and NF ventricular myocardium. Of note, in failing myocardium only ~15-16% of the genes are commonly regulated compared with NF samples. In addition both cardiomyopathies are clearly distinct on the transcriptome level. Comparison of the expression patterns between the failing RV and LV using a paired t-test revealed a lack of major differences between LV and RV gene expression in ARVC hearts. Our study is the first analysis of specific ARVC-related RV and LV gene expression patterns in terminal failing human hearts.

The complete genome sequence of the acarbose producer Actinoplanes sp. SE50/110.

BACKGROUND: Actinoplanes sp. SE50/110 is known as the wild type producer of the alpha-glucosidase inhibitor acarbose, a potent drug used worldwide in the treatment of type-2 diabetes mellitus. As the incidence of diabetes is rapidly rising worldwide, an ever increasing demand for diabetes drugs, such as acarbose, needs to be anticipated. Consequently, derived Actinoplanes strains with increased acarbose yields are being used in large scale industrial batch fermentation since 1990 and were continuously optimized by conventional mutagenesis and screening experiments. This strategy reached its limits and is generally superseded by modern genetic engineering approaches. As a prerequisite for targeted genetic modifications, the complete genome sequence of the organism has to be known. RESULTS: Here, we present the complete genome sequence of Actinoplanes sp. SE50/110 [GenBank:CP003170], the first publicly available genome of the genus Actinoplanes, comprising various producers of pharmaceutically and economically important secondary metabolites. The genome features a high mean G + C content of 71.32% and consists of one circular chromosome with a size of 9,239,851 bp hosting 8,270 predicted protein coding sequences. Phylogenetic analysis of the core genome revealed a rather distant relation to other sequenced species of the family Micromonosporaceae whereas Actinoplanes utahensis was found to be the closest species based on 16S rRNA gene sequence comparison. Besides the already published acarbose biosynthetic gene cluster sequence, several new non-ribosomal peptide synthetase-, polyketide synthase- and hybrid-clusters were identified on the Actinoplanes genome. Another key feature of the genome represents the discovery of a functional actinomycete integrative and conjugative element. CONCLUSIONS: The complete genome sequence of Actinoplanes sp. SE50/110 marks an important step towards the rational genetic optimization of the acarbose production. In this regard, the identified actinomycete integrative and conjugative element could play a central role by providing the basis for the development of a genetic transformation system for Actinoplanes sp. SE50/110 and other Actinoplanes spp. Furthermore, the identified non-ribosomal peptide synthetase- and polyketide synthase-clusters potentially encode new antibiotics and/or other bioactive compounds, which might be of pharmacologic interest.

A single-cell genomics pipeline for environmental microbial eukaryotes.

Single-cell sequencing of environmental microorganisms is an essential component of the microbial ecology toolkit. However, large-scale targeted single-cell sequencing for the whole-genome recovery of uncultivated eukaryotes is lagging. The key challenges are low abundance in environmental communities, large complex genomes, and cell walls that are difficult to break. We describe a pipeline composed of state-of-the art single-cell genomics tools and protocols optimized for poorly studied and uncultivated eukaryotic microorganisms that are found at low abundance. This pipeline consists of seven distinct steps, beginning with sample collection and ending with genome annotation, each equipped with quality review steps to ensure high genome quality at low cost. We tested and evaluated each step on environmental samples and cultures of early-diverging lineages of fungi and Chromista/SAR. We show that genomes produced using this pipeline are almost as good as complete reference genomes for functional and comparative genomics for environmental microbial eukaryotes.

Global gene expression analysis in nonfailing and failing myocardium pre- and postpulsatile and nonpulsatile ventricular assist device support.

Mechanical unloading by ventricular assist devices (VAD) leads to significant gene expression changes often summarized as reverse remodeling. However, little is known on individual transcriptome changes during VAD support and its relationship to nonfailing hearts (NF). In addition no data are available for the transcriptome regulation during nonpulsatile VAD support. Therefore we analyzed the gene expression patterns of 30 paired samples from VAD-supported (including 8 nonpulsatile VADs) and 8 nonfailing control hearts (NF) using the first total human genome array available. Transmural myocardial samples were collected for RNA isolation. RNA was isolated by commercial methods and processed according to chip-manufacturer recommendations. cRNA were hybridized on Affymetrix HG-U133 Plus 2.0 arrays, providing coverage of the whole human genome Array. Data were analyzed using Microarray Analysis Suite 5.0 (Affymetrix) and clustered by Expressionist software (Genedata). We found 352 transcripts were differentially regulated between samples from VAD implantation and NF, whereas 510 were significantly regulated between VAD transplantation and NF (paired t-test P < 0.001, fold change >or=1.6). Remarkably, only a minor fraction of 111 transcripts was regulated in heart failure (HF) and during VAD support. Unsupervised hierarchical clustering of paired VAD and NF samples revealed separation of HF and NF samples; however, individual differentiation of VAD implantation and VAD transplantation was not accomplished. Clustering of pulsatile and nonpulsatile VAD did not lead to robust separation of gene expression patterns. During VAD support myocardial gene expression changes do not indicate reversal of the HF phenotype but reveal a distinct HF-related pattern. Transcriptome analysis of pulsatile and nonpulsatile VAD-supported hearts did not provide evidence for a pump mode-specific transcriptome pattern.

Strain-specific quantification of root colonization by plant growth promoting rhizobacteria Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 in non-sterile soil and field conditions.

Bacillus amyloliquefaciens QST713 and B. firmus I-1582 are bacterial strains which are used as active ingredients of commercially-available soil application and seed treatment products Serenade® and VOTiVO®, respectively. These bacteria colonize plant roots promoting plant growth and offering protection against pathogens/pests. The objective of this study was to develop a qPCR protocol to quantitate the dynamics of root colonization by these two strains under field conditions. Primers and TaqMan® probes were designed based on genome comparisons of the two strains with publicly-available and unpublished bacterial genomes of the same species. An optimized qPCR protocol was developed to quantify bacterial colonization of corn roots after seed treatment. Treated corn seeds were planted in non-sterile soil in the greenhouse and grown for 28 days. Specific detection of bacteria was quantified weekly, and showed stable colonization between ~104-105 CFU/g during the experimental period for both bacteria, and the protocol detected as low as 103 CFU/g bacteria on roots. In a separate experiment, streptomycin-resistant QST713 and rifampicin-resistant I-1582 strains were used to compare dilution-plating on TSA with the newly developed qPCR method. Results also indicated that the presence of natural microflora and another inoculated strain does not affect root colonization of either one of these strains. The same qPCR protocol was used to quantitate root colonization by QST713 and I-1582 in two corn and two soybean varieties grown in the field. Both bacteria were quantitated up to two weeks after seeds were planted in the field and there were no significant differences in root colonization in either bacteria strain among varieties. Results presented here confirm that the developed qPCR protocol can be successfully used to understand dynamics of root colonization by these bacteria in plants growing in growth chamber, greenhouse and the field.

Diverse Marinimicrobia bacteria may mediate coupled biogeochemical cycles along eco-thermodynamic gradients.

Microbial communities drive biogeochemical cycles through networks of metabolite exchange that are structured along energetic gradients. As energy yields become limiting, these networks favor co-metabolic interactions to maximize energy disequilibria. Here we apply single-cell genomics, metagenomics, and metatranscriptomics to study bacterial populations of the abundant "microbial dark matter" phylum Marinimicrobia along defined energy gradients. We show that evolutionary diversification of major Marinimicrobia clades appears to be closely related to energy yields, with increased co-metabolic interactions in more deeply branching clades. Several of these clades appear to participate in the biogeochemical cycling of sulfur and nitrogen, filling previously unassigned niches in the ocean. Notably, two Marinimicrobia clades, occupying different energetic niches, express nitrous oxide reductase, potentially acting as a global sink for the greenhouse gas nitrous oxide.

Tuning fresh: radiation through rewiring of central metabolism in streamlined bacteria.

Most free-living planktonic cells are streamlined and in spite of their limitations in functional flexibility, their vast populations have radiated into a wide range of aquatic habitats. Here we compared the metabolic potential of subgroups in the Alphaproteobacteria lineage SAR11 adapted to marine and freshwater habitats. Our results suggest that the successful leap from marine to freshwaters in SAR11 was accompanied by a loss of several carbon degradation pathways and a rewiring of the central metabolism. Examples for these are C1 and methylated compounds degradation pathways, the Entner-Doudouroff pathway, the glyoxylate shunt and anapleuretic carbon fixation being absent from the freshwater genomes. Evolutionary reconstructions further suggest that the metabolic modules making up these important freshwater metabolic traits were already present in the gene pool of ancestral marine SAR11 populations. The loss of the glyoxylate shunt had already occurred in the common ancestor of the freshwater subgroup and its closest marine relatives, suggesting that the adaptation to freshwater was a gradual process. Furthermore, our results indicate rapid evolution of TRAP transporters in the freshwater clade involved in the uptake of low molecular weight carboxylic acids. We propose that such gradual tuning of metabolic pathways and transporters toward locally available organic substrates is linked to the formation of subgroups within the SAR11 clade and that this process was critical for the freshwater clade to find and fix an adaptive phenotype.

Genome-wide selective sweeps and gene-specific sweeps in natural bacterial populations.

Multiple models describe the formation and evolution of distinct microbial phylogenetic groups. These evolutionary models make different predictions regarding how adaptive alleles spread through populations and how genetic diversity is maintained. Processes predicted by competing evolutionary models, for example, genome-wide selective sweeps vs gene-specific sweeps, could be captured in natural populations using time-series metagenomics if the approach were applied over a sufficiently long time frame. Direct observations of either process would help resolve how distinct microbial groups evolve. Here, from a 9-year metagenomic study of a freshwater lake (2005-2013), we explore changes in single-nucleotide polymorphism (SNP) frequencies and patterns of gene gain and loss in 30 bacterial populations. SNP analyses revealed substantial genetic heterogeneity within these populations, although the degree of heterogeneity varied by >1000-fold among populations. SNP allele frequencies also changed dramatically over time within some populations. Interestingly, nearly all SNP variants were slowly purged over several years from one population of green sulfur bacteria, while at the same time multiple genes either swept through or were lost from this population. These patterns were consistent with a genome-wide selective sweep in progress, a process predicted by the 'ecotype model' of speciation but not previously observed in nature. In contrast, other populations contained large, SNP-free genomic regions that appear to have swept independently through the populations prior to the study without purging diversity elsewhere in the genome. Evidence for both genome-wide and gene-specific sweeps suggests that different models of bacterial speciation may apply to different populations coexisting in the same environment.

Phylogeny and physiology of candidate phylum 'Atribacteria' (OP9/JS1) inferred from cultivation-independent genomics.

The 'Atribacteria' is a candidate phylum in the Bacteria recently proposed to include members of the OP9 and JS1 lineages. OP9 and JS1 are globally distributed, and in some cases abundant, in anaerobic marine sediments, geothermal environments, anaerobic digesters and reactors and petroleum reservoirs. However, the monophyly of OP9 and JS1 has been questioned and their physiology and ecology remain largely enigmatic due to a lack of cultivated representatives. Here cultivation-independent genomic approaches were used to provide a first comprehensive view of the phylogeny, conserved genomic features and metabolic potential of members of this ubiquitous candidate phylum. Previously available and heretofore unpublished OP9 and JS1 single-cell genomic data sets were used as recruitment platforms for the reconstruction of atribacterial metagenome bins from a terephthalate-degrading reactor biofilm and from the monimolimnion of meromictic Sakinaw Lake. The single-cell genomes and metagenome bins together comprise six species- to genus-level groups that represent most major lineages within OP9 and JS1. Phylogenomic analyses of these combined data sets confirmed the monophyly of the 'Atribacteria' inclusive of OP9 and JS1. Additional conserved features within the 'Atribacteria' were identified, including a gene cluster encoding putative bacterial microcompartments that may be involved in aldehyde and sugar metabolism, energy conservation and carbon storage. Comparative analysis of the metabolic potential inferred from these data sets revealed that members of the 'Atribacteria' are likely to be heterotrophic anaerobes that lack respiratory capacity, with some lineages predicted to specialize in either primary fermentation of carbohydrates or secondary fermentation of organic acids, such as propionate.

Effects of sample treatments on genome recovery via single-cell genomics.

Single-cell genomics is a powerful tool for accessing genetic information from uncultivated microorganisms. Methods of handling samples before single-cell genomic amplification may affect the quality of the genomes obtained. Using three bacterial strains we show that, compared to cryopreservation, lower-quality single-cell genomes are recovered when the sample is preserved in ethanol or if the sample undergoes fluorescence in situ hybridization, while sample preservation in paraformaldehyde renders it completely unsuitable for sequencing.

Reconstructing each cell's genome within complex microbial communities-dream or reality?

As the vast majority of microorganisms have yet to be cultivated in a laboratory setting, access to their genetic makeup has largely been limited to cultivation-independent methods. These methods, namely metagenomics and more recently single-cell genomics, have become cornerstones for microbial ecology and environmental microbiology. One ultimate goal is the recovery of genome sequences from each cell within an environment to move toward a better understanding of community metabolic potential and to provide substrate for experimental work. As single-cell sequencing has the ability to decipher all sequence information contained in an individual cell, this method holds great promise in tackling such challenge. Methodological limitations and inherent biases however do exist, which will be discussed here based on environmental and benchmark data, to assess how far we are from reaching this goal.

The candidate phylum Poribacteria by single-cell genomics: new insights into phylogeny, cell-compartmentation, eukaryote-like repeat proteins, and other genomic features.

The candidate phylum Poribacteria is one of the most dominant and widespread members of the microbial communities residing within marine sponges. Cell compartmentalization had been postulated along with their discovery about a decade ago and their phylogenetic association to the Planctomycetes, Verrucomicrobia, Chlamydiae superphylum was proposed soon thereafter. In the present study we revised these features based on genomic data obtained from six poribacterial single cells. We propose that Poribacteria form a distinct monophyletic phylum contiguous to the PVC superphylum together with other candidate phyla. Our genomic analyses supported the possibility of cell compartmentalization in form of bacterial microcompartments. Further analyses of eukaryote-like protein domains stressed the importance of such proteins with features including tetratricopeptide repeats, leucin rich repeats as well as low density lipoproteins receptor repeats, the latter of which are reported here for the first time from a sponge symbiont. Finally, examining the most abundant protein domain family on poribacterial genomes revealed diverse phyH family proteins, some of which may be related to dissolved organic posphorus uptake.

Improving the genome annotation of the acarbose producer Actinoplanes sp. SE50/110 by sequencing enriched 5'-ends of primary transcripts.

Actinoplanes sp. SE50/110 is the producer of the alpha-glucosidase inhibitor acarbose, which is an economically relevant and potent drug in the treatment of type-2 diabetes mellitus. In this study, we present the detection of transcription start sites on this genome by sequencing enriched 5'-ends of primary transcripts. Altogether, 1427 putative transcription start sites were initially identified. With help of the annotated genome sequence, 661 transcription start sites were found to belong to the leader region of protein-coding genes with the surprising result that roughly 20% of these genes rank among the class of leaderless transcripts. Next, conserved promoter motifs were identified for protein-coding genes with and without leader sequences. The mapped transcription start sites were finally used to improve the annotation of the Actinoplanes sp. SE50/110 genome sequence. Concerning protein-coding genes, 41 translation start sites were corrected and 9 novel protein-coding genes could be identified. In addition to this, 122 previously undetermined non-coding RNA (ncRNA) genes of Actinoplanes sp. SE50/110 were defined. Focusing on antisense transcription start sites located within coding genes or their leader sequences, it was discovered that 96 of those ncRNA genes belong to the class of antisense RNA (asRNA) genes. The remaining 26 ncRNA genes were found outside of known protein-coding genes. Four chosen examples of prominent ncRNA genes, namely the transfer messenger RNA gene ssrA, the ribonuclease P class A RNA gene rnpB, the cobalamin riboswitch RNA gene cobRS, and the selenocysteine-specific tRNA gene selC, are presented in more detail. This study demonstrates that sequencing of enriched 5'-ends of primary transcripts and the identification of transcription start sites are valuable tools for advanced genome annotation of Actinoplanes sp. SE50/110 and most probably also for other bacteria.

Ecology and evolution of viruses infecting uncultivated SUP05 bacteria as revealed by single-cell- and meta-genomics.

Viruses modulate microbial communities and alter ecosystem functions. However, due to cultivation bottlenecks, specific virus-host interaction dynamics remain cryptic. In this study, we examined 127 single-cell amplified genomes (SAGs) from uncultivated SUP05 bacteria isolated from a model marine oxygen minimum zone (OMZ) to identify 69 viral contigs representing five new genera within dsDNA Caudovirales and ssDNA Microviridae. Infection frequencies suggest that ∼1/3 of SUP05 bacteria is viral-infected, with higher infection frequency where oxygen-deficiency was most severe. Observed Microviridae clonality suggests recovery of bloom-terminating viruses, while systematic co-infection between dsDNA and ssDNA viruses posits previously unrecognized cooperation modes. Analyses of 186 microbial and viral metagenomes revealed that SUP05 viruses persisted for years, but remained endemic to the OMZ. Finally, identification of virus-encoded dissimilatory sulfite reductase suggests SUP05 viruses reprogram their host's energy metabolism. Together, these results demonstrate closely coupled SUP05 virus-host co-evolutionary dynamics with the potential to modulate biogeochemical cycling in climate-critical and expanding OMZs.

Stop codon reassignments in the wild.

The canonical genetic code is assumed to be deeply conserved across all domains of life with very few exceptions. By scanning 5.6 trillion base pairs of metagenomic data for stop codon reassignment events, we detected recoding in a substantial fraction of the >1700 environmental samples examined. We observed extensive opal and amber stop codon reassignments in bacteriophages and of opal in bacteria. Our data indicate that bacteriophages can infect hosts with a different genetic code and demonstrate phage-host antagonism based on code differences. The abundance and diversity of genetic codes present in environmental organisms should be considered in the design of engineered organisms with altered genetic codes in order to preclude the exchange of genetic information with naturally occurring species.

Comparative single-cell genomics reveals potential ecological niches for the freshwater acI Actinobacteria lineage.

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.