The extracellular proteases of the phytopathogenic bacterium Xanthomonas campestris

The culture liquids of three Xanthomonas campestris pv. campestris strains were found to possess proteolytic activity. The culture liquid of strain B-611 with the highest proteolytic activity was fractionated by salting-out with ammonium sulfate, gel filtration, and ion-exchange chromatography. The electrophoretic analysis of active fractions showed the presence of two proteases in the culture liquid of strain B-611, the major of which being serine protease. The treatment of cabbage seedlings with the proteases augmented the activity of peroxidase in the cabbage roots by 28%.     

Xanthomonas campestris pv. campestris possesses a single gluconeogenic pathway that is required for virulence

Disruption of ppsA, a key gene in gluconeogenesis, of Xanthomonas campestris pv. campestris resulted in the failure of the pathogen to grow in medium with pyruvate or C4-dicarboxylates as the sole carbon source and a significant reduction in virulence, indicating that X. campestris pv. campestris possesses only the malic enzyme-PpsA route in gluconeogenesis, which is required for virulence.     

Bactericidal activity of glycinecin A, a bacteriocin derived from Xanthomonas campestris pv. glycines, on phytopathogenic Xanthomonas campestris pv. vesicatoria cells

The ability of glycinecin A, a bacteriocin derived from Xanthomonas campestris pv. glycines 8ra, to kill closely related bacteria has been demonstrated previously by our group. In the present study, we aimed at determining the glycinecin A-induced cause of death. Treatment with glycinecin A caused slow dissipation of membrane potential and rapid depletion of the pH gradient. Glycinecin A treatment also induced leakage of potassium ions from X. campestris pv. vesicatoria YK93-4 cells and killed sensitive bacterial cells in a dose-dependent manner. Sensitive cells were killed within 2 h of incubation, most likely due to the potassium ion efflux caused by glycinecin A. These results suggest that the bactericidal mechanism of action of glycinecin A is correlated with the permeability of membranes to hydroxyl and potassium ions, leading to the lethal activity of the bacteriocin on the target bacteria.     

The role of PilZ domain proteins in the virulence of Xanthomonas campestris pv. campestris

Cyclic di‐GMP [(bis‐(3′–5′)‐cyclic di‐guanosine monophosphate)] is an almost ubiquitous second messenger in bacteria that is implicated in the regulation of a range of functions that include developmental transitions, aggregative behaviour, adhesion, biofilm formation and virulence. Comparatively little is known about the mechanism(s) by which cyclic di‐GMP exerts these various regulatory effects. PilZ has been identified as a cyclic di‐GMP binding protein domain; proteins with this domain are involved in regulation of specific cellular processes, including the virulence of animal pathogens. Here we have examined the role of PilZ domain proteins in virulence and the regulation of virulence factor synthesis in Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot of crucifers. The Xcc genome encodes four proteins (XC0965, XC2249, XC2317 and XC3221) that have a PilZ domain. Mutation of XC0965, XC2249 and XC3221 led to a significant reduction of virulence in Chinese radish. Mutation of XC2249 and XC3221 led to a reduction in motility whereas mutation of XC2249 and XC0965 affected extracellular enzyme production. All mutant strains were unaffected in biofilm formation in vitro. The reduction of virulence following mutation of XC3221 could not be wholly attributed to an effect on motility as mutation of pilA, which abolishes motility, has a lesser effect on virulence.     

Genomic approaches in Xanthomonas campestris pv. vesicatoria allow fishing for virulence genes

Xanthomonas campestris pv. vesicatoria is an economically important pathogen of pepper and tomato and has been established as a model organism to study bacterial infection strategies. In the last two decades, intensive genetic and molecular analyses led to the isolation of many genes that play a role in the intimate molecular relationship with the host plant. Essential for pathogenicity is a type III protein secretion system, which delivers bacterial effector proteins into the host cell. Currently, the genome of X. campestris pv. vesicatoria is being sequenced. The availability of genomic sequence information will pave the way for the identification of new bacterial virulence factors by bioinformatic approaches. In this article, we will present preliminary data from the genomic sequence analysis and describe recent and novel studies to identify bacterial type III effector genes.     

The zinc uptake regulator Zur is essential for the full virulence of Xanthomonas campestris pv. campestris

Zur is a regulator of the high-affinity zinc uptake system in many bacteria. In Xanthomonas campestris pv. campestris 8004, a putative protein encoded by the open reading frame designated as XC1430 shows 42% amino acid similarity with the Zur of Escherichia coli. An XC1430-disrupted mutant 1430nk was constructed by homologous suicide plasmid integration. 1430nk failed to grow in rich medium supplemented with Zn2+ at a concentration of 400 microM and in nonrich medium supplemented with Zn2+ at a concentration of 110 microM, whereas the wild-type strain grew well in the same conditions. In rich medium with 400 microM Zn2+, 1430nk accumulated significantly more Zn2+ than the wild-type strain. 1430nk showed a reduction in virulence on the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.) and produced less extracellular polysaccharide (EPS) than did the wild-type strain in the absence of added zinc. These results revealed that XC1430 is a functional member of the Zur regulator family that controls zinc homeostasis, EPS production, and virulence in X. campestris pv. campestris.     

Identification and DNA sequence of a pathogenicity gene of Xanthomonas campestris pv. campestris

A region of Xanthomonas campestris pv. campestris DNA containing at least two pathogenicity genes was identified. Mutants in one gene were clearly reduced in pathogenicity while mutants in the other were only moderately reduced. Both classes of mutants were prototrophic and motile, and had wild-type levels of extracellular enzymes and extracellular polysaccharide. They also grew in vitro and in planta at the same rate as the wild type. Experiments involving one of the clear pathogenicity mutants indicated that the recovery of mutant cells from turnip seedlings 24 hr after inoculation was lower than for the wild type. This may be due to cell death as a result of action by some preformed or induced plant factor. From DNA sequencing an open reading frame was identified that encompassed the site of the mutations giving a clear reduction in pathogenicity. The predicted protein sequence had no homology with other proteins in the computer data base.     

Culture medium for Xanthomonas campestris pv. oryzae

Y uan , W. 1990. Culture medium for Xanthomonas campestris pv. oryzae. Journal of Applied Bacteriology 69 , 798–805.
Studies on nutrient requirements of four Chinese strains of Xanthomonas campestris pv. oryzae in a modified Watanabe's medium led to the development of a new synthetic medium containing sucrose, sodium glutamate, methionine, KH₂PO₄, NH₄C1 and iron chelated with EDTA. The concentration of each ingredient was optimized based on the number of colonies and time required for their appearance. Various concentrations of some nutrients were compared based upon their effects on growth of the pathogen strains and 34 contaminants from rice materials. Tryp-tone enhanced the growth of X. c. oryzae more than that of many contaminants, including Erwinia herbicola . Peptone stimulated growth of X. c. oryzae without promoting excessive contamination. When compared with other media used for X. c. oryzae , the new culture medium enriched with tryptone and peptone gave the highest recovery and earliest appearance of colonies of Chinese strains of this bacterium.     

Molecular evolution of virulence in natural field strains of Xanthomonas campestris pv. vesicatoria

The avrBs2 avirulence gene of the bacterial plant pathogen Xanthomonas campestris pv. vesicatoria triggers disease resistance in pepper plants containing the Bs2 resistance gene and contributes to bacterial virulence on susceptible host plants. We studied the effects of the pepper Bs2 gene on the evolution of avrBs2 by characterizing the molecular basis for virulence of 20 X. campestris pv. vesicatoria field strains that were isolated from disease spots on previously resistant Bs2 pepper plants. All field strains tested were complemented by a wild-type copy of avrBs2 in their ability to trigger disease resistance on Bs2 plants. DNA sequencing revealed four mutant alleles of avrBs2, two of which consisted of insertions or deletions of 5 nucleotides in a repetitive region of avrBs2. The other two avrBs2 alleles were characterized by point mutations with resulting single amino acid changes (R403P or A410D). We generated isogenic X. campestris pv. vesicatoria strains by chromosomal avrBs2 gene exchange to study the effects of these mutations on the dual functions of avrBs2 in enhancing bacterial virulence and inducing plant resistance by in planta bacterial growth experiments. The deletion of 5 nucleotides led to loss of avrBs2-induced resistance on Bs2 pepper plants and abolition of avrBs2-mediated enhancement of fitness on susceptible plants. Significantly, the point mutations led to minimal reduction in virulence function of avrBs2 on susceptible pepper plants, with either minimal (R403P allele) or an intermediate level of (A410D allele) triggering of resistance on Bs2 plants. Consistent with the divergent selection pressures on avrBs2 exerted by the Bs2 resistance gene, our results show that avrBs2 is evolving to decrease detection by the Bs2 gene while at the same time maintaining its virulence function.     

Nucleotide sequence of the engXCA gene encoding the major endoglucanase of Xanthomonas campestris pv. campestris

The nucleotide sequence of the gene (engXCA) encoding the major extracellular endoglucanase (ENGXCA) of the phytopathogenic bacterium Xanthomonas campestris pv. campestris (X. c. campestris) was determined and compared with the N-terminal amino acid (aa) sequence of the purified enzyme. An open reading frame of 1479 bp encoding 493 aa was identified, of which the N-terminal 25 aa represent a potential signal peptide. Determination of the exact position of a Tn5 insertion within engXCA, which did not reduce the encoded enzyme activity, indicated that the C-terminal region of the protein is not crucial for ENGXCA activity. Comparison of the complete deduced aa sequence with those deduced from other endoglucanase- and exoglucanase-encoding genes revealed a region with a high degree of homology, located towards the C terminus of the protein. These data indicate that the X. c. campestris ENGXCA may have a domain structure similar to that of many other bacterial and fungal cellulolytic enzymes. Hydrophobic cluster analysis was performed on the deduced aa sequence. Comparison of this analysis with those of 30 other cellulase sequences belonging to six different families indicated that the X. c. campestris enzyme can be classified in family A. The two aa residues which had previously been identified as 'potentially catalytic' within this family of cellulases, are conserved in the X. c. campestris ENGXCA.     

Isolation and characterization of a generalized transducing phage for Xanthomonas campestris pv. campestris.

We have isolated and characterized a lytic double-stranded DNA Xanthomonas campestris pv. campestris bacteriophage (XTP1) capable of mediating generalized transduction. The phage transduces chromosomal markers at frequencies of 10(-5) to 10(-6) transductants per PFU. We demonstrated its genetic utility by the isolation and cotransduction of linked transposon insertions to a nonselectable locus, xgl, required for the cleavage of 5-bromo-3-chloro-indoyl-beta-D-galactoside and showed that rif and str alleles in X. campestris are 75% linked. One-step growth experiments showed that the latent and rise periods were each 2 h and the average burst size was 35. The DNA genome is approximately 180 kb, presumably modified in a sequence-specific manner, and may be covalently attached to protein(s). Electron micrographs show the phage particle to have an icosahedral head and contractile tail with tail fibers uniquely attached to a location 40 nm proximal from the end of the tail.     

Biological role of xanthomonadin pigments in Xanthomonas campestris pv. campestris

Previous studies have indicated that the yellow pigments (xanthomonadins) produced by phytopathogenic Xanthomonas bacteria are unimportant during pathogenesis but may be important for protection against photobiological damage. We used a Xanthomonas campestris pv. campestris parent strain, single-site transposon insertion mutant strains, and chromosomally restored mutant strains to define the biological role of xanthomonadins. Although xanthomonadin mutant strains were comparable to the parent strain for survival when exposed to UV light; after their exposure to the photosensitizer toluidine blue and visible light, survival was greatly reduced. Chromosomally restored mutant strains were completely restored for survival in these conditions. Likewise, epiphytic survival of a xanthomonadin mutant strain was greatly reduced in conditions of high light intensity, whereas a chromosomally restored mutant strain was comparable to the parent strain for epiphytic survival. These results are discussed with respect to previous results, and a model for epiphytic survival of X. campestris pv. campestris is presented.     

DsbB is required for the pathogenesis process of Xanthomonas campestris pv. campestris

The DsbA/DsbB oxidation pathway is one of the two pathways that catalyze disulfide bond formation of proteins in the periplasm of gram-negative bacteria. It has been demonstrated that DsbA is essential for multiple virulence factors of several animal bacterial pathogens. In this article, we present genetic evidence to show that the open reading frame XC_3314 encodes a DsbB protein that is involved in disulfide bond formation in periplasm of Xanthomonas campestris pv. campestris, the causative agent of crucifer black rot disease. The dsbB mutant of X. campestris pv. campestris exhibited attenuation in virulence, hypersensitive response, cell motility, and bacterial growth in planta. Furthermore, mutation in the dsbB gene resulted in ineffective type II and type III secretion systems as well as flagellar assembly. These findings reveal that DsbB is required for the pathogenesis process of X. campestris pv. campestris.     

Defence-related enzymatic response in cabbage to Xanthomonas campestris pv. campestris

Black rot of cabbage caused by Xanthomonas campestris pv. campestris is one of the most important diseases of crucifers worldwide. Expression of defence-related enzymes in cabbage in response to X. campestris pv. campestris was investigated in the current experiment. Among the defence-related enzymes (phynylalanine ammonia lyase, peroxidase, polyphenol oxidase, superoxide dismutase [SOD] and chitinase) and quantity of phenolic compounds studied in the present investigation, phenylalanine ammonia lyase (PAL), the key enzyme in the phenylpropanoid pathway was the first enzyme suppressed at three days after inoculation in X. campestris pv. campestris-cabbage system. Correlation analysis indicated that PAL and phenolic compounds are the two most important compounds determining the susceptibility of cabbage to X. campestris pv. campestris. Induction of peroxidase isoform-1 (Rf value: 0.059) and SOD isoform-1 (Rf value: 0.179) three days after pathogen inoculation implicated the role of these isozymes in susceptible cabbage – X. campestris pv. campestris interaction. This study demonstrates the susceptibility of cabbage to X. campestris pv. campestris is a result of declination of PAL and phenolic contents at biochemical level as a manifestation of increase in bacterial population at the cellular level within the host tissues.     

Isolation of a Plasmid from Xanthomonas campestris pv. vignicola

A modified method is described for isolating high yields of plasmids without chromosome contamination from Xanthomonas campestris pv. vignicola (X. c. pv. vignicola), the causal agent of blight disease in Vigna species and also in Phaseolus vulgaris. Applying this method a plasmid of X, c. pv. vignicola was detected representing an estimated molecular weight of 95 megadaltons. Heat curing of the strain revealed that the detected plasmid had no effecton virulence but seemed to influence colony morphology.     

Phosphorylation of proteins in Xanthomonas campestris pv. oryzae

Protein phosphorylation was studied in Xanthomonas campestris pv. oryzae in vivo and in vitro. In vitro labelling showed that the protein kinases in this bacterium used both ATP and GTP as nucleotide substrates at nearly the same efficiency. At least 6 proteins were phosphorylated in vitro, including abundant species of p81, p44, and p32 with M _r of 81000, 44000, and 32000, respectively. Three types of phosphate-protein linkage were found in this bacterium: O-phosphate, N-phosphate and probably acyl phosphate. The p81 and p32 were phosphorylated at histidine. The p44 had mainly phosphoserine and a small part of phosphohistidine. The phosphorylation profile was variable depending on the growth conditions. Furthermore, by a virulent phage Xp10 infection the quantity of phosphorylation increased: for phosphohistinine more than 10-fold, and for phosphoserine about 3-fold. Thus, in this bacterium phosphorylation may be linked with a physiological regulation system and with Xp10 phage development.