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.
Highlights
Xanthomonas campestris pv. campestris (Xcc) is the pathogen of black rot disease, which is considered the most destructive disease of crucifers, including all cultivated varieties of brassicas worldwide (Vicente and Holub, 2013)
The GH35 proteins in Xcc are homologous to the Bga of X. axonopodis pv. manihotis (Figures 1B and Figure S1), which is a known β-galactosidase(Yang et al, 2003)
These data suggest that GH2 proteins have diverse functions but that GH35 proteins might have β-galactosidase activities in Xcc
Summary
Xanthomonas campestris pv. campestris (Xcc) is the pathogen of black rot disease, which is considered the most destructive disease of crucifers, including all cultivated varieties of brassicas worldwide (Vicente and Holub, 2013). There are only three annotated galactosidase genes (designated galA, galB, and galC) in the Xcc genome of 8004 and ATCC 33913 strains (da Silva et al, 2002; Qian et al, 2005). GalA and GalB are similar to glycosyl hydrolase (GH) family 2 enzymes and have no detectable activities in Xcc Xc17 (Yang et al, 2003). Manihotis Bga and has β-galactosidase activity in Xcc Xc17 (Yang et al, 2003). Mutations of galA, galB, and galC did not reduce the ability to grow on lactose or the βgalactosidase activity in Xcc Xc17L, a strain with elevated βgalactosidase activity, indicating that these three genes do not encode the main β-galactosidase in Xc17L (Yang et al, 2003).
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