Characterization of a unique chromosomal copper resistance gene cluster from Xanthomonas campestris pv. vesicatoria

We characterized the copper resistance genes in strain XvP26 of Xanthomonas campestris pv. vesicatoria, which was originally isolated from a pepper plant in Taiwan. The copper resistance genes were localized to a 7,652-bp region which, based on pulsed-field gel electrophoresis and Southern hybridization, was determined to be located on the chromosome. These genes hybridized only weakly, as determined by Southern analysis, to other copper resistance genes in Xanthomonas and Pseudomonas strains. We identified five open reading frames (ORFs) whose products exhibited high levels of amino acid sequence identity to the products of previously reported copper genes. Mutations in ORF1, ORF3, and ORF4 removed copper resistance, whereas mutations in ORF5 resulted in an intermediate copper resistance phenotype and insertions in ORF2 had no effect on resistance conferred to a copper-sensitive recipient in transconjugant tests. Based on sequence analysis, ORF1 was determined to have high levels of identity with the CopR (66%) and PcoR (63%) genes in Pseudomonas syringae pv. tomato and Escherichia coli, respectively. ORF2 and ORF5 had high levels of identity with the PcoS gene in E. coli and the gene encoding a putative copper-containing oxidoreductase signal peptide protein in Sinorhizobium meliloti, respectively. ORF3 and ORF4 exhibited 23% identity to the gene encoding a cation efflux system membrane protein, CzcC, and 62% identity to the gene encoding a putative copper-containing oxidoreductase protein, respectively. The latter two ORFs were determined to be induced following exposure to low concentrations of copper, while addition of Co, Cd, or Zn resulted in no significant induction. PCR analysis of 51 pepper and 34 tomato copper-resistant X. campestris pv. vesicatoria strains collected from several regions in Taiwan between 1987 and 2000 and nine copper-resistant strains from the United States and South America showed that successful amplification of DNA was obtained only for strain XvP26. The organization of this set of copper resistance genes appears to be uncommon, and the set appears to occur rarely in X. campestris pv. vesicatoria.     

Similarity between Copper Resistance Genes from Xanthomonas campestris and Pseudomonas syringae

Plasmid-borne copper resistance genes from copper-resistant strains of Xanthomonas campestris pv. vesicatoria from California, Florida, and Oklahoma shared structural similarities. A strain of X. campestris pv. campestris also contained plasmid-borne copper resistance genes similar to the resistance genes from X. campestris pv. vesicatoria. Furthermore, a region of the copper resistance genes from X. campestris pv. vesicatoria 07882 hybridized with copA, the first gene of the copper resistance operon (cop) of Pseudomonas syringae pv. tomato. A copper-inducible protein of similar size to CopA was detected by Western blot (immunoblot) analysis from the wild-type strain 07882 and from the cloned copper resistance genes of 07882 introduced into a copper-sensitive strain of X. campestris pv. vesicatoria. A low level of hybridization was observed with chromosomal DNA from other xanthomonads when the copper resistance genes from strain 07882 were used as probes.     

Molecular cloning, chromosomal mapping, and sequence analysis of copper resistance genes from Xanthomonas campestris pv. juglandis: homology with small blue copper proteins and multicopper oxidase.

Copper-resistant strains of Xanthomonas campestris pv. juglandis occur in walnut orchards throughout northern California. The copper resistance genes from a copper-resistant strain C5 of X. campestris pv. juglandis were cloned and located on a 4.9-kb ClaI fragment, which hybridized only to DNA of copper-resistant strains of X. campestris pv. juglandis, and was part of an approximately 20-kb region which was conserved among such strains of X. campestris pv. juglandis. Hybridization analysis indicated that the copper resistance genes were located on the chromosome. Plasmids conferring copper resistance were not detected in copper-resistant strains, nor did mating with copper-sensitive strains result in copper-resistant transconjugants. Copper resistance genes from X. campestris pv. juglandis shared nucleotide sequence similarity with copper resistance genes from Pseudomonas syringae pv. tomato, P. syringae, and X. campestris pv. vesicatoria. DNA sequence analysis of the 4.9-kb fragment from strain C5 revealed that the sequence had an overall G+C content of 58.7%, and four open reading frames (ORF1 to ORF4), oriented in the same direction. All four ORFs were required for full expression of copper resistance, on the basis of Tn3-spice insertional inactivation and deletion analysis. The predicted amino acid sequences of ORF1 to ORF4 showed 65, 45, 47, and 40% identity with CopA, CopB, CopC, and CopD, respectively, from P. syringae pv. tomato. The most conserved regions are ORF1 and CopA and the C-terminal region (166 amino acids from the C terminus) of ORF2 and CopB. The hydrophobicity profiles of each pair of predicted polypeptides are similar except for the N terminus of ORF2 and CopB. Four histidine-rich polypeptide regions in ORF1 and CopA strongly resembled the copper-binding motifs of small blue copper proteins and multicopper oxidases, such as fungal laccases, plant ascorbate oxidase, and human ceruloplasmin. Putative copper ligands of the ORF1 polypeptide product are proposed, indicating that the polypeptide of ORF1 might bind four copper ions: one type 1, one type 2, and two type 3.     

Copper Resistance Gene Homologs in Pathogenic and Saprophytic Bacterial Species from Tomato

Copper-resistant strains of Xanthomonas campestris pv. vesicatoria, Pseudomonas cichorii, Pseudomonas putida, Pseudomonas fluorescens, and a yellow Pseudomonas sp. were isolated from tomato plants or seeds. In Southern hybridizations, DNA from each strain showed homology with the copper resistance (cop) operon previously cloned from Pseudomonas syringae pv. tomato PT23. Homology was associated with plasmid and chromosomal DNA in X. compestris pv. vesicatoria, P. putida, and the yellow Pseudomonas sp. Homology was detected only in the chromosomal DNA of P. cichorii and P. fluorescens. Homology with cop was also detected in chromosomal DNA from copper-sensitive strains of P. cichorii, P. fluorescens, and P. syringae pv. tomato, suggesting that the cop homolog may be indigenous to certain Pseudomonas species and have some function other than copper resistance. No homology was detected in DNA from a copper-sensitive X. campestris pv. vesicatoria strain. Copper-inducible protein products were detected in each copper-resistant bacterium by immunoblot analysis with antibodies raised to the CopB protein from the cop operon. The role of the homologous DNA in copper resistance was confirmed for the X. campestris pv. vesicatoria strain by cloning and transferring the cop homolog to a copper-sensitive strain of X. campestris pv. vesicatoria. The possibility and implications of copper resistance gene exchange between different species and genera of pathogenic and saprophytic bacteria on tomato plants are discussed.     

Nutritional Similarity between Leaf-Associated Nonpathogenic Bacteria and the Pathogen Is Not Predictive of Efficacy in Biological Control of Bacterial Spot of Tomato

It has been demonstrated that for a nonpathogenic, leaf-associated bacterium, effectiveness in the control of bacterial speck of tomato is correlated with the similarity in the nutritional needs of the nonpathogenic bacterium and the pathogen Pseudomonas syringae pv. tomato. This relationship was investigated further in this study by using the pathogen Xanthomonas campestris pv. vesicatoria, the causal agent of bacterial spot of tomato, and a collection of nonpathogenic bacteria isolated from tomato foliage. The effects of inoculation of tomato plants with one of 34 nonpathogenic bacteria prior to inoculation with the pathogen X. campestris pv. vesicatoria were quantified by determining (i) the reduction in disease severity (number of lesions per square centimeter) in greenhouse assays and (ii) the reduction in leaf surface pathogen population size (log₁₀ of the number of CFU per leaflet) in growth chamber assays. Nutritional similarity between the nonpathogenic bacteria and X. campestris pv. vesicatoria was quantified by using either niche overlap indices (NOI) or relatedness in cluster analyses based upon in vitro utilization of carbon or nitrogen sources reported to be present in tomato tissues or in Biolog GN plates. In contrast to studies with P. syringae pv. tomato, nutritional similarity between the nonpathogenic bacteria and the pathogen X. campestris pv. vesicatoria was not correlated with reductions in disease severity. Nutritional similarity was also not correlated with reductions in pathogen population size. Further, the percentage of reduction in leaf surface pathogen population size was not correlated with the percentage of reduction in disease severity, suggesting that the epiphytic population size of X. campestris pv. vesicatoria is not related to disease severity and that X. campestris pv. vesicatoria exhibits behavior in the phyllosphere prior to lesion formation that is different from that of P. syringae pv. tomato.     

The avirulence gene avrBs1 from Xanthomonas campestris pv. vesicatoria encodes a 50-kD protein

A gene cloned from Xanthomonas campestris pv. vesicatoria race 2, avrBs1, specified avirulence on pepper cultivars containing the resistance gene Bs1. A series of exonuclease III deletions were made on a 3.2-kbp DNA fragment that determined full avirulence activity, observed as hypersensitive response (HR) induction. The deletion products were subcloned into the broad host range cloning vector pLAFR3, conjugated into a virulent X. c. pv. vesicatoria race 1 strain, 82-8, and scored for their ability to induce a HR on a pepper cultivar (ECW10R) containing the resistance gene Bs1. A span of approximately 1.8 kbp of DNA was necessary for full induction of the HR. The nucleotide sequence revealed two open reading frames (ORFs) capable of encoding proteins of 12.3 and 49.8 kD, designated ORF1 and ORF2, respectively. Deletions into ORF1 altered the HR-inducing activity to give an intermediate phenotype. Deletions into ORF2 completely destroyed activity. When the ORF2 coding region was driven by the lacZ promoter on plasmid pLAFR3 (placD), full avirulence activity was restored, indicating that ORF2 alone can induce the HR. Antisera raised to a beta-galactosidase-ORF2 fusion protein reacted with a 50-kD protein in X. c. pv. vesicatoria race 1 (placD) transconjugants. The deduced amino acid sequence of ORF2 had approximately 47% overall homology to the carboxyl terminus of the avirulence gene, avrA, isolated from Pseudomonas syringae pv. glycinea race 6, and 86% homology over a region of 49 amino acids. P. s. pv. glycinea, however, did not induce an HR on ECW10R plants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of pXV10A, a Copper Resistance Plasmid in Xanthomonas campestris pv. vesicatoria

The efficacy of copper bactericides for control of Xanthomonas campestris pv. vesicatoria in eastern Oklahoma tomato fields was evaluated. Copper bactericides did not provide adequate control, and copper-resistant (Cu^r) strains of the pathogen were isolated. The Cu^r genes in these strains were located on a large indigenous plasmid designated pXV10A. The host range of pXV10A was investigated; this plasmid was efficiently transferred into 8 of 11 X. campestris pathovars. However, the transfer of pXV10A to other phytopathogenic genera was not detected. DNA hybridization experiments were performed to characterize the Cu^r genes on pXV10A. A probe containing subcloned Cu^r genes from X. campestris pv. vesicatoria E3C5 hybridized to pXV10A; however, a subclone containing Cu^r genes from P. syringae pv. tomato PT23 failed to hybridize to pXV10A. Further DNA hybridization experiments were performed to compare pXV10A with pXvCu plasmids, a heterogenous group of Cu^r plasmids present in strains of X. campestris pv. vesicatoria from Florida. These studies indicated that the Cu^r genes on pXV10A and pXvCu plasmids share nucleotide sequence homology and may have a common origin. Further experiments showed that these plasmids are distinctly different because pXV10A did not contain sequences homologous to IS476, an insertion sequence present on pXvCu plasmids.     

Outer Membrane Proteins and Lipopolysaccharides in Pathovars of Xanthomonas campestris

Variations in the outer membrane proteins (OMPs) and lipopolysaccharides (LPSs) of 54 isolates belonging to 16 different pathovars of Xanthomonas campestris were characterized. OMP samples prepared by sarcosyl extraction of cell walls and LPS samples prepared by proteinase K treatment of sonicated cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 4 M urea. In general, the OMP and LPS profiles within each pathovar were very similar but different from the profiles of other pathovars. Heterogeneity in OMP and LPS profiles was observed within X. campestris pv. campestris, X. campestris pv. translucens, and X. campestris pv. vesicatoria. LPSs were isolated from six X. campestris pathovars, which fell into two major groups on the basis of O antigenicity. The O antigens of X. campestris pv. begoniae, X. campestris pv. graminis, and X. campestris pv. translucens cross-reacted with each other; the other group consisted of X. campestris pv. campestris, X. campestris pv. pelargonii, and X. campestris pv. vesicatoria. A chemical analysis revealed a significant difference between the compositions of the neutral sugars of the LPSs of those two groups; the LPSs of the first group contained xylose and a 6-deoxy-3-O-methyl hexose, whereas the LPSs of the other group lacked both sugars.     

Characterization of a Copper Resistance Plasmid Conserved in Copper-Resistant Strains of Pseudomonas syringae pv. tomato

A 35-kilobase plasmid was conserved among 12 copper-resistant strains of Pseudomonas syringae pv. tomato. Restriction patterns of this plasmid from each strain were identical, and a cloned copper resistance gene from 1 strain hybridized to the same location on the 35-kilobase plasmid of all 12 strains.     

The pilA gene of Xanthomonas campestris pv. citri is required for infection by the filamentous phage cf

Host factors that are important for infection of Xanthomonas campestris pv. citri by the filamentous bacteriophage cf were investigated by transposon mutagenesis with Tn5tac1. A mutant, XT501, that was resistant to cf infection was recovered, showing that the gene inactivated by the transposon is required for infection by the phage but not for cf replication or assembly. A 1.7-kb SacI-ApaI DNA fragment from XT501 containing the bacterial DNA flanking one end of the transposon was cloned and shown to be required for cf infection. Nucleotide sequence analysis of the 1.7-kb fragment reveals the presence of an ORF that encodes a protein of 146 amino acids. This protein shows 42% identity to the type 4 prepilin encoded by the pilA genes of other bacteria. The pilA gene of X. campestris pv. citri is thus essential for infection by the bacteriophage cf.     

gly Gene Cloning and Expression and Purification of Glycinecin A, a Bacteriocin Produced by Xanthomonas campestris pv. glycines 8ra

Glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines, inhibits the growth of X. campestris pv. vesicatoria. We have cloned and expressed the genes encoding glycinecin A in Escherichia coli. Recombinant glycinecin A was purified from cell extracts by ammonium sulfate precipitation followed by chromatography on Q-Sepharose, Mono Q (ion exchange), and size exclusion columns. Purified glycinecin A is composed of two polypeptides, is active over a wide pH range (6 to 9), and is stable at temperatures up to 60°C. Glycinecin A is a heterodimer consisting of 39- and 14-kDa subunits, as revealed through size exclusion chromatography and cross-linking analysis. Two genes, glyA and glyB, encoding the 39- and 14-kDa subunits, respectively, were identified based on the N-terminal sequences of the subunits. From the nucleotide sequences of glyA and glyB, we conclude that both genes are translated as bacteriocin precursors that include N-terminal leader sequences. When expressed in E. coli, recombinant glycinecin A was found primarily in cell extracts. In contrast, most glycinecin A from Xanthomonas was found in the culture media. E. coli transformed with either glyA or glyB separately did not show the bacteriocin activity.     

The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice

The nucleotide sequence was determined for the genome of Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, a bacterium that causes bacterial blight in rice (Oryza sativa L.). The genome is comprised of a single, 4 941 439 bp, circular chromosome that is G + C rich (63.7%). The genome includes 4637 open reading frames (ORFs) of which 3340 (72.0%) could be assigned putative function. Orthologs for 80% of the predicted Xoo genes were found in the previously reported X.axonopodis pv. citri (Xac) and X.campestris pv. campestris (Xcc) genomes, but 245 genes apparently specific to Xoo were identified. Xoo genes likely to be associated with pathogenesis include eight with similarity to Xanthomonas avirulence (avr) genes, a set of hypersensitive reaction and pathogenicity (hrp) genes, genes for exopolysaccharide production, and genes encoding extracellular plant cell wall-degrading enzymes. The presence of these genes provides insights into the interactions of this pathogen with its gramineous host.     

Phenotypic Switching Affecting Chemotaxis, Xanthan Production, and Virulence in Xanthomonas campestris

The chemotaxis towards sucrose and yeast extract of nine strains of Xanthomonas campestris representing pathovars campestris, armoraciae, translucens, vesicatoria, and pelargonii was analyzed by using swarm plates. Unexpectedly, each of these strains formed small or reduced swarms typical of nonmotile or nonchemotactic bacteria. With time, however, chemotactic cells appeared on the swarm plates as blebs of bacteria. These cells were strongly chemotactic and were concomitantly deficient in exopolysaccharide production. The switch from the wild type (exopolysaccharide producing and nonchemotactic) to the swarmer type (exopolysaccharide deficient and chemotactic) appeared irreversible ex planta in bacteriological medium. However, in radish leaves swarmer-type strains of X. campestris pv. campestris were able to revert to the wild type. Swarmer-type derivatives of two X. campestris pv. campestris wild-type isolates showed reduced virulence and growth in the host plants cauliflower and radish. However, exocellular complementation of X. campestris pv. campestris Hrp (nonpathogenic) mutant was achieved by coinoculation with a swarmer-type strain.     

DNA probes for detection of copper resistance genes in Xanthomonas campestris pv. vesicatoria.

The copper resistance (Cur) genes encoded on pXV10A, a 190-kb plasmid in Xanthomonas campestris pv. vesicatoria XV10, were isolated on a 44-kb cosmid clone designated pCuR1. Tn5 mutagenesis of pCuR1 indicated that a 4.0-kb region was required for copper resistance. Three restriction fragments located within the 4.0-kb region demonstrated high specificity for the Cur genes present in X. campestris pv. vesicatoria and will be useful in monitoring the presence of these genes in the environment.     

An hrcU-homologous gene mutant of Xanthomonas campestris pv. glycines 8ra that lost pathogenicity on the host plant but was able to elicit the hypersensitive response on nonhosts.

Transposon mutagenesis was used to isolate nonpathogenic mutants of Xanthomonas campestris pv. glycines 8ra, which causes bacterial pustule disease in soybean. A 6.1-kb DNA region in which a mutation gave loss of pathogenicity was isolated and found to carry six open reading frames (ORFs). Four ORFs had homology with hrcU, hrcV, hrcR, and hrcS genes of Ralstonia solanacearum and X. campestris pv. vesicatoria. One nonpathogenic mutant, X. campestris pv. glycines H80, lost pathogenicity on soybean but was able to elicit the hypersensitive response (HR) on nonhost pepper and tomato plants. This mutant still multiplied as well as the wild type in the leaves or cotyledons of soybean. Although the DNA and amino acid sequences showed high homology with known hrp genes, the hrcU-homolog ORF is not required for HR induction on nonhost plants, pepper and tomato, or for the multiplication of bacteria in the host plant. This gene was only required for the pathogenic symptoms of X. campestris pv. glycines 8ra on soybean.     

DNA probes for detection of copper resistance genes in Xanthomonas campestris pv. vesicatoria.

The copper resistance (Cur) genes encoded on pXV10A, a 190-kb plasmid in Xanthomonas campestris pv. vesicatoria XV10, were isolated on a 44-kb cosmid clone designated pCuR1. Tn5 mutagenesis of pCuR1 indicated that a 4.0-kb region was required for copper resistance. Three restriction fragments located within the 4.0-kb region demonstrated high specificity for the Cur genes present in X. campestris pv. vesicatoria and will be useful in monitoring the presence of these genes in the environment.     

Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria

Summary The avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria was cloned and found to be localized on a self-transmissable plasmid. Genetic analysis of an avrBs3 insertion mutation revealed that avrBs3 constitutes a single locus, specifying the resistant phenotype on pepper plants. Southern blot experiments showed that no DNA sequences homologous to avrBs3 were present in other races of X. c. pv. vesicatoria, which are unable to induce a hypersensitive reaction on ECW-30R. However, the DNA of several different pathovars of X. campestris hybridized to the avrBs3 probe. A deletion analysis defined a region of 3.6–3.7 kb essential for avrBs3 activity. The nucleotide sequence of this region was determined. A 3561 nucleotide open reading frame (ORF1), encoding a 125000 dalton protein, was found in the 3.7 kb region that was sufficient for avrBs3 activity. A second long ORF (2351 nucleotides) was identified on the other strand. A remarkable feature of both ORFs is the presence of 17 direct repeats of 102 bp which share 91%–100% homology with each other.     

Antimicrobial Resistance among Enterococci from Pigs in Three European Countries

Enterococci from pigs in Denmark, Spain, and Sweden were examined for susceptibility to antimicrobial agents and copper and the presence of selected resistance genes. The greatest levels of resistance were found among isolates from Spain and Denmark compared to those from Sweden, which corresponds to the amounts of antimicrobial agents used in food animal production in those countries. Similar genes were found to encode resistance in the different countries, but the tet(L) and tet(S) genes were more frequently found among isolates from Spain. A recently identified transferable copper resistance gene was found in all copper-resistant isolates from the different countries.     

Nucleotide sequence and organization of copper resistance genes from Pseudomonas syringae pv. tomato.

The nucleotide sequence of a 4.5-kilobase copper resistance determinant from Pseudomonas syringae pv. tomato revealed four open reading frames (ORFs) in the same orientation. Deletion and site-specific mutational analyses indicated that the first two ORFs were essential for copper resistance; the last two ORFs were required for full resistance, but low-level resistance could be conferred in their absence. Five highly conserved, direct 24-base repeats were found near the beginning of the second ORF, and a similar, but less conserved, repeated region was found in the middle of the first ORF.