Global expression analysis of Deinococcus radiodurans\u27 response to ionizing radiation: irrE is a novel regulator of this response

IRS24 is a strain of Deinococcus radiodurans carrying mutations in two loci, uvrA and irrE, rendering it sensitive to the lethal effects of UV and ionizing radiation. These sensitivities can be reversed by introducing the wild type irrE allele back into IRS24 via natural transformation. The irrE allele was localized to a 970bp region of D. radiodurans R1 Chromosome I containing one putative open reading frame, DR0167, and 179bp of upstream sequence. Subsequent sequence analysis of the irrE allele in IRS24 revealed a transition mutation at codon 111 of DR0167 (IrrE) resulting in an arginine to cysteine amino acid substitution. IrrE was also inactivated by transposon mutagenesis in the wild type strain, R1. The insertion mutant, LSU2030, has a more pronounced sensitivity to both UV and ionizing radiation suggesting that IRS24\u27s IrrE retains some activity. BLASTp analysis of IrrE reveals only minimal similarity to proteins currently available in protein sequence databases. A weak helix-turn-helix motif was identified within this protein that may indicate a capacity to bind DNA and, perhaps, a potential role for IrrE in gene regulation. To test whether the mutation in IrrE causes a regulatory deficiency, we examined the pattern of transcription following ionizing radiation, comparing LSU2030 and R1 using DNA microarray technology. Our analysis has determined that IrrE is a transcriptional activator that controls expression of many genes including recA. A recent investigation has shown that, as in E. coli\u27s response to stress, RecA is necessary for proteolytic cleavage of the LexA repressor in D. radiodurans. However, unlike the E. coli paradigm, deinococcal RecA is not controlled by LexA. Functional IrrE appears to be necessary for recA induction and in mounting an effective response to exogenous stress. This analysis along with examination of the transcriptional changes exhibited by R1 and a lexA-defective strain following ionizing radiation has focused our attention to a subset of 12 genes that are induced during D. radiodurans\u27 response to ionizing radiation and recovery from prolonged desiccation. These genes appear to be critical to this species\u27 ability to survive both stresses and may be involved in DNA double strand break repair

Evidence of Extensive DNA Transfer between Bacteroidales Species within the Human Gut

ABSTRACT The genome sequences of intestinal Bacteroidales strains reveal evidence of extensive horizontal gene transfer. In vitro studies of Bacteroides and other bacteria have addressed mechanisms of conjugative transfer and some phenotypic outcomes of these DNA acquisitions in the recipient, such as the acquisition of antibiotic resistance. However, few studies have addressed the horizontal transfer of genetic elements between bacterial species coresident in natural microbial communities, especially microbial ecosystems of humans. Here, we examine the genomes of Bacteroidales species from two human adults to identify genetic elements that were likely transferred among these Bacteroidales while they were coresident in the intestine. Using seven coresident Bacteroidales species from one individual and eight from another, we identified five large chromosomal regions, each present in a minimum of three of the coresident strains at near 100% DNA identity. These five regions are not found in any other sequenced Bacteroidetes genome at this level of identity and are likely all integrative conjugative elements (ICEs). Such highly similar and unique regions occur in only 0.4% of phylogenetically representative mock communities, providing strong evidence that these five regions were transferred between coresident strains in these subjects. In addition to the requisite proteins necessary for transfer, these elements encode proteins predicted to increase fitness, including orphan DNA methylases that may alter gene expression, fimbriae synthesis proteins that may facilitate attachment and the utilization of new substrates, putative secreted antimicrobial molecules, and a predicted type VI secretion system (T6SS), which may confer a competitive ecological advantage to these strains in their complex microbial ecosystem

Preserving Genome Integrity: The DdrA Protein of Deinococcus radiodurans R1

The bacterium Deinococcus radiodurans can withstand extraordinary levels of ionizing radiation, reflecting an equally extraordinary capacity for DNA repair. The hypothetical gene product DR0423 has been implicated in the recovery of this organism from DNA damage, indicating that this protein is a novel component of the D. radiodurans DNA repair system. DR0423 is a homologue of the eukaryotic Rad52 protein. Following exposure to ionizing radiation, DR0423 expression is induced relative to an untreated control, and strains carrying a deletion of the DR0423 gene exhibit increased sensitivity to ionizing radiation. When recovering from ionizing-radiation-induced DNA damage in the absence of nutrients, wild-type D. radiodurans reassembles its genome while the mutant lacking DR0423 function does not. In vitro, the purified DR0423 protein binds to single-stranded DNA with an apparent affinity for 3′ ends, and protects those ends from nuclease degradation. We propose that DR0423 is part of a DNA end-protection system that helps to preserve genome integrity following exposure to ionizing radiation. We designate the DR0423 protein as DNA damage response A protein

High-Quality Draft Genome Sequence of Vagococcus lutrae Strain LBD1, Isolated from the Largemouth Bass Micropterus salmoides

Vagococci are usually isolated from marine hosts and occasionally from endodontic infections. Using 16S rRNA gene comparison, the closest relatives are members of the genera Enterococcus and Carnobacterium. A draft sequence of Vagococcus lutrae was generated to clarify the relationship of Vagococcus to these and other related low-G+C Gram-positive bacteria

Relating the metatranscriptome and metagenome of the human gut

Although the composition of the human microbiome is now wellstudied, the microbiota’s \u3e8 million genes and their regulation remain largely uncharacterized. This knowledge gap is in part because of the difficulty of acquiring large numbers of samples amenable to functional studies of the microbiota. We conducted what is, to our knowledge, one of the first human microbiome studies in a well-phenotyped prospective cohort incorporating taxonomic, metagenomic, and metatranscriptomic profiling at multiple body sites using self-collected samples. Stool and saliva were provided by eight healthy subjects, with the former preserved by three different methods (freezing, ethanol, and RNAlater) to validate self-collection. Within-subject microbial species, gene, and transcript abundances were highly concordant across sampling methods, with only a small fraction of transcripts (\u3c5%) displaying between-method variation. Next, we investigated relationships between the oral and gut microbial communities, identifying a subset of abundant oral microbes that routinely survive transit to the gut, but with minimal transcriptional activity there. Finally, systematic comparison of the gut metagenome and metatranscriptome revealed that a substantial fraction (41%) of microbial transcripts were not differentially regulated relative to their genomic abundances. Of the remainder, consistently underexpressed pathways included sporulation and amino acid biosynthesis, whereas up-regulated pathways included ribosome biogenesis and methanogenesis. Across subjects, metatranscriptional profiles were significantly more individualized than DNA-level functional profiles, but less variable than microbial composition, indicative of subject-specific whole-community regulation. The results thus detail relationships between community genomic potential and gene expression in the gut, and establish the feasibility of metatranscriptomic investigations in subject-collected and shipped samples

Populations of latent Mycobacterium tuberculosis lack a cell wall: Isolation, visualization, and whole-genome characterization

AbstractObjective/BackgroundMycobacterium tuberculosis (MTB) causes active tuberculosis (TB) in only a small percentage of infected people. In most cases, the infection is clinically latent, where bacilli can persist in human hosts for years without causing disease. Surprisingly, the biology of such persister cells is largely unknown. This study describes the isolation, identification, and whole-genome sequencing (WGS) of latent TB bacilli after 782days (26months) of latency (the ability of MTB bacilli to lie persistent).MethodsThe in vitro double-stress model of latency (oxygen and nutrition) was designed for MTB culture. After 26months of latency, MTB cells that persisted were isolated and investigated under light and atomic force microscopy. Spoligotyping and WGS were performed to verify the identity of the strain.ResultsWe established a culture medium in which MTB bacilli arrest their growth, reduce their size (0.3–0.1μm), lose their acid fastness (85–90%) and change their shape. Spoligopatterns of latent cells were identical to original H37Rv, with differences observed at spacers two and 14. WGS revealed only a few genetic changes relative to the already published H37Rv reference genome. Among these was a large 2064-bp insertion (RvD6), which was originally detected in both H37Ra and CDC1551, but not H37Rv.ConclusionHere, we show cell-wall free cells of MTB bacilli in their latent state, and the biological adaptation of these cells was more phenotypic in nature than genomic. These cell-wall free cells represent a good model for understanding the nature of TB latency

Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes

We have developed a process for transcriptome analysis of bacterial communities that accommodates both intact and fragmented starting RNA and combines efficient rRNA removal with strand-specific RNA-seq. We applied this approach to an RNA mixture derived from three diverse cultured bacterial species and to RNA isolated from clinical stool samples. The resulting expression profiles were highly reproducible, enriched up to 40-fold for non-rRNA transcripts, and correlated well with profiles representing undepleted total RNA