Post-transcriptional regulation of target genes by the sRNA FnrS in Neisseria gonorrhoeae.

Small non-coding RNAs (sRNAs) are well-established post-transcriptional regulators of gene expression in bacteria that respond to a variety of environmental stimuli. They usually act by base-pairing with their target mRNAs, which is commonly facilitated by the RNA chaperone Hfq. In this study we initiated the analysis of the sRNA FnrS of Neisseria gonorrhoeae, which is induced under anaerobic conditions. We identified four putative FnrS target genes using bioinformatics approaches and validated these target genes using translational reporter gene fusions in both Escherichia coli and N. gonorrhoeae, thereby demonstrating their downregulation by direct base-pairing between the respective mRNA and FnrS. We demonstrate deregulation of target mRNAs upon deletion of fnrS and provide evidence that the isc gene cluster required for iron-sulfur cluster biosynthesis, which harbours iscS, which is a direct target of FnrS, is coordinately downregulated by the sRNA. By mutational analysis we show that, surprisingly, three distinct regions of FnrS are employed for interaction with different target genes.

Regulation and RNA-binding properties of Hfq-like RNA chaperones in Bacillus anthracis.

BACKGROUND: Small RNAs (sRNAs) are important modulators of gene expression in bacteria. Regulation by sRNAs is yet to be established in Bacillus anthracis. Here, regulation and RNA-binding properties of Hfq-like RNA chaperones in B. anthracis are investigated. METHODS: Transcript levels were measured by RT-PCR. Proteins were cloned, purified, and their ability to bind sRNA was seen by EMSA. Regulators of Hfq1 were identified by in silico analysis and validated by EMSA. Conserved sRNAs were identified by homology search and their ability to bind Hfq1 was seen by EMSA. Residues crucial for sRNA binding were identified by mutational studies. RESULTS: hfq1 and hfq3 showed expression during exponential phase on BHI medium, while hfq2 showed no expression. Hfq1 and Hfq3 formed hexamer and sRNA-Hfq complex, not Hfq2. In silico prediction and EMSA confirmed AbrB binding to the promoter of Hfq1. Homology search identified 7 sRNAs in B. anthracis. The sRNA CsfG showed binding to Hfq1 via residues Y24, F29, Q30, R32, K56, and H57. CONCLUSIONS: Hfq1 and Hfq3 can function as RNA chaperones in B. anthracis. The transition phase repressor AbrB might be responsible for the growth-dependent expression of Hfq1. The sporulation-specific sRNA CsfG binds to Hfq1 via its distal surface and requires an intact hexameric structure for forming CsfG-Hfq1 complex. GENERAL SIGNIFICANCE: This is the first report demonstrating the regulation and functional properties of Hfq-like RNA chaperones in B. anthracis and paves the path towards establishing sRNA-based regulation in B. anthracis.

Delineating the effect of host environmental signals on a fully virulent strain of Bacillus anthracis using an integrated transcriptomics and proteomics approach.

Pathogenic bacteria sense the host environment and regulate expression of virulence-related genes. Environmental signals like temperature, bicarbonate/CO2 and glucose induce toxin production in Bacillus anthracis, but the mechanisms by which these signals contribute to virulence and overall physiological adaptation remains elusive. An integrated, systems level investigation using transcriptomics and iTRAQ-based proteomics was done to assess the effect of temperature, bicarbonate/CO2 and glucose on B. anthracis. Significant changes observed in amino acid, carbohydrate, energy and nucleotide metabolism indicates events of metabolic readjustments by environmental factors. Directed induction of genes involved in polyamine biosynthesis and iron metabolism revealed the redirection of cellular metabolite pool towards iron uptake. Protein levels of glycolytic enzymes, ptsH and Ldh along with transcripts involved in immune evasion (mprF, bNOS, Phospholipases and asnA), cell surface remodeling (rfbABCD, antABCD, and cls) and utilization of lactate (lutABC) and inositol showed constant repression under environmental perturbations. Discrepancies observed in mRNA/protein level of genes involved in glycolysis, protein synthesis, stress response and nucleotide metabolism hinted at the existence of additional regulatory layers and illustrated the utility of an integrated approach. The above findings might assist in the identification of novel adaptive strategies of B. anthracis during host associated survival and pathogenesis. BIOLOGICAL SIGNIFICANCE: In this study, the changes observed at both transcript and protein level were quantified and integrated to understand the effect of host environmental factors (host temperature, bicarbonate and glucose) in shaping the physiology and adaptive strategies of a fully virulent strain of B. anthracis for efficient survival and virulence in its host. Perturbations affecting toxin production were found to concordantly affect vital metabolic pathways and several known as well as novel virulence factors. These changes act as a valuable asset for generating testable hypotheses that can be further verified by detailed molecular and mutant studies to identify novel adaptive strategies of B. anthracis during infection. Adaptation of an integrated transcriptomics and proteomics approach also led to the identification of discrepancies between mRNA/protein levels among genes across major functional categories. Few of these discrepancies have been previously reported in literature for model organisms. However their existence in B. anthracis and that too as a result of growth perturbations have not been reported till date. These findings demonstrate a substantial role of regulatory processes post mRNA synthesis via post transcriptional, translational or protein degradation mechanisms. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Exploring the metabolic network of the epidemic pathogen Burkholderia cenocepacia J2315 via genome-scale reconstruction.

BACKGROUND: Burkholderia cenocepacia is a threatening nosocomial epidemic pathogen in patients with cystic fibrosis (CF) or a compromised immune system. Its high level of antibiotic resistance is an increasing concern in treatments against its infection. Strain B. cenocepacia J2315 is the most infectious isolate from CF patients. There is a strong demand to reconstruct a genome-scale metabolic network of B. cenocepacia J2315 to systematically analyze its metabolic capabilities and its virulence traits, and to search for potential clinical therapy targets. RESULTS: We reconstructed the genome-scale metabolic network of B. cenocepacia J2315. An iterative reconstruction process led to the establishment of a robust model, iKF1028, which accounts for 1,028 genes, 859 internal reactions, and 834 metabolites. The model iKF1028 captures important metabolic capabilities of B. cenocepacia J2315 with a particular focus on the biosyntheses of key metabolic virulence factors to assist in understanding the mechanism of disease infection and identifying potential drug targets. The model was tested through BIOLOG assays. Based on the model, the genome annotation of B. cenocepacia J2315 was refined and 24 genes were properly re-annotated. Gene and enzyme essentiality were analyzed to provide further insights into the genome function and architecture. A total of 45 essential enzymes were identified as potential therapeutic targets. CONCLUSIONS: As the first genome-scale metabolic network of B. cenocepacia J2315, iKF1028 allows a systematic study of the metabolic properties of B. cenocepacia and its key metabolic virulence factors affecting the CF community. The model can be used as a discovery tool to design novel drugs against diseases caused by this notorious pathogen.