Genomic insights to SAR86, an abundant and uncultivated marine bacterial lineage

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

Going Deeper: Metagenome of a Hadopelagic Microbial Community

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

Genetic analysis of RNA polymerase/transcriptional activator interactions at thepepT promoter of S. typhimurium

The role of the alpha subunit of RNA polymerase (encoded by rpoA) in transcriptional activation by the global regulatory protein FNR has been investigated using a genetic approach. We have identified five rpoA mutations which decrease the FNR-dependent anaerobic induction of the pepT gene of S. typhimurium. Each of these mutations (G311E, G311R, L289F, R317H, W320ter) affects an amino acid in the C-terminal region of the alpha subunit. Phenotypic characterization shows that four of the five mutations have relatively specific effects on positively regulated transcription of the pepT gene and other FNR-dependent genes. We propose that the C-terminal region of the alpha subunit is involved in direct interactions with FNR. We reasoned that suppressor analysis might be a useful approach to identifying regions of FNR that are involved in interactions with RNA polymerase. Reversion of the G311E and G311R mutations led to the isolation of six mutations (E47K, T80I, A172V, A172T, V208A, and V208G) in the transcriptional activator FNR which restore or partially restore anaerobic induction of pepT in the presence of each of the five rpoA mutations. A172 and V208 both lie in the DNA binding domain of FNR suggesting that the mutations at these positions might restore activation by affecting DNA binding directly or indirectly. The T80I and E47K mutations lie elsewhere in the protein and are unlikely to affect DNA binding. We suggest that the T80 and E47 regions of FNR might be involved in direct interactions between FNR and the alpha subunit or another subunit of RNA polymerase. In addition, we have characterized the pepT promoter region. Mutational studies and primer extension analysis demonstrate that pepT is transcribed from two promoters, an FNR-dependent, anaerobically induced promoter (P1), and a constitutive promoter (P2) conferring low level expression. A mutant P1 promoter containing a consensus-10 sequence was shown to be regulated by CRP-cAMP in addition to FNR. The potABCD operon was shown to be divergently transcribed from pepT, and the potABCD promoter was identified.U of I OnlyETDs are only available to UIUC Users without author permissio

Atypical Role for PhoU in Mutagenic Break Repair under Stress in Escherichia coli.

Mechanisms of mutagenesis activated by stress responses drive pathogen/host adaptation, antibiotic and anti-fungal-drug resistance, and perhaps much of evolution generally. In Escherichia coli, repair of double-strand breaks (DSBs) by homologous recombination is high fidelity in unstressed cells, but switches to a mutagenic mode using error-prone DNA polymerases when the both the SOS and general (σS) stress responses are activated. Additionally, the σE response promotes spontaneous DNA breakage that leads to mutagenic break repair (MBR). We identified the regulatory protein PhoU in a genetic screen for functions required for MBR. PhoU negatively regulates the phosphate-transport and utilization (Pho) regulon when phosphate is in excess, including the PstB and PstC subunits of the phosphate-specific ABC transporter PstSCAB. Here, we characterize the PhoU mutation-promoting role. First, some mutations that affect phosphate transport and Pho transcriptional regulation decrease mutagenesis. Second, the mutagenesis and regulon-expression phenotypes do not correspond, revealing an apparent new function(s) for PhoU. Third, the PhoU mutagenic role is not via activation of the σS, SOS or σE responses, because mutations (or DSBs) that restore mutagenesis to cells defective in these stress responses do not restore mutagenesis to phoU cells. Fourth, the mutagenesis defect in phoU-mutant cells is partially restored by deletion of arcA, a gene normally repressed by PhoU, implying that a gene(s) repressed by ArcA promotes mutagenic break repair. The data show a new role for PhoU in regulation, and a new regulatory branch of the stress-response signaling web that activates mutagenic break repair in E. coli

Model of regulation of the Pho regulon.

<p>Figure based on conclusions, models and interpretations of Hsieh and Wanner [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123315#pone.0123315.ref019" target="_blank">19</a>].</p