多元轉錄調控因子Clp對Xanthomonas campestris pv. campestris β-lactamase表現之調控

Abstract

The Gram-negative plant pathogen Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers. The presence of a β-lactamase gene has been demonstrated to be widespread among Xcc. Recently, it was found that the bla gene expression can be regulated by a global transcriptional regulator, Clp (cAMP receptor protein-like protein). In this study, we explored to understand the mechanism of Xcc β-lactamase expression regulated by Clp. At first, we found that the growth rate of Xc17 clp mutant, TC817, was faster than that of Xc17 and could be suppressed by ampicillin, but no growth suppression was found in Xc17. Next, we investigated the effects of XCC1539 (ampDI), XCC3805 (ampDII), nagZ, ampR, and bla genes on Xc17 β-lactamase expression by RT-PCR and Western blotting. AmpDI and AmpDII, suggested to be a negative regulator in bla expression, are amidase 2 that can hydrolyze the products of peptidoglycan. AmpR is a transcriptional regulator of bla expression. Based on this study, we found that (1) Bla expression was induced by ampicillin in TC817, however, it was constitutively expressed in Xc17, (2) the transcript level of ampDII in Xc17 was higher than that in TC817. Bla expression was not able to be induced by ampicillin in TC817 overexpressed with AmpDII. Although there are two putative Clp-binding sites upstream of ampDII, its promoter activity is very weak in wild type and clp mutant, (3) the induction ability of Bla by ampicillin was suppressed when MurG protein was overexpressied in TC817, (4) the transcription and translation levels of ampR were low in Xc17, but high in TC817. Nevertheless, adding ampicillin to TC187 medium would reduce the expression of ampR, (5) no significant differences were found of the expression of ampDI and nagZ between Xc17 and TC817. We also analyzed the protein expression of TC817 with or without ampicillin for culture by two-dimensional gel electrophoresis. We detected several differential protein spots, and further identifications are reguired. Based on the results of this study we suggested a model for the Clp-regulated β-lactamase expression in Xc17. The loss of Xcc Clp protein will exert a global influence on gene expression, including peptidoglycan synthesis genes, like ampDII, murG, and ampR. Thus, the levels of inducer and repressor for AmpR could be changed, and the rafio of inactive/active form of AmpR adjueted, leading to various levels of Bla expression.