Study On The Rna Chaperone Hfq In Xanthomonas Campestris Pathovar Campestris

Xanthomonas campestris pv.campestris(Xcc)is the causal agent of black rot disease of many important cruciferous crops including broccoli,cabbage,cauliflower and is a model bacteria for studying the molecular mechanism of plant-pathogen interactions.Previous studies in our lab revealed that HfqXcx,a protein displaying high amino acid sequence similarity to the bacterial RNA chaperone Hfq,possibly plays an important role in the virulence and cell motility in Xcc strain 8004(Xcc 8004).The aim of this work is to clarify the biological and biochemical function of HfqXcc,and to understand the mechanism by which HfqXcc functioning.Phenotypic analysis of the hfqXcc deletion mutant and its complementation strain showed that deletion of hfqXcc resulted in dramatically reduced virulence,significantly decreased virulence factor production,impaired cell motility,and decreased high osmotic stress tolerance.These phenotypes can be complemented by hfq in trans,demonstrating that HfqXcc plays a critical role in virulence,virulence factor production,cell motility and stress tolerance of Xcc.We further found that deletion of hfqXcc resulted in a severe decrease in the accumulation of the small RNA sRXcc-141,although the transcription of sRXcc-141 has not been affected,indicating that HfqXcc stabilizes sRXcc-141 inside the cell.Furthermore,Co-IP experiment revealed that HfqXcc specifically bind to sRXcc-141 in vivo.These results together demonstrate that HfqXcc acts as an RNA chaperone in Xcc.Hfq is a small protein composed of about 100 amino acid residues.Bioinformatics analysis show that the 1?66 amino acid sequence at the N terminus region of Hfq is highly conserved,while the amino acid residues at C terminus region(67?100)is highly variable in different bacteria.Previous studies showed that deletion of the C terminus region of Hfq did not affect its biological functions.In contrast,our results show that the C terminus region of HfqXcc is indispensable for its function.To understand why deletion of hfqXcc lead to the observed phenotype changes in Xcc,we carried out transcriptome and proteome analysis to identify gene expression changes caused by hfqXcc deletion and accordingly determine the regulon of HfqXcc Transcriptome analysis show that 51 genes are regulated by HfqXcc(23 genes are positively regulated and 28 genes are negatively regulated by HfqXcc)under the condition of growing in the rich medium.The 51 HfqXcc-regulated genes includes 10 functional catalogues such as pathogenesis and adaptation,cell membrane and cell structure,energy and carbon metabolism,signal transduction,translation,transportation.And proteome analysis show that the expression of 157 proteins were regulated by HfqXcc(122 proteins are positively regulated and 35 are negatively regulated by HfqXcc).The 157 HfqXcc-regulated proteins includes 18 functional catalogues including intermediate metabolism,energy and carbon metabolism,pathogenesis and adaptation,translation and transport,cell membrane and cell structure,cell processes,coenzyme factors and carrier synthesis,fatty acid and phospholipid metabolism,regulation and transcription.These results demonstrated that HfqXcc acts as a global regulator in Xcc.But,unfortunately,the results of transcriptome and proteome analysis can not provide a useful information for reconstruction of the direct pathway that HfqXcc functioning.Based on the fact that deletion of hfqXcc resulted in decreased extracellular polysaccharide(EPS)production,and that gum operon has been shown to be essential for EPS synthesis,we used Northern blotting analysis to compare the mRNA level of gum operon in wild type strain and the hfqxcc deletion mutant.The result show that compared to the wild-type strain,the mRNA level of all gum genes was severely reduced in the hfqxcc deletion mutant,demonstrating that Hfqxcc positively regulates the expression of the gum operon.Since HfqXcc neither directly bind to the promoter of the gum operon nor to the 5'-UTR of the gum operon mRNA,it is very likely that HfqXcc regulates the gum operon indirectly.In summary,this work investigated the biological and biochemical function of HfqXcc as well as the mechanism by which HfqXcc functioning in Xcc.The data demonstrated that HfqXcc acts as an RNA chaperone and plays an important role in virulence,cell motility and stress tolerance of Xcc.These results broaden our understanding of the virulence regulatory network of Xcc.