Determination of the small RNA GcvB regulon in the Gram-negative bacterial pathogen Pasteurella multocida and identification of the GcvB seed binding region

Pasteurella multocida is a Gram-negative bacterium 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. Small RNA (sRNA) molecules are critical regulators that act by binding to specific mRNA targets, often in association with the RNA chaperone protein Hfq. In this study, transcriptomic analysis of the P. multocida strain VP161 revealed a putative sRNA with high identity to GcvB from Escherichia coli and Salmonella enterica serovar Typhimurium. High-throughput quantitative liquid proteomics was used to compare the proteomes of the P. multocida VP161 wild-type strain, a gcvB mutant, and a GcvB overexpression strain. These analyses identified 46 proteins that displayed significant differential production after inactivation of gcvB, 36 of which showed increased production. Of the 36 proteins that were repressed by GcvB, 27 were predicted to be involved in amino acid biosynthesis or transport. Bioinformatic analyses of putative P. multocida GcvB target mRNAs identified a strongly conserved 10 nucleotide consensus sequence, 5′-AACACAACAT-3′, with the central eight nucleotides identical to the seed binding region present within GcvB mRNA targets in E. coli and S. Typhimurium. Using a defined set of seed region mutants, together with a two-plasmid reporter system that allowed for quantification of sRNA–mRNA interactions, this sequence was confirmed to be critical for the binding of the P. multocida GcvB to the target mRNA, gltA.