Identification Of A Pair Of Hypothetical Two -Component Signal Transduction System Genes In Xanthomonas Campestris PV. Campestris

Xanthomonas campestris pv. campestris(Xcc), belonging to the y-subdivision of the proteobacteria, can infect almost all members of the crucifer family in the worldwide, which is one of the model organisms in studying plant-pathogen interactions. Two-component signal transduction system (TCSs) is one of the important signal transduction systems in bacteria. Typically, this system is comprised of a histidine kinase (HK) that receives the input stimuli and a response regulator (RR) that effects an appropriate change in cellular physiology. Genome analysis showed that Xcc 8004 strain has a total of 111 two-component signal transduction system genes. There are still many genes functions of two-component signal transduction systems unclear, including XC3067 and XC3068, which consist of a pair of hypothetical two-component signal transduction system genes. Bioinformatics analysis showed that XC3068 located in the genome next to XC3067, has 14 bp overlapping with each other, at the 5'-end of XC3067 and the 3'-end of XC3068. They were predicted to be transcribed in the same direction. XC3067 was annotated to encode a histidine kinase/response regulator hybrid protein. And XC3068 encode a sensor protein. In the previous study, the Tn5gusA5 insertion in XC3068 gene impacted on pathogenicity. In order to study the biological functions of the two-component signal transduction system XC3067 and XC3068, we constructed XC3067 and XC3068 deletion mutants DM3067, DM3068 and the double mutant DM3067/3068 by using genes homologous double exchange. The results showed that the growth of DM3067, DM3068 and DM3067/3068 were identical to the wild-type both in minimal medium (MMX) and nutrient medium (NYG). And the production of extracellular polysaccharide, extracellular protease, extracellular amylase, extracellular cellulose and motility of the mutants were not influenced. In order to study sensing the environment signal in Xcc 8004, we test some stress, including osmotic pressure, organic solvent and protein denaturant. The results indicated that compared with the wild-type, DM3067, DM3068 and DM3067/3068 could only affect the adaptation to protein denaturant, but not affect osmotic pressure and organic solvent. The result of pathogenicity test in host plant Chinese radish showed that the virulence of DM3068 and DM3067/3068 decreased. Hypersensitive response test in non-host pepper showed that DM3067, DM3068 and DM3067/3068 had decreased hypersensitive response. The complementation strains of DM3067 and DM3068 can restore the phenotypes to the wild-type strain. The results suggest that XC3068 might contribute to the pathogenicity of the wild-type strain. XC3067 and XC3068 were required in hypersensitive response and protein denaturant tolerance of the wild-type strain. The reverse transcription polymerase chain reaction (RT-PCR) results showed that XCS067 and XC3068 were located in the same transcription unit, suggesting that they are likely to form a two-component signal transduction system. The RT-PCR results also showed that the expression of XC3067 and XC3068 in MMX medium were same to that in NYG medium. Both hrpG and hrpX could not regulate the expression of XC3067 and XC3068 in MMX medium.This study demonstrated that XC3068 is required for the full virulence of Xcc 8004. XC3067 and XC3068 are related to hypersensitive response and protein denaturing agent resistance. RT-PCR results showed that XC3067 and XC3068 were located in the same transcription unit. And the expression of XC3067 and XC3068 were not induced in MMX medium and were not regulated by hrpG and hrpX in MMX medium.