Bacteriocins of phytopathogenic Corynebacterium species.

The majority (85% of all strains tested) of 12 phytopathogenic Corynebacterium species produced bacteriocin(s) on nutrient broth--yeast extract (NBY) medium. All C. nebraskense, C. michiganense, C. insidiosum, C. oortii, and C. iranicum strains produced bacteriocin(s). The optimal conditions for production of 23 distinct bacteriocins by eight species of Corynebacterium generally were 20 degrees C and 4 days of incubation on NBY or on modified Burkholder's agar that lacked peptone (MBAL). Production in liquid was marginal and not augmented by adding mitomycin C. Bacteriocins generally had little effect on other strains within a species but were inhibitory to other species. Most bacteriocins appeared to be bactericidal proteins resistant to heat (75 to 80 degrees C, 30 min) but sensitive to proteolytic enzymes. Some strains of C. nebraskense, C. michiganense, C. insidiosum, and C. flaccumfaciens produced two bacteriocins which were clearly differentiated by varying or testing one or more of the following: conditions for production, the indicator, heat stability, and susceptibility to proteolysis. Within certain limitations, a convenient and reproducible typing scheme was devised for strain and species differentiation of most phytopathogenic corynebacteria.     

Deoxyribonucleic acid relatedness of 21 strains of Xanthomonas species and pathovars

The relationship of 17 Xanthomonas campestris pathotype strains, three additional X. campestris strains, and the type strain of Xanthomonas albilineans were examined by DNA-DNA hybridization tests. The results coupled with those of a previous study (Hildebrand et al. 1990) support the hypothesis that X. campestris does not constitute a single bacterial species. There were low levels of DNA-DNA reassociation among many of the different pathovars examined. Six clusters of related pathovars were discerned. In addition, four of the pathovars were only distantly related to each other and to the six clusters. Xanthomonas albilineans was not closely related to any of the other xanthomonads tested.
Mapping and superimposing the botanical families of the host plants upon a three-dimensional genomic distance matrix of the xanthomonads confirms previous observations that pathovars that infect plants of the same botanical family do not necessarily belong to the same genomic group. Six legume-infecting pathovars cluster within one genomic group, but one pathovar, X. cam. pv. pisi is only distantly related to this group. There was also no genomic relationship between X. cam. pv. oryzicola and X. albilineans both of which infect Gramineae. Consequently, pathogenicity toward members of the same plant family is not a good indicator of the genomic relationships among xanthomonads nor is it a good taxonomic determinant.     

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%.     

Efficient transformation of thermotolerant methanol & strains of methylophilus spp. via electroporation

Plasmid transformation of three thermotolerant strains of methylotrophic bacterium Methylophilus spp. was investigated using three different methods. The plasmids could be transformed by electroporation and were stabily maintained in these methylotrophic bacteria. The calcium chloride (competent cell) method was successful only in the case of one of the three strains studied. Protoplast transformation of methylotrophic bacteria was not successful. The antibiotic resistance markers conferred resistance on the transformants, whereas the wild-type bacteria were sensitive to the antibiotics.     

Efficient integrative transformation of the phytopathogenic fungus Alternaria alternata mediated by the repetitive rDNA sequences

An attempt was made to transform Alternaria alternata protoplasts using a plasmid vector, pDH25, bearing the Escherichia coli hygromycin B (Hy) phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter. Transformants arose on a selective medium containing 100 μg Hy/ml. There were two types of transformants, forming large and small colonies on the selective medium. Transformation with one μg of the vector produced an average of 4.5 large colonies and 600 small ones. In large-colony transformants, the vector often integrated into the recipient chromosome in the form of highly rearranged tandem arrays. To increase transformation efficiency, fragments of the highly repetitive ribosomal RNA gene cluster (rDNA) of A. alternata were used to construct four new vectors for homologous recombination system. Use of these vectors gave higher transformation efficiency than the original plasmid. The best vector, pDH25r1a, gave rise to large-colony transformants at a frequency 20 times higher than pDH25. Transformation events in A. alternata with pDH25r1a occured by homologous recombination as a single crossover between the plasmid-borne rDNA segment and its homologue in the chromosome, often giving rise to tandemly repeated vector DNA.     

Antibacterial activity of plant extracts on phytopathogenic Xanthomonas campestris pathovars

Aqueous extracts from leaves of 30 higher plants, collected from different localities, were screened in vitro for antibacterial activity against different pathovars of the phytopathogenic bacterium, Xanthomonas campestris. Eight plant species showed antibacterial activity, based on the zone of inhibition in a diffusion assay. Significant antibacterial activity was observed in the aqueous extracts of Prosopis juliflora , Oxalis corniculata and Lawsonia inermis. The susceptibility of different pathovars of X. campestris to these plant extracts varied. The antibacterial activity of extracts of a few plants was comparable with that of the synthetic antibiotics, bacterimycin and streptocycline. The study indicates the potential of these plant extracts in the management of diseases caused by X. campestris in several important crop plants.     

Universal PCR primers for detection of phytopathogenic Agrobacterium strains.

Two PCR primer pairs, based on the virD2 and ipt genes, detected a wide variety of pathogenic Agrobacterium strains. The endonuclease domain of VirD2 protein, which cleaves transferred DNA (T-DNA) border sequences, is highly conserved; primer oligonucleotides specific for the endonuclease portion of virD2 detected all pathogenic strains of Agrobacterium tested. PCR primers corresponding to conserved sequences in ipt, the T-DNA-borne cytokinin synthesis gene, detected only Agrobacterium tumefaciens and distinguished it from Agrobacterium rhizogenes. The virD2 and ipt primer pairs did not interfere with each other when included in the same PCR amplification, and this permitted simultaneous detection of both genes in a single reaction. One nonpathogenic Agrobacterium radiobacter strain contained virD2 but not ipt; we speculate that this strain arose from a pathogenic progenitor through a deletion in the T-DNA. The virD2 primer pair appears to be universal for all pathogenic Agrobacterium species; used together, the primer sets reported here should allow unambiguous identification of Ti plasmid DNA in bacteria isolated from soil and plants.     

Multiple periplasmic catalases in phytopathogenic strains of Pseudomonas syringae.

Phytopathogenic strains of Pseudomonas syringae are exposed to plant-produced, detrimental levels of hydrogen peroxide during invasion and colonization of host plant tissue. When P. syringae strains were investigated for their capacity to resist H2O2, they were found to contain 10- to 100-fold-higher levels of total catalase activity than selected strains belonging to nonpathogenic related taxa (Pseudomonas fluorescens and Pseudomonas putida) or Escherichia coli. Multiple catalase activities were identified in both periplasmic and cytoplasmic fluids of exponential- and stationary-phase P. syringae cells. Two of these activities were unique to the periplasm of P. syringae pv. glycinea. During the stationary growth phase, the specific activity of cytoplasmic catalases increased four- to eightfold. The specific activities of catalases in both fluids from exponential-phase cells increased in response to treatment with 0.25 to 10 mM H2O2 but decreased when higher H2O2 concentrations were used. In stationary-growth phase cultures, the specific activities of cytoplasmic catalases increased remarkably after treatment with 0.25 to 50 mM H2O2. The growth of P. syringae into stationary phase and H2O2 treatment did not induce synthesis of additional catalase isozymes. Only the stationary-phase cultures of all of the P. syringae strains which we tested were capable of surviving high H2O2 stress at concentrations up to 50 mM. Our results are consistent with the involvement of multiple catalase isozymes in the reduction of oxidative stress during plant pathogenesis by these bacteria.     

Multiple periplasmic catalases in phytopathogenic strains of Pseudomonas syringae.

Phytopathogenic strains of Pseudomonas syringae are exposed to plant-produced, detrimental levels of hydrogen peroxide during invasion and colonization of host plant tissue. When P. syringae strains were investigated for their capacity to resist H2O2, they were found to contain 10- to 100-fold-higher levels of total catalase activity than selected strains belonging to nonpathogenic related taxa (Pseudomonas fluorescens and Pseudomonas putida) or Escherichia coli. Multiple catalase activities were identified in both periplasmic and cytoplasmic fluids of exponential- and stationary-phase P. syringae cells. Two of these activities were unique to the periplasm of P. syringae pv. glycinea. During the stationary growth phase, the specific activity of cytoplasmic catalases increased four- to eightfold. The specific activities of catalases in both fluids from exponential-phase cells increased in response to treatment with 0.25 to 10 mM H2O2 but decreased when higher H2O2 concentrations were used. In stationary-growth phase cultures, the specific activities of cytoplasmic catalases increased remarkably after treatment with 0.25 to 50 mM H2O2. The growth of P. syringae into stationary phase and H2O2 treatment did not induce synthesis of additional catalase isozymes. Only the stationary-phase cultures of all of the P. syringae strains which we tested were capable of surviving high H2O2 stress at concentrations up to 50 mM. Our results are consistent with the involvement of multiple catalase isozymes in the reduction of oxidative stress during plant pathogenesis by these bacteria.     

Integron variability in Xanthomonas arboricola pv. juglandis and Xanthomonas arboricola pv. pruni strains

The integron platform and the gene cassette arrays of 34 Xanthomonas arboricola pv. juglandis and of 47 Xanthomonas arboricola pv. pruni strains isolated from different geographical areas were screened to check their variability. Genetic variability of the strains was also tested by means of BOX-PCR. For two representative strains of the two pathovars, the integrase gene intI and part of the flanking gene ilvD were also cloned and sequenced. Whereas X. a. pv. pruni strains did not show relevant variability, six X. a. pv. juglandis strains isolated in Australia showed some differences in the gene sequences. The CLUSTALW algorithm indicated that the majority of the X. a. pv. juglandis strains are closely related to X. a. pv. pruni, whereas the X. a. pv. juglandis strains isolated in Australia were more similar to Xanthomonas hortorum pv. pelargonii. Similarly, the gene cassette array pattern of the Australian strains, as well as that of the oldest strain maintained in culture, was different from the other strains. Also, three X. a. pv. pruni strains showed a different cassette array pattern when compared with the majority of other strains but no relationships with geographical area of isolation or host plant was revealed. This study confirmed that in addition to species, integrons may generate diversity also within two X. arboricola pathovars.     

Characterization of stress-responsive genes, hrcA-grpE-dnaK-dnaJ, from phytopathogenic Xanthomonas campestris

Sequencing of a 6.4-kb DNA fragment, cloned from the plant pathogenic bacterium Xanthomonas campestris pv. campestris 17 revealed five ORFs whose deduced amino acid sequences show strong similarities to the bacterial HrcA, GrpE, DnaK, DnaJ, and PdxK. The four heat shock genes are organized in the order hrcA-grpE-dnaK-dnaJ, a genome organization found in many gram-positive bacteria, but only in one gram-negative species (Xylella fastidiosa). These observations suggest that the HrcA-CIRCE system, comprising at least four genes arranged in this order, already existed for the regulation of stress responses before bacteria diverged into gram-negative and gram-positive groups. Primer-extension results suggested the presence of promoters at the regions upstream of grpE and dnaK. In the presence of stress, heat or ethanol (4%), the X. campestris pv. campestris 17 grpE and dnaK promoters were induced two- to three-fold over controls. Since the grpE and dnaK promoters possess E. coli sigma(32) promoter-like sequences, they are functional in E. coli, although at levels much lower than in X. campestris pv. campestris 17. Furthermore, expression of the X. campestris pv. campestris 17 dnaK promoter in E. coli was elevated by the cloned X. campestris sigma(32) gene, indicating that the cognate sigma(32) works more efficiently for the X. campestris promoters.     

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.     

PCR detection of Ti and Ri plasmids from phytopathogenic Agrobacterium strains.

A universal primer set (VCF/VCR) for PCR analysis based on the sequences of the virC operon located on Ti and Ri plasmids was designed to detect these plasmids from phytopathogenic Agrobacterium strains. With the VCF (sequence, 5'-ATCATTTGTAGCGACT-3') and VCR (sequence, 5'-AGCTCAAACCTGCTTC-3') primer set, DNA fragments of 730 bp in length were amplified from cell lysates of 10 rhizogenic and 65 tumorigenic agrobacteria. DNA sequencing and Southern hybridization analysis confirmed that the amplified fragments corresponded to the target region. The PCR method is considered convenient for routine determination of the potential pathogenicity of Agrobacterium strains.     

Immunoelectrophorecal differences between phytopathogenic Fusarium species

Immunoelectrograms of certain tested Fusarium spp revealed that each has a characteristic pattern and showed the relatedness between F. oxysporum on the one hand and F. culmorum and F. semitectum on the other. The B₄ protein fraction is common to the former pair, while α_1a protein fraction links the second.     

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.     

Efficient integrative transformation of the phytopathogenic fungus Alternaria alternata mediated by the repetitive rDNA sequences

An attempt was made to transform Alternaria alternata protoplasts using a plasmid vector, pDH25, bearing the Escherichia coli hygromycin B (Hy) phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter. Transformants arose on a selective medium containing 100 μg Hy/ml. There were two types of transformants, forming large and small colonies on the selective medium. Transformation with one μg of the vector produced an average of 4.5 large colonies and 600 small ones. In large-colony transformants, the vector often integrated into the recipient chromosome in the form of highly rearranged tandem arrays. To increase transformation efficiency, fragments of the highly repetitive ribosomal RNA gene cluster (rDNA) of A. alternata were used to construct four new vectors for homologous recombination system. Use of these vectors gave higher transformation efficiency than the original plasmid. The best vector, pDH25r1a, gave rise to large-colony transformants at a frequency 20 times higher than pDH25. Transformation events in A. alternata with pDH25r1a occured by homologous recombination as a single crossover between the plasmid-borne rDNA segment and its homologue in the chromosome, often giving rise to tandemly repeated vector DNA.