Bacteria in the 16S rRNA clade SAR86 are among the most abundant uncultivated constituents of microbial assemblages in the surface ocean for which little genomic information is currently available. Bioinformatic techniques were used to assemble two nearly complete genomes from marine metagenomes and single-cell sequencing provided two more partial genomes. Recruitment of metagenomic data shows that these SAR86 genomes substantially increase our knowledge of non-photosynthetic bacteria in the surface ocean. Phylogenomic analyses establish SAR86 as a basal and divergent lineage of γ-proteobacteria, and the individual genomes display a temperature-dependent distribution. Modestly sized at 1.25–1.7 Mbp, the SAR86 genomes lack several pathways for amino-acid and vitamin synthesis as well as sulfate reduction, trends commonly observed in other abundant marine microbes. SAR86 appears to be an aerobic chemoheterotroph with the potential for proteorhodopsin-based ATP generation, though the apparent lack of a retinal biosynthesis pathway may require it to scavenge exogenously-derived pigments to utilize proteorhodopsin. The genomes contain an expanded capacity for the degradation of lipids and carbohydrates acquired using a wealth of tonB-dependent outer membrane receptors. Like the abundant planktonic marine bacterial clade SAR11, SAR86 exhibits metabolic streamlining, but also a distinct carbon compound specialization, possibly avoiding competition.

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Whole genome amplification by the multiple displacement amplification (MDA) method allows sequencing of genomes from single cells of bacteria that cannot be cultured. However, genome assembly is challenging because of highly non-uniform read coverage generated by MDA. We describe an improved assembly approach tailored for single cell Illumina sequences that incorporates a progressively increasing coverage cutoff. This allows variable coverage datasets to be utilized effectively with assembly of E. coli and S. aureus single cell reads capturing >91% of genes within contigs, approaching the 95% captured from a multi-cell E. coli assembly. We apply this method to assemble a single cell genome of the uncultivated SAR324 clade of Deltaproteobacteria, a cosmopolitan bacterial lineage in the global ocean. Metabolic reconstruction suggests that SAR324 is aerobic, motile and chemotaxic. These new methods enable acquisition of genome assemblies for individual uncultivated bacteria, providing cell-specific genetic information absent from metagenomic studies.

Microbial cells under growth-limiting stress can generate mutations by mechanisms distinct from those in rapidly growing cells. These mechanisms might be specific stress responses that increase mutation rates, potentially altering rates of evolution, or might reflect non-stress-specific processes in rare growing cells.In an Escherichia coli model system, both frameshift reversion mutations and gene amplifications occur as apparent starvation-induced mutations. Whereas frameshift reversion ("point mutation") requires recombination proteins, the SOS response, and error-prone DNA polymerase IV (DinB), amplification requires neither SOS nor pol IV. We report that both point mutation and amplification require the stationaryphase and general stress response transcription factor RpoS ( S ). Growth-dependent mutation does not. Alternative interpretations are excluded. The results imply, first, that point mutation and amplification are stress responses that occur in differentiated stationary-phase (not rare growing) cells and, second, that transient genetic instability, producing both point mutation and genome rearrangement, may be a previously unrecognized component of the RpoS-dependent general stress response.

The "dark matter of life" describes microbes and even entire divisions of bacterial phyla that have evaded cultivation and have yet to be sequenced. We present a genome from the globally distributed but elusive candidate phylum TM6 and uncover its metabolic potential. TM6 was detected in a biofilm from a sink drain within a hospital restroom by analyzing cells using a highly automated single-cell genomics platform. We developed an approach for increasing throughput and effectively improving the likelihood of sampling rare events based on forming small random pools of single-flow-sorted cells, amplifying their DNA by multiple displacement amplification and sequencing all cells in the pool, creating a "mini-metagenome." A recently developed single-cell assembler, SPAdes, in combination with contig binning methods, allowed the reconstruction of genomes from these mini-metagenomes. A total of 1.07 Mb was recovered in seven contigs for this member of TM6 (JCVI TM6SC1), estimated to represent 90% of its genome. High nucleotide identity between a total of three TM6 genome drafts generated from pools that were independently captured, amplified, and assembled provided strong confirmation of a correct genomic sequence. TM6 is likely a Gram-negative organism and possibly a symbiont of an unknown host (nonfree living) in part based on its small genome, low-GC content, and lack of biosynthesis pathways for most amino acids and vitamins. Phylogenomic analysis of conserved single-copy genes confirms that TM6SC1 is a deeply branching phylum. (1), which is accomplished using multiple displacement amplification (MDA) (2-5) of genomic DNA to obtain sufficient template. We applied a high-throughput strategy to capture and sequence genomes of bacteria from a biofilm in a hospital sink including pathogens, such as the oral periodontal pathogen (Porphyromonas gingivalis) (6) and uncultivated members (this study). Despite the fact that a typical person spends ∼90% of their time indoors (7), our knowledge of the microbial diversity of the indoor environment has only recently begun to be explored using culture-independent methods (8, 9). Biofilms within water distribution systems in particular are thought to be diverse microbial communities and potential reservoirs of disease-causing organisms in the indoor environment. Several pathogens including Escherichia coli, Legionella pneumophila (10-13), Vibrio cholerae (14), and Helicobacter pylori (15, 16) have been detected in biofilms within water distribution systems. A recent 16S rRNA gene (abbreviated henceforth as 16S unless otherwise stated) molecular survey also revealed significant loads of Mycobacterium avium in showerhead biofilms (17). Based on these findings, indoor environments can clearly serve as significant reservoirs of pathogenic bacteria, and therefore there is great interest in investigating the rare and abundant bacterial species within biofilms in these environments.One approach to capture uncultivated bacteria is to isolate single bacterial cells by fluorescent activat...

Bacterial virulence factors are typically surface-associated or secreted molecules that in Gram-negative bacteria must cross the outer membrane (OM). Protein translocation across the bacterial OM is not well understood. To elucidate this process we studied P pilus biogenesis in Escherichia coli. We present high-resolution electron micrographs of the OM usher PapC and show that it forms an oligomeric complex containing a channel approximately 2 nm in diameter. This is large enough to accommodate pilus subunits or the linear tip fibrillum of the pilus but not large enough to accommodate the final 6.8-nm-wide helical pilus rod. We show that P pilus rods can be unraveled into linear fibers by incubation in 50% glycerol. Thus, they are likely to pass through the usher in this unwound form. Packaging of these fibers into their final helical structure would only occur outside the cell, a process that may drive outward growth of the pilus organelles. The usher complex appears to be similar to complexes formed by members of the PulD͞pIV family of OM proteins, and thus these two protein families, previously thought to be unrelated, may share structural and functional homologies.

The paucity of sequence data from pelagic deep-ocean microbial assemblages has severely restricted molecular exploration of the largest biome on Earth. In this study, an analysis is presented of a large-scale 454-pyrosequencing metagenomic dataset from a hadopelagic environment from 6,000 m depth within the Puerto Rico Trench (PRT). A total of 145 Mbp of assembled sequence data was generated and compared to two pelagic deep ocean metagenomes and two representative surface seawater datasets from the Sargasso Sea. In a number of instances, all three deep metagenomes displayed similar trends, but were most magnified in the PRT, including enrichment in functions for two-component signal transduction mechanisms and transcriptional regulation. Overrepresented transporters in the PRT metagenome included outer membrane porins, diverse cation transporters, and di- and tri-carboxylate transporters that matched well with the prevailing catabolic processes such as butanoate, glyoxylate and dicarboxylate metabolism. A surprisingly high abundance of sulfatases for the degradation of sulfated polysaccharides were also present in the PRT. The most dramatic adaptational feature of the PRT microbes appears to be heavy metal resistance, as reflected in the large numbers of transporters present for their removal. As a complement to the metagenome approach, single-cell genomic techniques were utilized to generate partial whole-genome sequence data from four uncultivated cells from members of the dominant phyla within the PRT, Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes and Planctomycetes. The single-cell sequence data provided genomic context for many of the highly abundant functional attributes identified from the PRT metagenome, as well as recruiting heavily the PRT metagenomic sequence data compared to 172 available reference marine genomes. Through these multifaceted sequence approaches, new insights have been provided into the unique functional attributes present in microbes residing in a deeper layer of the ocean far removed from the more productive sun-drenched zones above.

Pathways of mutagenesis are induced in microbes under adverse conditions controlled by stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e. are stressed. Stress-induced mutagenesis in the Escherichia coli Lac assay occurs either by ‘point’ mutation or gene amplification. Point mutagenesis is associated with DNA double-strand-break (DSB) repair and requires DinB error-prone DNA polymerase and the SOS DNA-damage- and RpoS general-stress responses. We report that the RpoE envelope-protein-stress response is also required. In a screen for mutagenesis-defective mutants, we isolated a transposon insertion in the rpoE P2 promoter. The insertion prevents rpoE induction during stress, but leaves constitutive expression intact, and allows cell viability. rpoE insertion and suppressed null mutants display reduced point mutagenesis and maintenance of amplified DNA. Furthermore, σE acts independently of stress responses previously implicated: SOS/DinB and RpoS, and of σ32, which was postulated to affect mutagenesis. I-SceI-induced DSBs alleviated much of the rpoE phenotype, implying that σE promoted DSB formation. Thus, a third stress response and stress input regulate DSB-repair-associated stress-induced mutagenesis. This provides the first report of mutagenesis promoted by σE, and implies that extracytoplasmic stressors may affect genome integrity and, potentially, the ability to evolve.