Functinal Characterization Of The Omega Subunit Of The RNA Polymerase Of Xanthomonas Campestris Pv. Campestris

Omega(ω),the smallest subunit of bacterial RNA polymerase(RNAP),is encoded by rpoZ,and is wildely distributed and highly conserved in various bacteria species.Since few phenotypic alterations were obsurved in the rpoZ mutant,it is belived thatωis not important in bacteria.For this reason,up to date,the exact role ofωin bacteria including animal and plant bacterial pathogens is largely unknown.Xanthomonas campestris pv.campestris(here after Xcc)is the causal agent of the black rot disease of cruciferous plants,and is the model bacteria for studying the interaction between pathogen and its plant host.Genome sequencing and annotation showed that,in the genome of Xcc strain 8004 (Xcc8004),the gene rpoZ(ORF number XC0913)encodes theωsubunit of the Xcc RNAP.To investigte the function of the co subunit of Xcc RNAP,in this study,the nonpolar mutant of rpoZ gene,named NK0913,was constructed by homologous suicide plasmid pK18mob integration and the phenotypes of the mutant were analyed.The results showed that,(ⅰ)NK0913 shows a reduced growth rate in the rich medium NYG and the minimal medium MMX compare to the wild type strain 8004;(ⅱ)NK0913 can not induce a hypersensitive reaction(HR)in the nonhost plant pepper ECW-10R,and a significant reduced virulence in the host plant Chinese radish(Raphanus sativus L.var.radiculus Pers.);(ⅲ)NK0913 displyed a reduction in exopolysaccharides(EPS) production,extracellular enzyme(cellulose,protease and amylase)activity and cell motility;(ⅳ)although the wild type train does not form aggregates in LB medium,NK0913 grows in aggregated fashion in the medium.Introduction of the plasmid pLALR3 carrying rpoZ gene into NK0913 can restore all of the phenotypes of the mutant.,These results reveal that theωsubunit plays an important role in cell growth,pathogenesis,EPS production,extracellular enzyme activity and cell motility of Xcc.To investigate the exact role ofωsubunit,an RT-PCR and the GUS-promoter transcriptional fusion reporter analysis were performed to determine the regulatory relation between rpoZ gene and hrp genes.The result of GUS-promoter analysis showed that the expression of the hrp operons hrpB, hrpC and hrpF,and the two key hrp regulatory genes hrpG and hrpX is singnificantly reduced in the rpoZ mutant NK0913.The RT-PCR results showed that the expression of hrpB5,hpaP,hrcQ,hrpD6,hpaA,hpaB,hpa1,hpa2 and hrpW is reduced but the hrpF is inncreased in NK0913.These results suggested thatωsubunit is involved in virulence and HR through regulating the expression of hrp genes. We further used RT-PCR analysis to investigate the regulatory relation between rpoZ and the genes involved in flagellum synthesis.The results showed that the expression of motA,fliD,fliE and fliL is reduced in the rpoZ mutant NK0913,indicating thatωsubunit is involved in cell motility through regulating the expression of these flagellum synthesis-related genes.