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.     

Cloning and analysis of a 35.3-kilobase DNA region involved in exopolysaccharide production by Xanthomonas campestris pv. campestris.

Cosmid clones able to restore exopolysaccharide production in possibly insertion sequence element-induced surface mutants of Xanthomonas campestris pv. campestris were isolated. By fragment-specific Tn5-lac mutagenesis of one of the cosmids, pXCB1002, a new DNA region which is involved in exopolysaccharide biosynthesis and which is organized into at least 12 complementation groups was identified.     

野油菜黄单胞菌野油菜致病变种8004菌株wxcA基因与EPS的产量有关

在以前的工作中,采用转座子Tn5 gusA5对野油菜黄单胞菌野油菜致病变种(Xcc)8004菌株进行诱变,获得一批胞外多糖(EPS)合成减少的突变体,对这些突变体的Tn5 gusA5的插入位点进行分析后,发现有两株突变体是wxcA基因不同插入位点的突变体。以前认为wxcA基因与脂多糖(LPS)的O-抗原合成有关而与EPS的合成无关。为明确wxc4基因的功能,对8004菌株的wxcA基因进行缺失,获得的△wxcA突变体的EPS产量与野生型菌株相比,减少了50％,并且一段PCR合成的包含wxcA基因的DNA片段能反式互补△wxcA突变体,恢复突变体的EPS产量。这证实了8004菌株的wxcA基因与EPS的合成产量有关。     

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.     

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.     

野油菜黄单胞菌野油菜致病变种中一个与EPS合成有关的新基因的鉴定

用转座子Tn5gusA5对野油菜黄单胞菌野油菜致病变种(Xanthomonas campestris pv.campestris,简称Xcc)野生型菌株8004进行诱变,分离到一批胞外多糖(EPS)合成减少的突变体。采用TAIL-PCR(thermal asymmetric interlaced PCR)分析突变体的Tn5gusA5插入位点,发现其中一株编号为151D09的突变体的插入位点位于Xcc 8004菌株的基因组编号为XC3695的ORF内,该ORF功能尚未见报道。序列分析表明,该ORF演绎的编码产物与Serratia marcescens的kdtX基因和Klebsiella pneumoniae的waaE基因演绎的编码产物分别具有52%和50%的相似性,并具有第2家族糖基转移酶的功能域, 因此暂将该ORF命名为waxE基因。用同源双交换方法构建了waxE基因的缺失突变体,并采用PCR和Southern杂交的方法对突变体进行了验证。waxE基因缺失突变体在营养丰富培养基的生长繁殖不受影响,但其EPS产量与野生型菌株8004相比,降低35%左右,并且一段PCR合成的包含waxE基因的DNA片段能反式互补waxE基因缺失突变体,恢复缺失突变体的EPS产量,表明Xcc waxE基因与EPS的生物合成有关。     

Transformation of Xanthomonas campestris pathovar campestris with plasmid DNA

Procedures for the introduction of plasmid DNA into Gram-negative bacteria have been adapted and optimized to permit transformation of the plant pathogen Xanthomonas campestris pathovar campestris with the cloning vector pKT230 and other broad-host-range plasmids. The technique involves CaCl2-induced competence and heat shock and is similar to that routinely used for Escherichia coli. Wild-type X. c. campestris strains appear to restrict incoming unmodified DNA, so that plasmid DNA for transformation must be prepared from X. c. campestris (into which it has previously been introduced by conjugation). To overcome this disadvantage a restriction-deficient mutant has been isolated.     

Evaluation of Xanthomonas campestris survival in a soil microcosm system.

Xanthomonas campestris pv. campestris is a pathogen of cruciferous plants. We studied the survival of the wild type strain and mutant derivatives which are deficient in exopolysaccharide (EPS) or in extracellular protease synthesis in soil microcosms in order to test the hypothesis that, in this environment, adherence to soil particles and scavenging of nutrients are very important strategies for bacterial survival. In sterile soil microcosms, differences in survival were only observed between the EPS producer and its mutant. In non-sterile soil experiments, survival of Prt- mutant was similar to EPS- mutant, suggesting that both characteristics have a strong influence in survival in the presence of the natural bacterial community. Bacterial decrease represented by the slope of regression lines was higher in non-sterile soil microcosms due to the influence of biotic interactions.     

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.     

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.     

Comparison of two Xanthomonas campestris pathovar campestris genomes revealed differences in their gene composition

For the Xanthomonas campestris pathovar campestris wild-type strain B100 a plasmid-based clone library was constructed. The plasmids carried chromosomal fragments of 3-4 kb in size that were tagged in vitro with the artificial transposon KAN-2. More than 3000 of the transposon target sites were characterized by DNA sequencing. The sequences obtained were compared to the recently published genome of Xanthomonas campestris pathovar campestris strain ATCC 33913. Most of the sequenced clones derived from strain B100 matched the chromosomal sequence of strain ATCC 33913. An alignment to the circular map of this chromosome revealed that the similarities were statistically distributed over the entire genome of strain ATCC 33913. The similarity was obvious for protein coding sequences, as well as for mobile genetic elements. However, four regions in the genome of Xanthomonas campestris pathovar campestris strain ATCC 33913, ranging in size from 11 to 37 kb, were not represented in the sequenced clone library of Xanthomonas campestris pathovar campestris strain B100. On the other hand, 1.2% of the sequenced clones originating from Xanthomonas campestris pathovar campestris strain B100 showed no or insignificant similarities to the genome of strain ATCC 33913.     

Increase of xanthan production by cloning xps genes into wild-type Xanthomonas campestris

Previously, genomic banks of Xanthomonas campestris were constructed in Escherichia coli, using mobilizable broad-host-range cosmids as the vectors. Following conjugal transfer, genes involved in the biosynthesis of xanthan polysaccharide (XPS) were cloned by the ability to restore the mucoid phenotype to the non-mucoid mutants. In this study, all clones were transferred into the wild-type strain Xc17 to evaluate the effects of the cloned genes on XPS production. Most clones showed no significant effect; however, two plasmids, pP2401 and pP2201, caused 10 and 15% yield increases, respectively, compared with that of controls. While it was not clear how pP2201 caused the yield increase, the effect of pP2401 seemed to result from elevated phosphomannose isomerase activity. Since XPS synthesis in X. campestris is a very efficient process, only relatively small increases are to be expected; an enhancement of productivity by 10-15% is important to the commercial production of xanthan.     

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.     

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.     

Extracellular proteases from Xanthomonas campestris pv. campestris, the black rot pathogen

Two proteases (PRT1 and PRT2) were fractionated from culture supernatants of wild-type Xanthomonas campestris pv. campestris by cation-exchange chromatography on SP-5PW. Inhibitor experiments showed that PRT 1 was a serine protease which required calcium ions for activity or stability or both and that PRT 2 was a zinc-requiring metalloprotease. PRT 1 and PRT 2 showed different patterns of degradation of beta-casein. The two proteases comprised almost all of the extracellular proteolytic activity of the wild type. A protease-deficient mutant which lacked both PRT 1 and PRT 2 showed considerable loss of virulence in pathogenicity tests when bacteria were introduced into mature turnip leaves through cut vein endings. This suggests that PRT 1 and PRT 2 have a role in black rot pathogenesis.     

Extracellular proteases from Xanthomonas campestris pv. campestris, the black rot pathogen.

Two proteases (PRT1 and PRT2) were fractionated from culture supernatants of wild-type Xanthomonas campestris pv. campestris by cation-exchange chromatography on SP-5PW. Inhibitor experiments showed that PRT 1 was a serine protease which required calcium ions for activity or stability or both and that PRT 2 was a zinc-requiring metalloprotease. PRT 1 and PRT 2 showed different patterns of degradation of beta-casein. The two proteases comprised almost all of the extracellular proteolytic activity of the wild type. A protease-deficient mutant which lacked both PRT 1 and PRT 2 showed considerable loss of virulence in pathogenicity tests when bacteria were introduced into mature turnip leaves through cut vein endings. This suggests that PRT 1 and PRT 2 have a role in black rot pathogenesis.     

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.