Effect of parB on plasmid stability and gene expression in Xanthomonas campestris

The stabilization locus parB was subcloned into the broad host range plasmid pAP2, which contains the alpha-amylase gene from Bacillus subtilis, and introduced into Xanthomonas campestris pv campestris and X.c.pv manihotis. Analysis of the stability of plasmid pAP2 (parB-) and pAP23 (parB+) showed that the parB locus decreased significantly the plasmid loss rate mainly by X.c.pv campestris. The lower efficiency of stabilization in X.c.pv manihotis was probably due to the incompatibility system between the native plasmids and the newly introduced pAP23. Although parB had conferred higher stability, it determined a lower rate of alpha-amylase activity even by the strain Cm where its stabilization rate was higher.     

A gene involved in quinate metabolism is specific to one DNA homology group of Xanthomonas campestris

A gene involved in quinate metabolism was cloned from Xanthomonas campestris pv. juglandis strain C5. The gene, qumA, located on a 4. 2-kb KpnI-EcoRV fragment in plasmid pQM38, conferred quinate metabolic activity to X. c. pv. celebensis. Tn3-spice insertional analyses further located the qumA gene on a region of about 3.0 kb within pQM38. Nucleotide sequencing of this 3.0-kb fragment reveals that the coding region of qumA is 2373 bp, the deduced amino acid sequence of which closely resembles a pyrrolo-quinoline quinone-dependent quinate dehydrogenase of Acinetobacter calcoaceticus. A 0.7 kb SalI-PstI fragment internal to qumA was used as a probe to hybridize against total genomic DNA from 43 pathovars of X. campestris. The fragment hybridized only to total genomic DNA from the four pathovars of DNA homology group 6, X. c. pv. celebensis, X. c. pv. corylina, X. c. pv. juglandis and X. c. pv. pruni, and from X. c. pv. carotae, which belongs to DNA homology group 5. This 0.7 kb fragment was also used as a probe to hybridize BamHI-digested total genomic DNAs from the four pathovars of DNA homology group 6 and X. c. pv. carotae. The restriction fragment length polymorphism pattern of DNA homology group 6 was different from that of X. c. pv. carotae. The probe hybridized to a 5.7-kb BamHI fragment in all four pathovars of group 6 and to a 6.1-kb BamHI fragment in three of four pathovars. It hybridized only to a 9. 9-kb BamHI fragment in X. c. pv. carotae. Quinate metabolism has previously been reported as a phenotypic property specific to X. campestris DNA homology group 6. Accordingly, a combination of the quinate metabolism phenotypic test and Southern hybridization using a qumA-derived probe will be very useful in the identification of pathovars in DNA homology group 6.     

The Blight-Associated Epitope and DNA Fragment from Xanthomonas campestris pv. campestris are not Required for Blight

Abstract: It has been reported that all tested naturally occurring strains of Xanthomonas campestris pv. campestris that are known to be capable of inducing blight symptoms in cabbage react with MAb A11 and hybridize with a 5.4-kb DNA fragment (in plasmid pJC41), cloned from the Xanthomonas campestris species type strain, Xcc528^T, whereas all tested naturally occurring strains that do not cause blight react with MAb X21 and do not hybridize to pJC41. The roles of the 5.4-kb DNA in pJC41 and the epitope recognized by MAb A11 in the pathogenicity of X. c. campestris strains that cause blight were examined by mutational analyses. A 4.0-kb deletion of the pJC41 region on the Xcc528^T chromosome was created by marker exchange, but the derivatives were evidently not affected in their ability to elicit blight symptoms. Nitrosoguanidine was used to mutagenize two blight strains, Xcc528^T and CAM19, and mutants were selected that were not reactive to MAb A11. The MAb A11-negative mutant of Xcc528^T was reactive to MAb X21, but was evidently not affected in the ability to elicit blight symptoms. MAb A11-negative mutants of CAM19, however, were not reactive to MAb X21, and showed reduction or loss of virulence, which suggested the requirement for at least one of the two antigens (to MAbs A11 or X21) for pathogenicity. A genomic library of CAM19 was made and screened for genes responsible for the production of the A11 antigen. Cosmid clones were identified that restored MAb A11 reactivity to the mutants. None of these cosmids restored the virulence of the mutant strains that had lost virulence. Therefore, neither the blight-associated 5.4-kb DNA fragment nor the MAb A11 antibody marker were required for blight symptom elicitation or pathogenicity.     

Detection of Xanthomonas campestris pv. citri by the polymerase chain reaction method.

pFL1 is a pUC9 derivative that contains a 572-bp EcoRI insert cloned from plasmid DNA of Xanthomonas campestris pv. citri XC62. The nucleotide sequence of pFL1 was determined, and the sequence information was used to design primers for application of the polymerase chain reaction (PCR) to the detection of X. campestris pv. citri, the causal agent of citrus bacterial canker disease. Seven 18-bp oligonucleotide primers were designed and tested with DNA from X. campestris pv. citri strains and other strains of X. campestris associated with Citrus spp. as templates in the PCR. Four primer pairs directed the amplification of target DNA from X. campestris pv. citri strains but not from strains of X. campestris associated with a different disease, citrus bacterial spot. Primer pair 2-3 directed the specific amplification of target DNA from pathotype A but not other pathotypes of X. campestris pv. citri. A pH 9.0 buffer that contained 1% Triton X-100 and 0.1% gelatin was absolutely required for the successful amplification of the target DNA, which was 61% G+C. Limits of detection after amplification and gel electrophoresis were 25 pg of purified target DNA and about 10 cells when Southern blots were made after gel electrophoresis and probed with biotinylated pFL1. This level of detection represents an increase in sensitivity of about 100-fold over that of dot blotting with the same hybridization probe. PCR products of the expected sizes were amplified from DNA extracted from 7-month-old lesions from which viable bacteria could not be isolated. These products were confirmed to be specific for X. campestris pv. citri by Southern blotting. This PCR-based detection protocol will be a useful addition to current methods of detection of this pathogen, which is currently the target of international quarantine measures.     

Use of cloned DNA methylase genes to increase the frequency of transfer of foreign genes into Xanthomonas campestris pv. malvacearum.

In vitro-packaged cosmid libraries of DNA from the bacterium Xanthomonas campestris pv. malvacearum were restricted 200- to 1,000-fold when introduced into Mcr+ strains of Escherichia coli compared with restriction in the Mcr- strain HB101. Restriction was predominantly associated with the mcrBC+ gene in E. coli. A plasmid (pUFR052) encoding the XmaI and XmaIII DNA methylases was isolated from an X. campestris pv. malvacearum library by a screening procedure utilizing Mcr+ and Mcr- E. coli strains. Transfer of plasmids from E. coli strains to X. campestris pv. malvacearum by conjugation was enhanced by up to five orders of magnitude when the donor cells contained pUFR052 as well as the plasmid to be transferred. Subcloning of pUFR052 revealed that at least two regions of the plasmid were required for full modification activity. Use of such modifier plasmids is a simple, novel method that may allow the efficient introduction of genes into any organism in which restriction systems provide a potent barrier to such gene transfer.     

Clustering of mutations blocking synthesis of xanthan gum by Xanthomonas campestris.

Mutations that block the synthesis of xanthan gum by Xanthomonas campestris B1459S-4L-II were isolated as nonmucoid colonies after treatment with ethyl methanesulfonate. Complete libraries of DNA fragments from wild-type X. campestris were cloned into Escherichia coli by using a broad-host-range cosmid vector and then transferred into each mutant strain by conjugal mating. Cloned fragments that restored xanthan gum synthesis (Xgs+; mucoidy) were compared according to restriction pattern, DNA sequence homology, and complementation of a subset of Xgs- mutations. Groups of clones that contained overlapping homologous DNA were found to complement specific Xgs- mutations. The results suggest clustering of the genetic loci involved in xanthan synthesis. The clustering occurred within three unlinked regions. Two forms of complementation were observed. In most instances, independently isolated cosmid clones that complemented a single mutation were found to be partially homologous. Less frequent was the second form of complementation, in which two cosmid clones that lacked any homologous sequences restored the mucoid phenotype to a single mutant. Finally, xanthan production was measured for wild-type X. campestris carrying multiple plasmid copies of the cloned xanthan genes.     

Characterization of transposon-generated protease mutant of xanthomonas campestris pathovar glycine 8ra

Protease negative mutant of Xanthomonas campestris pathovar glycine 8ra (prt-mutant) was constructed by mutagenesis employing artificial transposon Omegon-Km. Transposon delivery was conducted through diparental conjugation using X. campestris pathovar glycine 8ra as recipient and Escherichia coli S17-1 carrying pJFF 3500 plasmid as the donor. The frequency of transconjugation was found 1.9 x 10(-7) per recipient. Enzyme analysis indicated the presence of mutant with lower protease activity than that of the wild-type. Genetic analysis employing pulsed-field gel electrophoresis (PFGE) of the genomic DNA digested with AseI or SpeI restriction endonuclease could significantly differentiate X. campestris pathovar glycine 8ra prt from the wild-type parent. The 9.85 kb pLR omega 6 plasmid was constructed from the genomic DNA of the prt mutant after being digested with KpnI restriction endonuclease and ligated with T4 DNA ligase.     

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.     

Genetic construction of Xanthomonas campestris and xanthan gum production from whey

Summary Plasmids pUR291 and pNZ521 containing lacZ gene, maturation protein and proteinase P genes, were transferred into X. campestris either by conjugation or by transformation. Plasmid pNZ521 was also conjugally transferred into X. campestris XMT1 a transformant carrying plasmid pUR291. All the constructed strains were evaluated for xanthan gum production in either a medium of 50% whey or the same medium supplemented with 1.5% lactose or 1.5% glucose. Mixed cultures either with transconjugants or with transformants were tested for xanthan gum production as well.     

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.     

The A protein of the filamentous bacteriophage Cf of Xanthomonas campestris pv. citri.

Filamentous bacteriophages have very strict host specificities. Experiments were performed to investigate whether the A protein of the filamentous phage Cf, which infects Xanthomonas campestris pv. citri but not X. campestris pv. oryzae, is involved in determining Cf's host specificity. The gene encoding the A protein of Cf was cloned and expressed in X. campestris pv. citri. The genomic DNA of another filamentous bacteriophage, Xf, which infects X. campestris pv. oryzae but not X. campestris pv. citri, was then introduced by electroporation into X. campestris pv. citri that had expressed the A protein of Cf. The progeny phages thus produced were able to infect both X. campestris pv. oryzae and X. campestris pv. citri, indicating that the A protein of Cf was incorporated into the viral particles of Xf and conferred upon Xf the ability to infect the host of Cf. Inactivation of the A protein gene abolished the infectivity of Cf. The results of this study indicate that the A protein of Cf is responsible for controlling the host specificity of Cf.     

Electrotransformation of Xanthomonas campestris by RF DNA of filamentous phage φLf

Conditions were optimized for electrotransformation of Xanthomonas campestris pv. campestris by the replicative form (RF) DNA of filamentous phase phi Lf. Early logarithmic cells were washed exhaustively with deionized water and subjected to a pulse at a field strength of 12.5 kV/cm with a 25 microF capacitor and a 400 omega resistor. An efficiency of 5.1 x 10(7) pfu per microgram RF DNA was obtained. Under the same conditions, the broad host range plasmid pLAFR1 (21.6 kb) transformed X. campestris strains at efficiencies around 10(5) pfu per microgram DNA prepared from XcP20H. The advantages of the protocol used in the present study are that the cells can be washed with water instead of complex buffer, and the DNA used can be prepared by the alkaline method of Birnboim & Doly without purification by ultracentrifugation.     

Xanthomonas campestris pv. campestris secretes the endoglucanases ENGXCA and ENGXCB: construction of an endoglucanase-deficient mutant for industrial xanthan production

Xanthomonas campestris pv. campestris secretes at least two cellulose-degrading endoglucanases. One of these endoglucanases is encoded by the engXCA gene of X. c. pv. campestris 8400 that was previously characterized by Gough et al. [Gene (1990) 89: 53-59]. An additional endoglucanase encoded by the engXCB gene was identified in X. c. pv. campestris 8400 and FC2. The engXCB gene product that was grouped into the endoglucanase family E contains a putative N-terminal signal peptide, suggesting a secretion by the type II secretion system. The ENGXCB protein contributed approximately 8% to the cellulase activity in xanthan preparations. Deletion of engXCA and engXCB resulted in a fivefold reduction of the cellulose-degrading activity in xanthan preparations. The cellulase activity determined in xanthan preparations of the engXCA-engXCB mutant was only slightly higher than the activity found in preparations that were subjected to heat treatment. Mutations in engXCA and engXCB did not affect the growth rate and xanthan production of X. c. pv. campestris FC2 under several cultivation conditions. The engXCA-engXCB deletion mutant is markerless, which makes this mutant a valuable strain for xanthan production and approaches aimed at inactivating further genes encoding extracellular enzymes.     

Identification of plant-induced genes of the bacterial pathogen Xanthomonas campestris pathovar campestris using a promoter-probe plasmid

A promoter-probe plasmid suitable for use in Xanthomonas campestris pathovar campestris (causal agent of crucifer black rot) was constructed by ligating a broad host range IncQ replicon into the promoter-probe plasmid pKK232-8, which contains a promoterless chloramphenicol acetyltransferase gene. Xanthomonas chromosomal DNA fragments were 'shotgun' cloned into a restriction site in front of this gene, and the resulting library was transferred en masse into Xanthomonas. Individual transconjugants possessing DNA insertions with promoter activity in plants were identified by virtue of their ability to infect chloramphenicol-treated turnip seedlings. Of 19 transconjugants identified in this way five were chloramphenicol resistant both in turnip seedlings and on agar plates. However the remaining 14 were only chloramphenicol resistant in planta, and thus apparently contained plant-inducible promoter fragments. Resistance to chloramphenicol was correlated with increased chloramphenicol acetyltransferase activity for the transconjugants assayed. The promoter fragments were used to isolate genomic clones from a library, and the role of the genes contained in these clones in pathogenicity is being investigated.     

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.     

Construction of lactose-utilizing Xanthomonas campestris and production of xanthan gum from whey

Xanthomonas campestris pv. campestris possesses a low level of beta-galactosidase and therefore is not able to grow and produce significant amounts of xanthan gum in a medium containing lactose as the sole carbon source. In this study, a beta-galactosidase expression plasmid was constructed by ligating an X. campestris phage phi LO promoter with pKM005, a ColE1 replicon containing Escherichia coli lacZY genes and the lpp ribosome-binding site. It was then inserted into an IncP1 broad-host-range plasmid, pLT, and subsequently transferred by conjugation to X. campestris 17, where it was stably maintained. The lacZ gene under the control of the phage promoter was expressed at a high level, enabling the cells to grow in a medium containing lactose. Production of xanthan gum in lactose or diluted whey by the engineered strain was evaluated, and it was found to produce as much xanthan gum in these substrates as the cells did in a medium containing glucose.     

Differential expression of conserved protease genes in crucifer-attacking pathovars of Xanthomonas campestris.

Strains of Xanthomonas campestris pathovars armoraciae and raphani, which cause leaf spotting diseases in brassicas, produce a major extracellular protease in liquid culture which was partially purified. The protease (PRT 3) was a zinc-requiring metalloenzyme and was readily distinguishable from the two previously characterized proteases (PRT 1 and PRT 2) of X. campestris pv. campestris by the pattern of degradation of beta-casein and sensitivity to inhibitors. PRT 3 was produced at a low level in the vascular brassica pathogen X. campestris pv. campestris (five strains tested), in which PRT 1 and PRT 2 predominate. In contrast, expression of PRT 1, a serine protease, could not be detected in the six tested strains of the leaf spotting mesophyll pathogens. However, all these strains had DNA fragments which hybridized to a prtA probe and which probably carry a functional prtA (the structural gene for PRT 1). The structural gene for PRT 3 (prtC) was cloned by screening a genomic library of X. campestris pv. raphani in a protease-deficient X. campestris pv. campestris strain. Subcloning and Tn5 mutagenesis located the structural gene to 1.2 kb of DNA. DNA fragments which hybridized to the structural gene were found in all strains of the crucifer-attacking X. campestris pathovars tested as well as in a number of other pathovars. Experiments in which the pattern of protease production of the pathovars was manipulated by introduction of cloned genes into heterologous pathovars suggested that no determinative relationship exists between the pattern of protease gene expression and the (vascular or mesophyllic) mode of pathogenesis.     

Efficacy of four semi-selective media for recovery of Xanthomonas campestris pv. campestris from tropical soils

R. FUKUI, R. ARIAS AND R. ALVAREZ. 1994. Four semi-selective media, CS20 ABN, aesculin—trehalose (ET), Fieldhouse—Sasser (FS), and starch—methionine (SM), were compared for efficacy in recovering Xanthomonas campestris pv. campestris from artificially and naturally infested soils. Recoveries of X. c. campestris from soils infested with relatively large populations were similar on the four media. The FS and ET media exhibited higher selectivity against background saprophytes, whereas enumeration of X. c. campestris on CS20 ABN or SM was often hampered by the overgrowth of background saprophytes. Among three starch-containing media (CS20 ABN, ET and FS media), the zones of starch hydrolysis, characteristic of colonies of X. c. campestris, were most distinctive for FS medium. This allowed easier identification of the target colonies among numerous non-target colonies in tests with soil containing smaller numbers of X. c. campestris. Although the starch zone was also distinctive on CS20 ABN, this medium was not as effective as FS because the starch zones were so large that neighbouring zones fused with each other and many saprophytes formed colonies within the zones. Overall, FS was most suitable for soil studies in terms of the consistent recovery of the pathogen, the selectivity against saprophytes, and the differentiation from non-target organisms.