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Abstract
Xanthomonas axonopodis pv. glycines (Xag) is a Gram-negative bacterium that causes bacterial pustule disease in soybean. To acclimate to new environments, the expression of genes in bacteria is controlled directly or indirectly by diverse transcriptional factors. Among them, LysR type transcriptional regulators are well-characterized and abundant in bacteria. In a previous study, comparative proteomic analysis revealed that LysR type carbohydrate-related transcriptional regulator in Xag (LcrX) was more abundant in XVM2, which is a minimal medium, compared with a rich medium. However, the functions of LcrX in Xag have not been characterized. In this study, we generated an LcrX-overexpressing strain, Xag(LcrX), and the knockout mutant strain, XagΔlcrX(EV), to elucidate the functions of LcrX. Bacterial multiplication of Xag(LcrX) in soybean was significantly impaired, indicating that LcrX is related to virulence. Comparative proteomic analysis revealed that LcrX is mainly involved in carbohydrate metabolism/transport and inorganic ion transport/metabolism. Based on the results of proteomics analysis, diverse phenotypic assays were carried out. A gel electrophoresis mobility shift assay demonstrated that LcrX specifically bound to the putative promoter regions of genes encoding putative fructose 1,6-bisphosphatase and protease. Through a 96-well plate assay under various conditions, we confirmed that the growth of Xag(LcrX) was dramatically affected in the presence of various carbon sources, while the growth of XagΔlcrX(EV) was only slightly changed. Biofilm formation activity was reduced in Xag(LcrX) but enhanced in XagΔlcrX(EV). The production of siderophores was also decreased in Xag(LcrX) but not altered in XagΔlcrX(EV). In contrast, LcrX was not associated with exopolysaccharide production, protease activity, or bacterial motility. These findings provide new insights into the functions of a carbohydrate-related transcriptional regulator in Xag.
Highlights
Xanthomonas axonopodis pv. glycines (Xag) is a Gram-negative plant pathogenic bacterium that causes bacterial pustule disease, one of the most devastating and economically important diseases in soybeans (Athinuwat et al, 2009)
High homology was observed between LysR type carbohydrate-related transcriptional regulator in Xag (LcrX) and CAJ25335 (297/300, 99%), AAM38319 (300/300 100%), and AXQ50657 (211/300, 70%) in X. campestris, X. citri, and Stenotrophomonas rhizophila, respectively (Figure 1C), indicating that LcrX is conserved in Xanthomonas spp. and closely related genera
LcrX exhibited low homology with CynR related to the cyanate metabolism (78/300, 26%) (Lamblin and Fuchs, 1994), OxyR involved in an oxidative stress (62/300, 20%), (Ochsner et al, 2000), and CcmR associated with the inorganic carbon concentrating mechanism (67/300, 22%) (Woodger et al, FIGURE 1 | Predicted three-dimensional structure and sequence alignment of LcrX. (A) Deduced amino acid sequence of LcrX where blue and red boxes indicate DNA-binding domain and LysR activation domain, respectively. (B) Predicted 3D structure obtained using the protein modeling server I-TASSER and PyMOL program. (C) Comparison of amino acid sequences of LcrX with its homologs in Xanthomonas spp. and Stenotrophomonas spp. using the ClustalOmega program
Summary
Xanthomonas axonopodis pv. glycines (Xag) is a Gram-negative plant pathogenic bacterium that causes bacterial pustule disease, one of the most devastating and economically important diseases in soybeans (Athinuwat et al, 2009). Pustules on the leaves appear with rugged surfaces and chlorotic halos. This disease reduced the size and quantity of seeds (Narvel et al, 2001; Darrasse et al, 2013). TFs directly or indirectly regulate expression of one or several genes and numerous genes, eventually leading to the global regulation of gene expression (Browning and Busby, 2004; Geertz and Maerkl, 2010). CRP is associated with global regulation of gene expression in E. coli by controlling 22 different TFs. global gene expression directly or indirectly regulated by TFs can control bacterial behaviors and phenotypic alterations
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