Project description:Objectives: To determine the transcripts that are differentially expressed in a hfq mutant. Hfq is an RNA chaperone that mediates many interactions between regultory RNAs and their mRNA targets. Analysis of the transcriptomes of the Pasteurella multocida wild-type strain and the Pasteurella multocida hfq mutant will allow for identification of genes controlled by hfq and the sRNAs with which it interacts.
Project description:Objectives: To determine the transcripts that are differentially expressed in a hfq mutant. Hfq is an RNA chaperone that mediates many interactions between regultory RNAs and their mRNA targets. Analysis of the transcriptomes of the Pasteurella multocida wild-type strain and the Pasteurella multocida hfq mutant will allow for identification of genes controlled by hfq and the sRNAs with which it interacts. Methods: RNA sequencing was employed to determine the transcriptomes of a wild-type Pasteurella multocida strain and a hfq mutant strain. Comparison of these two transcriptomes allows for determination of differentially expressed genes and therefore those genes controlled by Hfq and sRNAs with which it interacts.
Project description:Pasteurella multocida is a pathogen that causes a range of distinct diseases in livestock animals. Different P. multocida strains produce different capsule and lipopolysaccharide (LPS) structures. Different P. multocida diseases are associated with different capsule and LPS types, and little is known about what underpins this disease specificity. In this study, we utilised transposon-directed insertion site sequencing (TraDIS, also called Tn-Seq) to identify genes required for growth in rich media, and genes required for survival during systemic infections in BALB/c mice, for two diverse P. multocida strains, strain VP161 (capsule type A and LPS type L1) and strain M1404 (capsule type B and LPS type L2). Rich media analysis showed that both VP161 and M1404 shared 461 genes essential for growth in rich media, with comparison to the entire species showing that 95% of these rich media essential genes present in all publicly available closed P. multocida genomes. In vivo fitness analysis identified 63 and 94 genes important for VP161 and M1404 survival in BALB/c mice, respectively. Only 35 homologs were identified in both strains as important for survival, showing that conserved biological systems can be differentially important for different P. multocida strains. Investigation of proteins involved in the catabolite response showed that an active cyclic-adenosine monophosphate (cAMP) receptor protein (CRP) was required for maximal fitness in M1404. Furthermore, disrupting CRP or cAMP production also reduced capsule production in M1404, but increased capsule production in VP161, showing different strains of P. multocida have different regulatory systems for crucial virulence factors.
Project description:The Gram-negative pathogen Pasteurella multocida is responsible for many important animal diseases. While a number of P. multocida virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. One mechanism by which bacteria regulate transcript abundance and protein production is riboregulation, which involves the interaction of a small RNA (sRNA) with a target mRNA to alter transcript stability and/or translational efficiency. This interaction often requires stabilization by a ribosome binding protein such as ProQ or Hfq. In E. coli and other species, ProQ has been shown to play a critical role in stabilizing sRNA-mRNA interactions and preferentially binds to 3’ stem-loop regions of the mRNA transcripts, characteristic of intrinsic transcriptional terminators. The aim of this study was to determine the role of ProQ riboregulation in P. multocida and identify the RNA regions to which it binds. We assessed differentially expressed transcripts in a proQ mutant and identified sites of direct ProQ-RNA interaction using in vivo UV-crosslinking and analysis of cDNA (CRAC). These analyses demonstrated that ProQ binds to, and stabilises, ProQ-dependant sRNAs and transfer RNAs in P. multocida via adenosine enriched, highly structured sequences. The binding of ProQ to two RNA molecules was characterised and showed that ProQ bound within the coding sequence of the uncharacterized PmVP161_1121 and within the 3’ region of the sRNA Prrc13.