Genome-Wide Analysis of β-Galactosidases in Xanthomonas campestris pv. campestris 8004

Abstract

Bacterial β-galactosidase is involved in lactose metabolism and acts as a prevalent reporter enzyme used in studying the activities of prokaryotic and eukaryotic promoters. Xanthomonas campestris pv. campestris (Xcc) is the pathogen of black rot disease in crucifers. β-Galactosidase activity can be detected in Xcc culture, which makes Escherichia coli LacZ unable to be used as a reporter enzyme in Xcc. To systemically understand the β-galactosidase in Xcc and construct a β-galactosidase -deficient strain for promoter activity analysis using LacZ as a reporter, we here analyzed the putative β-galactosidases in Xcc 8004. As glycosyl hydrolase (GH) family 2 (GH2) and 35 (GH35) family enzymes were reported to have beta-galactosidase activities, we studied all of them encoded by Xcc 8004. When expressed in E. coli, only two of the enzymes, XC1214 and XC2985, were found to have β-galactosidase activity. When deleted from the Xcc 8004 genome, only the XC1214 mutant had no β-galactosidase activity, and other GH2 and GH35 gene deletions resulted in no significant reduction in β-galactosidase activity. Therefore, XC1214 is the main β-galactosidase in Xcc 8004. Notably, we have constructed a β-galactosidase-free strain that can be employed in gene traps using LacZ as a reporter in Xcc. The results reported herein should facilitate the development of high-capacity screening assays that utilize the LacZ reporter system in Xcc.

The RavA/VemR two‐component system plays vital regulatory roles in the motility and virulence of Xanthomonas campestris

“…However, RpoN2 was recently reported to be vital for the virulence of the Xc17 strain (Li, Wang, et al, 2020). To further determine the roles of rpoN2 , vemR , and fleQ in the interaction between Xcc and host plants, we constructed single (Δ rpoN2 , Δ vemR , Δ fleQ ), double (Δ rpoN2 /Δ vemR , Δ vemR /Δ fleQ , Δ rpoN2 /Δ fleQ ), and triple (Δ rpoN2 /Δ vemR /Δ fleQ ) deletion mutants in Xcc strain Δ lac8 (Δ lac8 , a derivative strain of Xcc 8004, has no β‐galactosidase activity but has the same other phenotypes as Xcc 8004, including virulence, motility, EPS production, biofilm formation, and growth rate; hereafter, we refer to Δ lac8 as the wild‐type [WT] strain) (Wang et al, 2018). Then, we compared the virulence of these strains on Brassica oleracea ‘Wenxin'.…”

“…To understand whether VemR and RavA play roles in the regulation of fliC expression, we used promoter‐reporter constructs to detect fliC expression levels. We constructed a fliC p ‐ lac Z fusion strain based on Δ lac8 (Wang et al, 2018) that could directly reflect the expression levels of fliC on plates containing X‐gal (5‐bromo‐4‐chloro‐3‐indolyl β‐ d ‐galactoside). When rpoN2 or fleQ was deleted, the colonies did not exhibit a blue colour (Figure ).…”

“…The promoter of fliC was fused with the lac Z gene and integrated into the β‐galactosidase‐deficient strain Δ lac8 to construct the reporter strain Δ lac8‐fliC‐lacZ (Wang et al, 2018). To construct the ravA deletion mutant, c.500 bp fragments flanking ravA were amplified and then cloned together into the Hin dIII/ Xba I sites of pK18 mobsacB (Schäfer et al, 1994).…”

“…β‐Galactosidase ( LacZ ) activity analysis was carried out as previously described (Griffith & Wolf, 2002; Smale, 2010; Wang et al, 2018). For plate assays, 1 µl of diluted cell culture was inoculated on NYG plates containing 40 mg/L X‐gal.…”