A multilocus sequence analysis of Xanthomonas campestris reveals a complex structure within crucifer-attacking pathovars of this species

Previous classification of Xanthomonas campestris has defined six pathovars (aberrans, armoraciae, barbareae, campestris, incanae, and raphani) that cause diseases on cruciferous plants. However, pathogenicity assays with a range of strains and different hosts identifies only three types of symptom: black rot, leaf spot and bacterial blight. These findings raise the question of the genetic relatedness between strains assigned to different pathovars or symptom phenotypes. Here we have addressed this issue by multilocus sequence analysis of 42 strains. The X. campestris species was polymorphic at the 8 loci analysed and had a high genetic diversity; 23 sequence types were identified of which 16 were unique. All strains that induce black rot (pathovars aberrans and campestris) were genetically close but split in two groups. Only three clonal complexes were found, all within pathovar campestris. The assignment of the genome-sequenced strain 756C to pathovar raphani suggested from disease symptoms was confirmed, although this group of strains was particularly polymorphic. Strains belonging to pathovars barbareae and incanae were closely related, but distinct from pathovar campestris. There is evidence of genetic exchanges of housekeeping genes within this species as deduced from a clear incongruence between individual gene phylogenies and from network structures from SplitsTree analysis. Overall this study showed that the high genetic diversity derived equally from recombination and point mutation accumulation. However, X. campestris remains a species with a clonal evolution driven by a differential adaptation to cruciferous hosts.     

Electrotransformation of three pathovars of Xanthomonas campestris

Procedures for electrotransformation have been adapted for three pathovars of Xanthomonas campestris: campestris, vesicatoria and manihotis. Three differently sized plasmids (51, 9.3 and 3.3 kb) at different concentrations (10, 30 and 50 ng/sample) and different field strengths (10, 12, 14 and 18 kV/cm) were used. The efficiency of transformation was dependent on the recipient strain and the plasmid introduced. In general, a field strength of 14 kV/cm as well as a concentration of 30 ng plasmid DNA/sample seemed to be adequate for most conditions. Only X. campestris pv. vesicatoria strain 479 required a higher field strength for better efficiency. The plasmid size was inversely related to the efficiency of transformation; up to 1.6 × 10¹⁰, 5.3 × 10⁷ and 3.7 × 10⁵ transformants/g DNA were obtained using the pXG31 (3.3 kb), pUFR027 (9.3 kb) and RP4 (51 kb) plasmids, respectively.     

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.     

Deoxyribonucleic acid relatedness of 24 xanthomonad strains representing 23 Xanthomonas campestris pathovars and Xanthomonas fragariae

Twenty Xanthomonas campestris pathotype strains, three non-pathotype strains, and one strain of X. fragariae were studied by S1 DNA:DNA hybridization tests. The results of these tests do not support the retention of X. campestris as a single species. DNA reassociation values among many of the strains were low. Three clusters of closely related strains were observed, but nine strains did not cluster. Xanthomonas campestris pv. secalis was more closely related to X. fragariae than to any other X. campestris pathovar. Mapping the host family upon a three-dimensional genomic distance matrix of the xanthomonads suggested that strains attacking the same plant family usually show some relationship, but only a distant one. Thus, pathogenicity toward members of the same host family is not a measure of the genomic relationships of xanthomonads.     

Identification of Xanthomonas campestris pv. campestris galactose utilization genes from transcriptome data

A 70 mer oligonucleotide microarray was constructed to analyze genome-wide expression profiles of Xanthomonas campestris pv. campestris B100, a plant-pathogenic bacterium that is industrially employed to produce the exopolysaccharide xanthan gum which has many applications as a stabilizing, thickening, gelling, and emulsifying agent in food, pharmaceutical, and cosmetic industries. As an application example, global changes of gene expression were monitored during growth of X. campestris pv. campestris B100 on two different carbon sources. Exponential growing bacterial cultures were incubated either for 1h or permanently in minimal medium supplemented with 1% galactose in comparison to growth in minimal medium supplemented with 1% glucose. Six genes were identified that were significantly increased in gene expression under both growth conditions. These genes were located in three distinguished chromosomal regions in operon-like gene clusters. Genes from these clusters encode secreted glycosidases, which were predicted to be specific for galactose-containing carbohydrates, as well as transport proteins probably located in the outer and inner cell membrane. Finally genes from one cluster code for cytoplasmic enzymes of a metabolic pathway specific for the breakdown of galactose to intermediates of glycolysis.     

Sequence and molecular analysis of the rpoA cluster genes from Xanthomonas campestris pv. campestris

The Xanthomonas campestris rpsM (S13)-rpsK (S11)-rpsD (S4)-rpoA (alpha)-rplQ (L17) cluster, encoding RNA polymerase alpha-subunit and four ribosomal proteins, reside in a 3164-bp DNA region. The N-terminal sequence of the authentic alpha-protein determined chemically matches that predicted from the nucleotide sequence. rplQ is monocistronic, instead of being co-transcribed with the other genes as in Escherichia coli. Antiserum against the His-tagged alpha-protein cross-reacted with the E. coli alpha-protein.     

The type IV pre-pilin leader peptidase of Xanthomonas campestris pv. campestris is functional without conserved cysteine residues

Type IV pre-pilin leader peptidase was demonstrated to be required for protein secretion, in addition to its involvement in biogenesis of type IV pili. The type IV pre-pilin leader peptidase gene of Xanthomonas campestris pv. campestris was located on a 3 kb Acc l fragment on account of its hybridization with the DNA fragment containing the type IV pre-pilin leader-peptidase gene pilD/xcpA of Pseudomonas aeruginosa . Sequencing of the cloned fragment revealed an open reading frame (ORF) (designated xpsO ) of 287 amino acid residues. A protein with an apparent molecular mass of approximately 32.5 kDa was synthesized in vitro from a DNA fragment containing the xpsO gene. The amino acid sequence shares 50% identity with that of PilD throughout the entire sequence. Among other type IV pre-pilin leader peptidases, XpsO is unique in not having the two conserved -CXXC- motifs in a cytoplasmic domain. Instead, new motifs were noted when the protein was compared with XpsE, which is another member of the extracellular protein-secretion machinery. When the xpsO gene was introduced into the pilD mutant of P. aeruginosa , both the sensitivity against infection with the pilus-specific phage PO4 and the ability to secrete extracellular protein were recovered. Furthermore, immunoblot analysis indicated that the P. aeruginosa pilin was apparently processed in vivo by the xpsO gene product.     

Cloning of a locus involved in pathogenicity and production of extracellular polysaccharide and protease in Xanthomonas campestris pv. campestris

Abstract A Tn5-induced mutant of Xanthomonas campestris pv campestris deficient in protease and extracellular polysaccharide production, and non-pathogenic to turnip seedlings, was isolated. Recombinant clones which restored all three characteristics concomitantly were obtained from a wild-type genomic library. One of the plasmid clones, pIJ3090, showed no hybridization to previously isolated clones involved In protease production. The mutant was not complemented by other known clones involved in pathogenicity, in xanthan gum production or in protease production.     

Cloning and characterization of pathogenicity genes from Xanthomonas campestris pv. glycines.

Nonpathogenic mutants of Xanthomonas campestris pv. glycines 8ra were generated with N-methyl-N-nitro-N'-nitrosoguanidine to identify and characterize pathogenicity genes of the bacterium. A total of 16 nonpathogenic mutants were isolated from 2,000 colonies. One mutant, NP1, was chosen for further study. NP1 did not multiply in soybean cotyledons. A genomic library of strain 8ra was constructed in the cosmid pLAFR3, and the cosmids were tested for complementation in NP1. One cosmid clone, pIH1, which contained a 31-kb insert, complemented mutant NP1. A restriction map of pIH1 was constructed, and deletion analyses identified a 10-kb HindIII fragment that restored pathogenicity to NP1. Southern hybridization analysis indicated that DNA sequences in the 10-kb HindIII fragment are conserved among other X. campestris pathovars tested. Three regions responsible for restoring pathogenicity have been identified by Tn3-HoHo1 mutagenesis. A 2.7-kb ClaI fragment was sequenced, and two possible open reading frames (ORF1 and ORF2) were found. Results indicated that ORF2 but not ORF1 may be expressed in Escherichia coli and in X. campestris pv. glycines. The carboxy terminus of the potential polypeptide encoded by ORF2 has an amino acid sequence similar to that of the gamma subunit of oxaloacetate decarboxylase, which is involved in sodium ion transport in Klebsiella pneumoniae.     

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.     

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.     

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.     

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.     

Cloning of genes involved in pathogenicity of Xanthomonas campestris pv. campestris using the broad host range cosmid pLAFR1

A genomic library was prepared in Escherichia coli from DNA of wild-type Xanthomonas campestris pv. campestris (aetiological agent of crucifer black rot), partially digested with endonuclease EcoRI, using the mobilisable broad host range cosmid vector pLAFR1. Recombinant plasmids contained inserts ranging in size from 19.1 to 32.3 kb (mean 26.6). Certain of the clones complemented E. coli auxotrophic markers. Using the narrow host range plasmid pRK2013 as a helper the pooled recombinant plasmids were transferred conjugally to X. c. campestris mutants, and clones were identified which restored yellow pigmentation to white mutants, prototrophy to amino acid auxotrophs and pathogenicity towards turnip plants to two non-pathogenic mutants. The lesion in one mutant (8288, complemented by the plasmid pIJ3000) is unknown. However mutant 8237 is defective in production of extracellular protease and polygalacturonate lyase and restoration of pathogenicity by complementation with the plasmid pIJ3020 concomitantly restored both enzyme levels to wild-type values.