Evaluation of pentachlorophenol-degrading potentiality of Pseudomonas sp. in a soil microcosm

Pseudomonas sp. strain IST103 obtained from a stable consortium was capable of degrading pentachlorophenol (PCP) as sole carbon and energy source. The PCP-degrading potentiality of the strain was determined by growth of bacteria in culture medium, utilization of PCP by high performance liquid chromatography (HPLC), chloride release and ring cleavage. The strain was applied in two set of soil microcosms containing 20 and 40% moisture, each having different concentrations, 0, 10, 100, 500, and 1000 mg/l, of PCP. The result showed significant utilization of PCP (77% in 45 days) and higher growth of bacterial strain when PCP was applied in 100 mg/l concentration at 40% moisture. Inhibitory effects on the growth of bacterial strain were seen in 500 and 1000 mg/l concentration.     

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.     

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 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.     

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.     

Identification of a pathogenicity locus in Xanthomonas campestris pv. vesicatoria

Summary Mutants of a tomato strain ofXanthomonas campestris pv.vesicatoria (XCV), causal agent of bacterial spot of tomato and pepper, were produced using the transposon Tn5 carried in the suicide plasmid pGS9. One prototrophic mutant, M461, was isolated which caused no visible reaction on tomato or pepper, but maintained the wild-type ability to induce a hypersensitive reaction (HR) on tobacco. This mutant showed similar growth characteristics to the wild-type in culture, but growth in planta was reduced. A genomic library of wild-type XCV was constructed in the broad host range cosmid vector pLAFR3. Clone p6AD4 restored pathogenicity to M461 on tomato and the ability to induce a HR on pepper. This clone contained ca. 22 kb of XCV DNA. The insertion in M461 was in a site corresponding to a 1.1 kbEcoRI fragment of p6AD4.     

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.     

十字花科黑腐病菌hrpG基因原核表达

在十字花科黑腐病菌(Xcc)中,hrp基因对寄主的致病性和非寄主的超敏反应中起核心作用,而hrpG对整个hrp基因簇起调控作用.HrpG为OmpR家族的双组分系统感受调控蛋白,含有两个结构域,分别是N端Response_reg和C端Trans reg_C.本研究利用表达载体pQE-30 Xa,成功构建了HrpG的表达重组子,在E.coli M15 [pREP4]中进行诱导表达.通过调节诱导温度、IPTG浓度和诱导时间最终确定在温度为20℃,IPTG浓度为0.8 mmol/L,诱导表达4 h.hrpG基因在宿主细胞E.coli M15获得高效可溶性表达.目前尚未有可溶性HrpG蛋白获得成功表达的报导,本研究中获得HrpG蛋白在大肠杆菌获得大量可溶性的表达,将为in vitro研究HrpG的生理活性,特异的结合位点和调控功能研究打下良好基础.     

Requirement of a mip-like gene for virulence in the phytopathogenic bacterium Xanthomonas campestris pv. campestris

Macrophage infectivity potentiators (Mips) are FKBP domain-containing proteins reported as virulence factors in several human pathogens, such as members of genera Legionella, Salmonella and Chlamydia. The putative peptidylprolyl cis-trans isomerase (PPIase) encoded by XC2699 of the plant bacterial pathogen Xanthomonas campestris pv. campestris 8004 exhibits a 49% similarity at the amino-acid level to the Mip protein of Legionella pneumophila. This mip-like gene, XC2699, was overexpressed in Escherichia coli and the purified (His)6-tagged Mip-like protein encoded by XC2699 exhibited a PPIase activity specifically inhibited by FK-506. A mutation in the mip-like gene XC2699 led to significant reductions in virulence and replication capacity in the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.). Furthermore, the production of exopolysaccharide and the activity of extracellular proteases, virulence factors of X. campestris pv. campestris, were significantly decreased in the mip-like mutant. These results reveal that the mip-like gene is involved in the pathogenesis of X. campestris pv. campestris through an effect on the production of these virulence factors.     

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.     

Identification of two fructose transport and phosphorylation pathways in Xanthomonas campestris pv. campestris.

Summary Fructose was shown to be phosphorylated by a specific phosphoenolpyruvatc-dependent phosphotransferase system (PTS) in Xanthomonas campestris pv. campestris. Transposon mutagenesis of X. campestris was performed and two mutants affected in growth on fructose were isolated. Both mutants were deficient in PTS activity. Comparison of the rate of uptake and phosphorylation of fructose in the wild-type and in the mutant strains revealed the presence of a second fructose permeation and phosphorylation pathway in this bacterium: an unidentified permease coupled to an ATP-dependent fructokinase. One of the two mutants was also deficient in fructokinase activity. Chromosomal DNA fragments containing the regions flanking the transposon insertion site were cloned from both mutant strains. Their physical study revealed that the insertion sites were separated by 1.4 kb, allowing the reconstruction of a wild-type DNA fragment which complemented one of the two mutants. The region flanking the transposon insertion site was sequenced in one of the mutants, showing that the transposon had interrupted the gene encoding the fructose Ell. The mutant strains also failed to utilize mannose, sucrose and mannitol, suggesting the existence of a branch point between the metabolism of fructose and of these latter carbohydrates.     

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 (S. G. Heu et al., Appl. Environ. Microbiol. 67:4105-4110, 2001). 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.     

Immunomagnetic isolation of Xanthomonas campestris pv. pelargonii

Immunomagnetic fishing was developed as an improved procedure for increasing the bacterial target to non-target recovery ratio in suspensions containing mixtures of target and non-target organisms. A cell suspension containing the target Xanthomonas campestris pv. pelargonii and non-target organisms, is treated with rabbit polyclonal antiserum against X.c. pv. pelargonii and incubated for 1 h. The suspension is then mixed with paramagnetic iron oxide particles coated with goat anti-rabbit antibodies (immunomagnetic particles). After incubation, the polished surface of a 14 mm diameter neodymium supermagnet is placed at the air-water interace and the magnetic particles are attracted to the magnet. After all visible magnetic particles have attached to the bottom of the magnet, the magnet is dipped in sterile buffer to remove non-target organisms. The magnet with attached magnetic particles is rubbed evenly over an agar surface to dislodge the particles and attached bacteria. Conventional immunomagnetic isolation (immunomagnetic attraction) and immunomagnetic fishing were compared, for the recovery of the target organism in geranium leaf washings spiked with X.c. pv. pelargonii. With immunomagnetic attraction and immunomagnetic fishing, bacterial non-target organisms were reduced to 11.4 and 1.5% of the initial population, respectively, whereas the target was only reduced to 63.7 and 53.8%.     

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.     

Rapid cell death in Xanthomonas campestris pv. glycines

Xanthomonas campestris pv. glycines strain AM2 (XcgAM2), the etiological agent of bacterial pustule disease of soybean, exhibited post-exponential rapid cell death (RCD) in LB medium. X. campestris pv. malvacearum NCIM 2310 and X. campestris NCIM 2961 also displayed RCD, though less pronouncedly than XcgAM2. RCD was not observed in Pseudomonas syringae pv. glycines, or Escherichia coli DH5alpha. Incubation of the post-exponential LB-grown XcgAM2 cultures at 4 degrees C arrested the RCD. RCD was also inhibited by the addition of starch during the exponential phase of LB-growing XcgAM2. Protease negative mutants of XcgAM2 were found to be devoid of RCD behavior observed in the wild type XcgAM2. While undergoing RCD, the organism was found to transform to spherical membrane bodies. The presence of membrane bodies was confirmed by using a membrane specific fluorescent label, 1,6-diphenyl 1,3,5-hexatriene (DPH), and also by visualizing these structures under microscope. The membrane bodies of XcgAM2 were found to contain DNA, which was devoid of the indigenous plasmids of the organism. The membrane bodies were found to bind annexin V indicative of the externalization of membrane phosphatidyl serine. Nicking of DNA in XcgAM2 cultures undergoing RCD in LB medium was also detected using a TUNEL assay. The RCD in XcgAM2 appeared to have features similar to the programmed cell death in eukaryotes.     

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.