Yield and rheological properties of exopolysaccharide from a local isolate: Xanthomonas axonopodis pv. vesicatoria

Background: The aim of the present study was to evaluate gum productivity of a local strain, Xanthomonas axonopodis pv. vesicatoria, isolated from pepper plant, and its rheological behavior for the first time compared to the standard strain, Xanthomonas campestris DSM 19000 (NRRL B-1459). The influence of operational conditions (agitation rate and inoculum volume) on gum production and rheological properties of gums from the Xanthomonas strains were investigated. Results: The isolated strain of Xanthomonas showed similar xanthan yield compared to the standard strain. Furthermore, this study clearly confirmed that gum yield depended on bacterial strain, agitation rate, and inoculum size. The most suitable conditions for the gum production in an orbital shaker in terms of agitation rate and inoculum size were 180 rpm and 5%, respectively, resulting in an average production of 10.96 and 11.19 g/L for X. axonopodis pv.vesicatoria and X. campestris DSM 19000, respectively. Regarding the rheological properties, Ostwald-de-Waele and power law models were used to describe flow and oscillatory behavior of the gum solutions, respectively. Consistency of the novel gum solution remarkably was much higher than the commercial xanthan gum solution. Flow and oscillatory behavior and their temperature ramps showed that weak gel-like structure could be obtained with less gum concentrations when the novel gum was used. Conclusion: Therefore, yield and technological properties of the aqueous solutions of the exopolysaccharide synthesized by X. axonopodis pv. vesicatoria were observed to be more suitable for industrial production.

Functional Analysis of the Ferric Uptake Regulator Gene fur in Xanthomonas vesicatoria.

Iron is essential for the growth and survival of many organisms. Intracellular iron homeostasis must be maintained for cell survival and protection against iron toxicity. The ferric uptake regulator protein (Fur) regulates the high-affinity ferric uptake system in many bacteria. To investigate the function of the fur gene in Xanthomonas vesicatoria (Xv), we generated a fur mutant strain, fur-m, by site-directed mutagenesis. Whereas siderophore production increased in the Xv fur mutant, extracellular polysaccharide production, biofilm formation, swimming ability and quorum sensing signals were all significantly decreased. The fur mutant also had significantly reduced virulence in tomato leaves. The above-mentioned phenotypes significantly recovered when the Xv fur mutation allele was complemented with a wild-type fur gene. Thus, Fur either negatively or positively regulates multiple important physiological functions in Xv.

Functional and proteomic analyses reveal that wxcB is involved in virulence, motility, detergent tolerance, and biofilm formation in Xanthomonas campestris pv. vesicatoria.

The bacterial envelope possesses diverse functions, including protection against environmental stress and virulence factors for host infection. Here, we report the function of wxcB in Xanthomonas campestris pv. vesicatoria (Xcv), a causal agent of bacterial leaf spot disease in tomato and pepper. To characterize roles of wxcB, we generated a knockout mutant (XcvΔwxcB) and found that the virulence of the mutant was weaker than that of the wild type in tomato plants. To predict the mechanism affected by wxcB, we compared protein expressions between the wild type and the mutant. Expression of 152 proteins showed a greater than 2-fold difference. Proteins involved in motility and cell wall/membrane were the most abundant. Through phenotypic assays, we further demonstrated that the mutant displayed reduced motility and tolerance to treatment, but it showed increased biofilm formation. Interestingly, the LPS profile was unchanged. These results lead to new insights into the functions of wxcB that is associated with cell wall/membrane functions, which contributes to pathogen virulence.

Regulation of cell wall-bound invertase in pepper leaves by Xanthomonas campestris pv. vesicatoria type three effectors.

Xanthomonas campestris pv. vesicatoria (Xcv) possess a type 3 secretion system (T3SS) to deliver effector proteins into its Solanaceous host plants. These proteins are involved in suppression of plant defense and in reprogramming of plant metabolism to favour bacterial propagation. There is increasing evidence that hexoses contribute to defense responses. They act as substrates for metabolic processes and as metabolic semaphores to regulate gene expression. Especially an increase in the apoplastic hexose-to-sucrose ratio has been suggested to strengthen plant defense. This shift is brought about by the activity of cell wall-bound invertase (cw-Inv). We examined the possibility that Xcv may employ type 3 effector (T3E) proteins to suppress cw-Inv activity during infection. Indeed, pepper leaves infected with a T3SS-deficient Xcv strain showed a higher level of cw-Inv mRNA and enzyme activity relative to Xcv wild type infected leaves. Higher cw-Inv activity was paralleled by an increase in hexoses and mRNA abundance for the pathogenesis-related gene PRQ. These results suggest that Xcv suppresses cw-Inv activity in a T3SS-dependent manner, most likely to prevent sugar-mediated defense signals. To identify Xcv T3Es that regulate cw-Inv activity, a screen was performed with eighteen Xcv strains, each deficient in an individual T3E. Seven Xcv T3E deletion strains caused a significant change in cw-Inv activity compared to Xcv wild type. Among them, Xcv lacking the xopB gene (Xcv ΔxopB) caused the most prominent increase in cw-Inv activity. Deletion of xopB increased the mRNA abundance of PRQ in Xcv ΔxopB-infected pepper leaves, but not of Pti5 and Acre31, two PAMP-triggered immunity markers. Inducible expression of XopB in transgenic tobacco inhibited Xcv-mediated induction of cw-Inv activity observed in wild type plants and resulted in severe developmental phenotypes. Together, these data suggest that XopB interferes with cw-Inv activity in planta to suppress sugar-enhanced defense responses during Xcv infection.

Characterization of HrpB2 from Xanthomonas campestris pv. vesicatoria identifies protein regions that are essential for type III secretion pilus formation.

The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to translocate effector proteins into plant cells. T3S depends on HrpB2, which is essential for assembly of the extracellular T3S pilus and is itself weakly secreted. To characterize the role of HrpB2, we used a transposon mutagenesis approach, which led to the insertion of pentapeptide-encoding sequences into hrpB2. Complementation studies with HrpB2 mutant derivatives revealed that the N-terminal region of HrpB2 tolerates pentapeptide insertions, whereas insertions in the regions spanning amino acids 60-74 and 93-130, respectively, resulted in a loss of bacterial pathogenicity and T3S, including secretion of HrpB2 itself. The C-terminal region (amino acids 93-130) of HrpB2 contains a conserved VxTLxK amino acid motif that is also present in predicted inner rod proteins from animal-pathogenic bacteria and is required for the contribution of HrpB2 to pilus assembly and T3S. Electron microscopy and fractionation studies revealed that HrpB2 is not a component of the extracellular pilus structure but localizes to the bacterial periplasm and the outer membrane. We therefore propose that the essential contribution of HrpB2 to T3S and pilus assembly is linked to its possible function as a periplasmic component of the T3S system at the base of the pilus.

Expression of Xanthomonas campestris pv. vesicatoria type III effectors in yeast affects cell growth and viability.

The gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of spot disease in tomato and pepper. X. campestris pv. vesicatoria pathogenicity depends on a type III secretion system delivering effector proteins into the host cells. We hypothesized that some X. campestris pv. vesicatoria effectors target conserved eukaryotic cellular processes and examined phenotypes induced by their expression in yeast. Out of 21 effectors tested, 14 inhibited yeast growth in normal or stress conditions. Viability assay revealed that XopB and XopF2 attenuated cell proliferation, while AvrRxo1, XopX, and XopE1 were cytotoxic. Inspection of morphological features and DNA content of yeast cells indicated that cytotoxicity caused by XopX and AvrRxo1 was associated with cell-cycle arrest at G0/1. Interestingly, XopB, XopE1, XopF2, XopX, and AvrRxo1 that inhibited growth in yeast also caused phenotypes, such as chlorosis and cell death, when expressed in either host or nonhost plants. Finally, the ability of several effectors to cause phenotypes in yeast and plants was dependent on their putative catalytic residues or localization motifs. This study supports the use of yeast as a heterologous system for functional analysis of X. campestris pv. vesicatoria type III effectors, and sets the stage for identification of their eukaryotic molecular targets and modes of action.

Membrane topology of conserved components of the type III secretion system from the plant pathogen Xanthomonas campestris pv. vesicatoria.

Type III secretion (T3S) systems play key roles in the assembly of flagella and the translocation of bacterial effector proteins into eukaryotic host cells. Eleven proteins which are conserved among gram-negative plant and animal pathogenic bacteria have been proposed to build up the basal structure of the T3S system, which spans both inner and outer bacterial membranes. We studied six conserved proteins, termed Hrc, predicted to reside in the inner membrane of the plant pathogen Xanthomonas campestris pv. vesicatoria. The membrane topology of HrcD, HrcR, HrcS, HrcT, HrcU and HrcV was studied by translational fusions to a dual alkaline phosphatase-beta-galactosidase reporter protein. Two proteins, HrcU and HrcV, were found to have the same membrane topology as the Yersinia homologues YscU and YscV. For HrcR, the membrane topology differed from the model for the homologue from Yersinia, YscR. For our data on three other protein families, exemplified by HrcD, HrcS and HrcT, we derived the first topology models. Our results provide what is believed to be the first complete model of the inner membrane topology of any bacterial T3S system and will aid in elucidating the architecture of T3S systems by ultrastructural analysis.