Transcriptional regulation and molecular characterization of the manA gene encoding the biofilm dispersing enzyme mannan endo-1,4-beta-mannosidase in Xanthomonas campestris

Abstract

Exopolysaccharide and several extracellular enzymes of Xanthomonas campestris pv. campestris (Xcc), the causative agent of black rot in crucifers, are important virulence determinants. It is known that Clp (cAMP receptor protein-like protein) and RpfF (an enoyl-CoA hydratase homologue required for the synthesis of diffusible signal factor, DSF) regulate the production of these determinants. Addition of DSF or Xcc extracellular protein containing partially purified mannanase (EC 3.2.1.78, encoded by manA) can disperse the cell aggregates formed by rpfF mutant. In this study, nucleotide G 64 nt upstream of the manA translation start codon was determined as the transcription initiation site by the 5' RACE technique. Transcriptional fusion assays showed that manA transcription is positively regulated by Clp and RpfF and induced by locust bean gum. The manA coding region was cloned and expressed in E. coli as recombinant ManA (rManA). The rManA was purified by affinity chromatography, and its biochemical properties were characterized. The rManA had a pH optimum at 7.0 (0.1 M Hepes) and a temperature optimum at about 37 degrees C. Sequence and mutational analyses demonstrated that Xcc manA encodes the major mannanase, a member of family 5 of glycosyl hydrolases. This study not only extends previous work on Clp and RpfF regulation by showing that they both influence the expression of manA in Xcc, but it also for the first time characterizes Xanthomonas mannanase at the protein level.