Expression and function of clpS and clpA in Xanthomonas campestris pv. campestris

Abstract

ATP-dependent proteases (FtsH, Lon, and Clp family proteins) are ubiquitous in bacteria and play essential roles in many regulatory cell processes. Xanthomonas campestris pv. campestris is a Gramnegative pathogen and can cause black rot diseases in crucifers. The genome of X. campestris pv. campestris have several clp genes, namely clpS, clpA, clpX, clpP, clpQ, and clpY. Among them, only clpX and clpP were known to be required for pathogenicity. Here, we focused on two uncharacterized clp genes (clpS and clpA) that encode the adaptor (ClpS) and ATPase subunit (ClpA) of the ClpAP protease complex, respectively. Transcriptional analysis revealed that expression of both clpS and clpA is induced by heat shock. Inactivation of clpA but not clpS resulted in susceptibility to high temperature and revealed an attenuation of virulence on the host plant. The altered phenotypes of the clpA mutant could be complemented in trans. Site-directed mutagenesis revealed that K223 and K504 are critical amino acid residues for ClpA function in heat tolerance. The clpA mutant revealed different protein expression pro les as compared to the wild type in response to heat stress. Taken together, we characterized two clp genes (clpS and clpA) by examining their expression and function including stress tolerance and pathogenicity. We demonstrated that both clpS and clpA are expressed in a temperature dependent manner, and clpA is required for survival at high temperature and full virulence of X. campestris pv. campestris. This is the rst time that clpS and clpA has been characterized in Xanthomonas.