Project description:cDNA microarray analysis to identify genes regulated by the RNA chaperone, Hfq. Four experiments were performed: 1/ Hfq+ vs Hfq- strains. 269 significantly differentially regulated genes were identified by SAM (Statistical Analysis of Microarrays), of which 120 changed more than 1.5 fold (48 increased and 72 decreased in hfq-). Amongst other genes, these experiments identified significant regulation of the sigma E and sigma 32 regulons. However, only genes induced by sigma E were similarly induced in hfq-; 8 operons repressed by sigma E were not repressed in hfq-. 2/ wt vs delta rseA. RseA is the antisigma factor for sigmaE. This comparison results in elevated steady-state levels of sigma E, and confirmed induction and repression of target regulon members. 3/ hfq+ vs hfq+ rpoE overexpression. RpoE encoding sigma E was overexpressed in an hfq+ background, confirming normal regulation of the sigma E regulon. 4/ hfq+ vs hfq- rpoE overexpression. Sigma E was overexpressed in an hfq- background. This demonstrated that 8 operons normally repressed by sigma E require hfq for this repression. The simple conclusion is that sigma E regulates small RNAs that, together with Hfq, bind target mRNAs and results in their rapid degradation. This study is detailed in Guisbert et al 2007 (J Bacteriol, 189:1963-73) Keywords: Genetic modification
Project description:Pseudomonas chlororaphis strain 30-84 is an effective biological control agent against take-all disease of wheat. In this study, we conducted an RNA-seq analysis by comparing the wild type strain with a Hfe deficient mutant. RNA-seq analysis identified over 900 genes differentially regulated by Hfq.
Project description:Pseudomonas chlororaphis strain 30-84 is an effective biological control agent against take-all disease of wheat. In this study, we conducted an RNA-seq analysis by comparing the wild type strain with a Hfe deficient mutant. RNA-seq analysis identified over 900 genes differentially regulated by Hfq. A total of 4 samples were analyzed in AB medium + 2% casamino acids, Pseudomonas chlororaphis wild type strain (2 replicates); Pseudomonas chlororaphis ZN mutant (2 replicates).
Project description:Co-immunoprecipitation with endogenous Hfq3xFlag in exponential, stationary, non-induced and virulence induced conditions, followed by RNA-sequencing, revealed 1697 mRNAs and 208 ncRNAs associated with Hfq. We identified 56 new ncRNAs and validated 3 Hfq-dependent trans sRNAs on by Northern blot analysis. Interestingly, 55% of the ncRNAs were encoded antisense to a protein coding sequence. Abundance of 4 asRNAs and their corresponding target mRNAs was altered upon hfq, indicating a substantial influence of Hfq on asRNA-target interactions.
Project description:Co-immunoprecipitation with endogenous Hfq3xFlag in exponential, stationary, non-induced and virulence induced conditions, followed by RNA-sequencing, revealed 1697 mRNAs and 208 ncRNAs associated with Hfq. We identified 56 new ncRNAs and validated 3 Hfq-dependent trans sRNAs on by Northern blot analysis. Interestingly, 55% of the ncRNAs were encoded antisense to a protein coding sequence. Abundance of 4 asRNAs and their corresponding target mRNAs was altered upon hfq, indicating a substantial influence of Hfq on asRNA-target interactions. 8 cDNA libraries were sequenced. A. tumefaciens hfq3xFlag and hfqWT (control) strains were grown to stationary and exponential growth phase and under non-induced and virulence induced conditions.
Project description:The small RNAs associated with protein Hfq constitute one of the largest classes of post-transcriptional regulators known to date. Most previously investigated members of this class are encoded by conserved free-standing genes. Here, deep sequencing of Hfq-bound transcripts from multiple stages of growth of Salmonella Typhimurium revealed a plethora of new small RNA species from within mRNA loci, including DapZ which overlaps with the 3’ region of the biosynthetic gene, dapB. Synthesis of the DapZ small RNA is independent of DapB protein synthesis, and controlled by HilD, the master regulator of Salmonella invasion genes. DapZ carries a short G/U-rich domain similar to that of the globally acting GcvB small RNA, and uses GcvB-like seed pairing to repress translation of the major ABC transporters, DppA and OppA. This exemplifies double functional output from an mRNA locus by the production of both a protein and an Hfq-dependent trans-acting RNA. Our atlas of Hfq targets suggests that the 3’ regions of mRNA genes constitute a rich reservoir to feed the Hfq network with new regulatory small RNAs.
Project description:In this study, we used gel-free nanoLC-MS/MS-based proteomics to compare the protein profile of B. pertussis wild type and an isogenic hfq defective mutant strain under control an iron limited conditions. We found that Hfq affects the abundance of 302 proteins, which represents 8% of the total B. pertussis coding sequence. The absence of Hfq induced changes in the abundance of proteins involved in metabolic pathways, stress response and virulence. Hfq was also found involved in B. pertussis adaptation to iron starvation, one of the main stresses this pathogen faces inside the host. Altogether, these results indicate that B. pertussis Hfq is involved in bacterial physiological processes as well as bacterial pathogenesis.
Project description:Ribosome biogenesis is a complex and intricate process involving multiple factors. A plethora of auxiliary proteins assist the correct assembly of rRNA and ribosomal proteins into mature ribosomes. Defects in this process cause dysfunctional ribosomes which may alter protein synthesis and impair cell fitness. Despite its importance, yet we are lacking mechanistic details of ribosome biogenesis. Here we show that the widely-conserved RNA chaperone Hfq, which is an important post-transcriptional regulator in sRNA-mRNA basepairing in Escherichia coli, plays a critical role in rRNA processing and consequently ribosome assembly. Hfq binds the 17S rRNA precursor and facilitates its correct processing and folding to mature 16S rRNA. Strikingly, Hfq depletion decreases the pool of mature 70S ribosomes. Mutations in residues located in the distal face of Hfq render reduced ribosome levels in contrast to mutations in the proximal and rim surfaces which govern interactions with the sRNAs. Our results indicate that the described Hfq-dependent regulation of ribosomes is independent of its function as sRNA-regulator. Furthermore, we observed that inactivation of Hfq compromises translation efficiency and fidelity, both features of aberrantly assembled ribosomes. Our work expands the functions of Hfq beyond its function in small RNA-mediated regulation and unveil a novel role of Hfq as crucial auxiliary factor in ribosome biogenesis and translation.