Extracellular Amylase Is Required for Full Virulence and Regulated by the Global Posttranscriptional Regulator RsmA in<i>Xanthomonas campestris</i>Pathovar<i>campestris</i>

Abstract

As with many phytopathogenic bacteria, the virulence of <i>Xanthomonas campestris</i> pv. <i>campestris</i>, the causal agent of black rot disease in cruciferous plants, relies on secretion of a suite of extracellular enzymes that includes cellulase (endoglucanase), pectinase, protease, and amylase. Although the role in virulence of a number of these enzymes has been assessed, the contribution of amylase to <i>X. campestris</i> pv. <i>campestris</i> virulence has yet to be established. In this work, we investigated both the role of extracellular amylase in <i>X. campestris</i> pv. <i>campestris</i> virulence and the control of its expression. Deletion of <i>XC3487</i> (here renamed <i>amyA_Xcc</i>), a putative amylase-encoding gene from the genome of <i>X. campestris</i> pv. <i>campestris</i> strain 8004, resulted in a complete loss of extracellular amylase activity and significant reduction in virulence. The extracellular amylase activity and virulence of the <i>amyA_Xcc</i> mutant could be restored to the wild-type level by expressing <i>amyA_Xcc</i> in trans. These results demonstrated that <i>amyA_Xcc</i> is responsible for the extracellular amylase activity of <i>X. campestris</i> pv. <i>campestris</i> and indicated that extracellular amylase plays an important role in <i>X. campestris</i> pv. <i>campestris</i> virulence. We also found that the expression of <i>amyA_Xcc</i> is strongly induced by starch and requires activation by the global posttranscriptional regulator RsmA. RsmA binds specifically to the 5'-untranslated region of <i>amyA_Xcc</i> transcripts, suggesting that RsmA regulates <i>amyA_Xcc</i> directly at the posttranscriptional level. Unexpectedly, in addition to posttranscriptional regulation, the use of a transcriptional reporter demonstrated that RsmA also regulates <i>amyA_Xcc</i> expression at the transcriptional level, possibly by an indirect mechanism.