A putative glucose 6-phosphate isomerase has pleiotropic functions on virulence and other mechanisms in Acidovorax citrulli.

Acidovorax citrulli (Ac) is a causal agent of watermelon bacterial fruit blotch (BFB) disease. Because resistance cultivars/lines have not yet been developed, it is imperative to elucidate Ac's virulence factors and their mechanisms to develop resistant cultivars/lines in different crops, including watermelon. The glucose-6-phosphate isomerase (GPI) is a reversible enzyme in both glycolysis and gluconeogenesis pathways in living organisms. However, the functions of GPI are not characterized in Ac. In this study, we determined the roles of GpiAc (GPI in Ac) by proteomic and phenotypic analyses of the mutant lacking GPI. The mutant displayed significantly reduced virulence to watermelon in two different virulence assays. The mutant's growth patterns were comparable to the wild-type strain in rich medium and M9 with glucose but not with fructose. The comparative proteome analysis markedly identified proteins related to virulence, motility, and cell wall/membrane/envelope. In the mutant, biofilm formation and twitching halo production were reduced. We further demonstrated that the mutant was less tolerant to osmotic stress and lysozyme treatment than the wild-type strain. Interestingly, the tolerance to alkali conditions was remarkably enhanced in the mutant. These results reveal that GpiAc is involved not only in virulence and glycolysis/gluconeogenesis but also in biofilm formation, twitching motility, and tolerance to diverse external stresses suggesting the pleiotropic roles of GpiAc in Ac. Our study provides fundamental and valuable information on the functions of previously uncharacterized glucose 6-phosphate isomerase and its virulence mechanism in Ac.

Proteomic and Phenotypic Analyses of a Putative YggS Family Pyridoxal Phosphate-Dependent Enzyme in Acidovorax citrulli.

Acidovorax citrulli (Ac) is a phytopathogenic bacterium that causes bacterial fruit blotch (BFB) in cucurbit crops, including watermelon. However, there are no effective methods to control this disease. YggS family pyridoxal phosphate-dependent enzyme acts as a coenzyme in all transamination reactions, but its function in Ac is poorly understood. Therefore, this study uses proteomic and phenotypic analyses to characterize the functions. The Ac strain lacking the YggS family pyridoxal phosphate-dependent enzyme, AcΔyppAc(EV), virulence was wholly eradicated in geminated seed inoculation and leaf infiltration. AcΔyppAc(EV) propagation was inhibited when exposed to L-homoserine but not pyridoxine. Wild-type and mutant growth were comparable in the liquid media but not in the solid media in the minimal condition. The comparative proteomic analysis revealed that YppAc is primarily involved in cell motility and wall/membrane/envelop biogenesis. In addition, AcΔyppAc(EV) reduced biofilm formation and twitching halo production, indicating that YppAc is involved in various cellular mechanisms and possesses pleiotropic effects. Therefore, this identified protein is a potential target for developing an efficient anti-virulence reagent to control BFB.

Comparative Proteomic Analysis for a Putative Pyridoxal Phosphate-Dependent Aminotransferase Required for Virulence in Acidovorax citrulli.

Acidovorax citrulli (Ac) is the causative agent of bacterial fruit blotch disease in watermelon. Since resistant cultivars have not yet been developed, the virulence factors/mechanisms of Ac need to be characterized. This study reports the functions of a putative pyridoxal phosphate-dependent aminotransferase (PpdaAc) that transfers amino groups to its substrates and uses pyridoxal phosphate as a coenzyme. It was observed that a ppdaAc knockout mutant had a significantly reduced virulence in watermelon when introduced via germinated-seed inoculation as well as leaf infiltration. Comparative proteomic analysis predicted the cellular mechanisms related to PpdaAc. Apart from causing virulence, the PpdaAc may have significant roles in energy production, cell membrane, motility, chemotaxis, post-translational modifications, and iron-related mechanisms. Therefore, it is postulated that PpdaAc may possess pleiotropic effects. These results provide new insights into the functions of a previously unidentified PpdaAc in Ac.

Two DNA Methyltransferases for Site-Specific 6mA and 5mC DNA Modification in Xanthomonas euvesicatoria.

Xanthomonas euvesicatoria (Xe) is a gram-negative phytopathogenic bacterium that causes bacterial spot disease in tomato/pepper leading to economic losses in plantations. DNA methyltransferases (MTases) are critical for the survival of prokaryotes; however, their functions in phytopathogenic bacteria remain unclear. In this study, we characterized the functions of two putative DNA MTases, XvDMT1 and XvDMT2, in Xe by generating XvDMT1- and XvDMT2-overexpressing strains, Xe(XvDMT1) and Xe(XvDMT2), respectively. Virulence of Xe(XvDMT2), but not Xe(XvDMT1), on tomato was dramatically reduced. To postulate the biological processes involving XvDMTs, we performed a label-free shotgun comparative proteomic analysis, and results suggest that XvDMT1 and XvDMT2 have distinct roles in Xe. We further characterized the functions of XvDMTs using diverse phenotypic assays. Notably, both Xe(XvDMT1) and Xe(XvDMT2) showed growth retardation in the presence of sucrose and fructose as the sole carbon source, with Xe(XvDMT2) being the most severely affected. In addition, biofilm formation and production of exopolysaccharides were declined in Xe(XvDMT2), but not Xe(XvDMT1). Xe(XvDMT2) was more tolerant to EtOH than Xe(XvDMT1), which had enhanced tolerance to sorbitol but decreased tolerance to polymyxin B. Using single-molecule real-time sequencing and methylation-sensitive restriction enzymes, we successfully predicted putative motifs methylated by XvDMT1 and XvDMT2, which are previously uncharacterized 6mA and 5mC DNA MTases, respectively. This study provided new insights into the biological functions of DNA MTases in prokaryotic organisms.

Proteomic and Phenotypic Analyses of a Putative Glycerol-3-Phosphate Dehydrogenase Required for Virulence in Acidovorax citrulli.

Acidovorax citrulli (Ac) is the causal agent of bacterial fruit blotch (BFB) in watermelon, a disease that poses a serious threat to watermelon production. Because of the lack of resistant cultivars against BFB, virulence factors or mechanisms need to be elucidated to control the disease. Glycerol-3-phosphate dehydrogenase is the enzyme involved in glycerol production from glucose during glycolysis. In this study, we report the functions of a putative glycerol-3-phosphate dehydrogenase in Ac (GlpdAc) using comparative proteomic analysis and phenotypic observation. A glpdAc knockout mutant, AcΔglpdAc(EV), lost virulence against watermelon in two pathogenicity tests. The putative 3D structure and amino acid sequence of GlpdAc showed high similarity with glycerol-3-phosphate dehydrogenases from other bacteria. Comparative proteomic analysis revealed that many proteins related to various metabolic pathways, including carbohydrate metabolism, were affected by GlpdAc. Although AcΔglpdAc(EV) could not use glucose as a sole carbon source, it showed growth in the presence of glycerol, indicating that GlpdAc is involved in glycolysis. AcΔglpdAc(EV) also displayed higher cell-to-cell aggregation than the wild-type bacteria, and tolerance to osmotic stress and ciprofloxacin was reduced and enhanced in the mutant, respectively. These results indicate that GlpdAc is involved in glycerol metabolism and other mechanisms, including virulence, demonstrating that the protein has pleiotropic effects. Our study expands the understanding of the functions of proteins associated with virulence in Ac.

Putative Bifunctional Chorismate Mutase/Prephenate Dehydratase Contributes to the Virulence of Acidovorax citrulli.

Acidovorax citrulli (Ac) is a plant pathogenic bacterium that causes bacterial fruit blotch (BFB) in cucurbit crops. Despite its importance in the cucurbit industry, resistant cultivars/lines against BFB have not yet been identified. Therefore, there is a need to characterize the virulence factors/mechanisms in Ac to control the disease. Chorismate mutase, a key enzyme in the shikimate pathway, produces aromatic amino acids. Here, we report the functions of putative bifunctional chorismate mutase/prephenate dehydratase in Ac (CmpAc) determined by proteomic analysis and phenotypic assays. Ac strain lacking CmpAc, AcΔcmpAc(EV), were significantly less virulent on watermelon in the germinated-seed inoculation and leaf infiltration assays. Sequence analysis revealed that CmpAc possesses two distinct domains: chorismate mutase and prephenate dehydratase, indicating that CmpAc is a bifunctional protein. Auxotrophic assays demonstrated that CmpAc is required for the biosynthesis of phenylalanine, but not tyrosine. The comparative proteomic analysis revealed that CmpAc is mostly involved in cell wall/membrane/envelop biogenesis. Furthermore, AcΔcmpAc(EV) showed reduced twitching halo production and enhanced biofilm formation. In addition, AcΔcmpAc(EV) was less tolerant to osmotic stress but more tolerant to antibiotics (polymyxin B). Thus, our study provides new insights into the functions of a putative bifunctional protein related to virulence in Ac.