The role of PilZ domain proteins in the virulence of Xanthomonas campestris pv. campestris

Abstract

Cyclic di-GMP [(bis-(3'-5')-cyclic di-guanosine monophosphate)] is an almost ubiquitous second messenger in bacteria that is implicated in the regulation of a range of functions that include developmental transitions, aggregative behaviour, adhesion, biofilm formation and virulence. Comparatively little is known about the mechanism(s) by which cyclic di-GMP exerts these various regulatory effects. PilZ has been identified as a cyclic di-GMP binding protein domain; proteins with this domain are involved in regulation of specific cellular processes, including the virulence of animal pathogens. Here we have examined the role of PilZ domain proteins in virulence and the regulation of virulence factor synthesis in Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot of crucifers. The Xcc genome encodes four proteins (XC0965, XC2249, XC2317 and XC3221) that have a PilZ domain. Mutation of XC0965, XC2249 and XC3221 led to a significant reduction of virulence in Chinese radish. Mutation of XC2249 and XC3221 led to a reduction in motility whereas mutation of XC2249 and XC0965 affected extracellular enzyme production. All mutant strains were unaffected in biofilm formation in vitro. The reduction of virulence following mutation of XC3221 could not be wholly attributed to an effect on motility as mutation of pilA, which abolishes motility, has a lesser effect on virulence.

Figure 1

The effects of mutation of genes encoding the PilZ domain proteins XC0965, XC2249, XC2317 and XC3221 on the virulence of Xcc to Chinese radish. The virulence of each mutant was tested by measurement of the lesion length after bacteria were introduced into the vascular system of Chinese radish by leaf clipping. Values given are the means and SD of 60 measurements. Also shown are the effects of mutation of rpfG, which encodes an HD‐GYP domain regulatory protein and pilA encoding the major pilin. (A) Representative virulence assays for (from left to right) Xcc wild‐type strain 8004, negative control (H₂O), XC3221::Tn5gusA5 mutant, pilA mutant (XC3823::Tn5gusA5) and rpfG deletion mutant. (B) Mutation of XC3221, XC2249 and XC0965 gave a significant reduction in virulence in repeated tests, although mutation of XC2317 had no effect. Introduction of the cloned genes restores virulence of these mutants to wild‐type levels, but had no influence on the XC2317 mutant, which retained wild‐type virulence.

Figure 2

Mutation of genes encoding PilZ domain proteins has effects on motility and the synthesis of extracellular enzyme virulence factors by Xcc. (A) XC0965 and XC2317 mutants have lower levels of extracellular endoglucanase than wild‐type; the complemented strains XC0965/pXC0965 and XC2317/pXC2317 have wild‐type levels. The effects of mutation of XC0965 and XC2317 are much less than those seen after mutation of rpfG (which is XC2335), whereas mutation of pilA (which is XC3823) has no effect on endoglucanase activity. (B) Mutation of XC2317 led to a small reduction in protease production whereas mutation of XC0965, XC2249 and XC3221 had no apparent effect. (C) Mutation of XC2249 and XC3221 influence motility on 0.5% Eiken agar‐NYG plates. These effects are reversed by complementation with the cloned XC2249 and XC3221 genes respectively.

Figure 3

Effect of mutation of hrpG and hrpX on the expression of genes encoding PilZ domain proteins. Transcript levels of all genes were measured by RT‐PCR. Bacterial strains were grown to an OD at 600 nm of 1.5 in MME medium before RNA extraction. All RT‐PCR results were normalized using the Ct values obtained for the 16S rRNA amplifications run in the same plate. The relative amounts of selected genes and relative levels of gene transcripts were determined by a standard curve (i.e. Ct values plotted against logarithm of the DNA copy number). The results shown are averages of three repetitions.