Whole genome mapping of 5' RNA ends in bacteria by tagged sequencing : A comprehensive view in Enterococcus faecalis

Enterococcus faecalis is the third cause of nosocomial infections. To obtain the first comprehensive view of transcriptional organizations in this bacterium, we used a modified RNA-seq approach enabling to discriminate primary from processed 5'RNA ends. We also validated our approach by confirming known features in Escherichia coli. We mapped 559 transcription start sites and 352 processing sites in E. faecalis. A blind motif search retrieved canonical features of SigA- and SigN-dependent promoters preceding TSSs mapped. We discovered 95 novel putative regulatory RNAs, small- and antisense RNAs, and 72 transcriptional antisense organisations. Presented data constitute a significant insight into bacterial RNA landscapes and a step towards the inference of regulatory processes at transcriptional and post-transcriptional levels in a comprehensive manner

Commensal bacteria augment Staphylococcus aureus infection by inactivation of phagocyte-derived reactive oxygen species

Staphylococcus aureus is a human commensal organism and opportunist pathogen, causing potentially fatal disease. The presence of non-pathogenic microflora or their components, at the point of infection, dramatically increases S. aureus pathogenicity, a process termed augmentation. Augmentation is associated with macrophage interaction but by a hitherto unknown mechanism. Here, we demonstrate a breadth of cross-kingdom microorganisms can augment S. aureus disease and that pathogenesis of Enterococcus faecalis can also be augmented. Co-administration of augmenting material also forms an efficacious vaccine model for S. aureus. In vitro, augmenting material protects S. aureus directly from reactive oxygen species (ROS), which correlates with in vivo studies where augmentation restores full virulence to the ROS-susceptible, attenuated mutant katA ahpC. At the cellular level, augmentation increases bacterial survival within macrophages via amelioration of ROS, leading to proliferation and escape. We have defined the molecular basis for augmentation that represents an important aspect of the initiation of infection

Decoration of the enterococcal polysaccharide antigen EPA is essential for virulence, cell surface charge and interaction with effectors of the innate immune system

Enterococcus faecalis is an opportunistic pathogen with an intrinsically high resistance to lysozyme, a key effector of the innate immune system. This high level of resistance requires a complex network of transcriptional regulators and several genes (oatA, pgdA, dltA and sigV) acting synergistically to inhibit both the enzymatic and cationic antimicrobial peptide activities of lysozyme. We sought to identify novel genes modulating E. faecalis resistance to lysozyme. Random transposon mutagenesis carried out in the quadruple oatA/pgdA/dltA/sigV mutant led to the identification of several independent insertions clustered on the chromosome. These mutations were located in a locus referred to as the enterococcal polysaccharide antigen (EPA) variable region located downstream of the highly conserved epaA-epaR genes proposed to encode a core synthetic machinery. The epa variable region was previously proposed to be responsible for EPA decorations, but the role of this locus remains largely unknown. Here, we show that EPA decoration contributes to resistance towards charged antimicrobials and underpins virulence in the zebrafish model of infection by conferring resistance to phagocytosis. Collectively, our results indicate that the production of the EPA rhamnopolysaccharide backbone is not sufficient to promote E. faecalis infections and reveal an essential role of the modification of this surface polymer for enterococcal pathogenesis

Time-Resolved Transcriptome Analysis of Bacillus subtilis Responding to Valine, Glutamate, and Glutamine

Microorganisms can restructure their transcriptional output to adapt to environmental conditions by sensing endogenous metabolite pools. In this paper, an Agilent customized microarray representing 4,106 genes was used to study temporal transcript profiles of Bacillus subtilis in response to valine, glutamate and glutamine pulses over 24 h. A total of 673, 835, and 1135 amino-acid-regulated genes were identified having significantly changed expression at one or more time points in response to valine, glutamate, and glutamine, respectively, including genes involved in cell wall, cellular import, metabolism of amino-acids and nucleotides, transcriptional regulation, flagellar motility, chemotaxis, phage proteins, sporulation, and many genes of unknown function. Different amino acid treatments were compared in terms of both the global temporal profiles and the 5-minute quick regulations, and between-experiment differential genes were identified. The highlighted genes were analyzed based on diverse sources of gene functions using a variety of computational tools, including T-profiler analysis, and hierarchical clustering. The results revealed the common and distinct modes of action of these three amino acids, and should help to elucidate the specific signaling mechanism of each amino acid as an effector

Distinct Roles of ComK1 and ComK2 in Gene Regulation in Bacillus cereus

The B. subtilis transcriptional factor ComK regulates a set of genes coding for DNA uptake from the environment and for its integration into the genome. In previous work we showed that Bacillus cereus expressing the B. subtilis ComK protein is able to take up DNA and integrate it into its own genome. To extend our knowledge on the effect of B. subtilis ComK overexpression in B. cereus we first determined which genes are significantly altered. Transcriptome analysis showed that only part of the competence gene cluster is significantly upregulated. Two ComK homologues can be identified in B. cereus that differ in their respective homologies to other ComK proteins. ComK1 is most similar, while ComK2 lacks the C-terminal region previously shown to be important for transcription activation by B. subtilis ComK. comK1 and comK2 overexpression and deletion studies using transcriptomics techniques showed that ComK1 enhances and ComK2 decreases expression of the comG operon, when B. subtilis ComK was overexpressed simultaneously

Spo0A∼P Imposes a Temporal Gate for the Bimodal Expression of Competence in Bacillus subtilis

ComK transcriptionally controls competence for the uptake of transforming DNA in Bacillus subtilis. Only 10%–20% of the cells in a clonal population are randomly selected for competence. Because ComK activates its own promoter, cells exceeding a threshold amount of ComK trigger a positive feedback loop, transitioning to the competence ON state. The transition rate increases to a maximum during the approach to stationary phase and then decreases, with most cells remaining OFF. The average basal rate of comK transcription increases transiently, defining a window of opportunity for transitions and accounting for the heterogeneity of competent populations. We show that as the concentration of the response regulator Spo0A∼P increases during the entry to stationary phase it first induces comK promoter activity and then represses it by direct binding. Spo0A∼P activates by antagonizing the repressor, Rok. This amplifies an inherent increase in basal level comK promoter activity that takes place during the approach to stationary phase and is a general feature of core promoters, serving to couple the probability of competence transitions to growth rate. Competence transitions are thus regulated by growth rate and temporally controlled by the complex mechanisms that govern the formation of Spo0A∼P. On the level of individual cells, the fate-determining noise for competence is intrinsic to the comK promoter. This overall mechanism has been stochastically simulated and shown to be plausible. Thus, a deterministic mechanism modulates an inherently stochastic process

A simple and efficient method to search for selected primary transcripts: non-coding and antisense RNAs in the human pathogen Enterococcus faecalis

Enterococcus faecalis is a commensal bacterium and a major opportunistic human pathogen. In this study, we combined in silico predictions with a novel 5′RACE-derivative method coined ‘5′tagRACE’, to perform the first search for non-coding RNAs (ncRNAs) encoded on the E. faecalis chromosome. We used the 5′tagRACE to simultaneously probe and characterize primary transcripts, and demonstrate here the simplicity, the reliability and the sensitivity of the method. The 5′tagRACE is complementary to tiling arrays or RNA-sequencing methods, and is also directly applicable to deep RNA sequencing and should significantly improve functional studies of bacterial RNA landscapes. From 45 selected loci of the E. faecalis chromosome, we discovered and mapped 29 novel ncRNAs, 10 putative novel mRNAs and 16 antisense transcriptional organizations. We describe in more detail the oxygen-dependent expression of one ncRNA located in an E. faecalis pathogenicity island, the existence of an ncRNA that is antisense to the ncRNA modulator of the RNA polymerase, SsrS and provide evidences for the functional interplay between two distinct toxin–antitoxin modules

Quantitative proteomic analysis of the influence of lignin on biofuel production by Clostridium acetobutylicum ATCC 824

Background: Clostridium acetobutylicum has been a focus of research because of its ability to produce high-value compounds that can be used as biofuels. Lignocellulose is a promising feedstock, but the lignin–cellulose–hemicellulose biomass complex requires chemical pre-treatment to yield fermentable saccharides, including cellulose-derived cellobiose, prior to bioproduction of acetone–butanol–ethanol (ABE) and hydrogen. Fermentation capability is limited by lignin and thus process optimization requires knowledge of lignin inhibition. The effects of lignin on cellular metabolism were evaluated for C. acetobutylicum grown on medium containing either cellobiose only or cellobiose plus lignin. Microscopy, gas chromatography and 8-plex iTRAQ-based quantitative proteomic technologies were applied to interrogate the effect of lignin on cellular morphology, fermentation and the proteome. Results: Our results demonstrate that C. acetobutylicum has reduced performance for solvent production when lignin is present in the medium. Medium supplemented with 1 g L−1 of lignin led to delay and decreased solvents production (ethanol; 0.47 g L−1 for cellobiose and 0.27 g L−1 for cellobiose plus lignin and butanol; 0.13 g L−1 for cellobiose and 0.04 g L−1 for cellobiose plus lignin) at 20 and 48 h, respectively, resulting in the accumulation of acetic acid and butyric acid. Of 583 identified proteins (FDR < 1 %), 328 proteins were quantified with at least two unique peptides. Up- or down-regulation of protein expression was determined by comparison of exponential and stationary phases of cellobiose in the presence and absence of lignin. Of relevance, glycolysis and fermentative pathways were mostly down-regulated, during exponential and stationary growth phases in presence of lignin. Moreover, proteins involved in DNA repair, transcription/translation and GTP/ATP-dependent activities were also significantly affected and these changes were associated with altered cell morphology. Conclusions: This is the first comprehensive analysis of the cellular responses of C. acetobutylicum to lignin at metabolic and physiological levels. These data will enable targeted metabolic engineering strategies to optimize biofuel production from biomass by overcoming limitations imposed by the presence of lignin