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.     

oriC region and replication termination site,dif, of the Xanthomonas campestris pv. campestris 17 chromosome

A 13-kb DNA fragment containing oriC and the flanking genes thdF, orf900, yidC, rnpA, rpmH, oriC, dnaA, dnaN, recF, and gyrB was cloned from the gram-negative plant pathogen Xanthomonas campestris pv. campestris 17. These genes are conserved in order with other eubacterial oriC genes and code for proteins that share high degrees of identity with their homologues, except for orf900, which has a homologue only in Xylella fastidiosa. The dnaA/dnaN intergenic region (273 bp) identified to be the minimal oriC region responsible for autonomous replication has 10 pure AT clusters of four to seven bases and only three consensus DnaA boxes. These findings are in disagreement with the notion that typical oriCs contain four or more DnaA boxes located upstream of the dnaA gene. The X. campestris pv. campestris 17 attB site required for site-specific integration of cloned fragments from filamentous phage phiLf replicative form DNA was identified to be a dif site on the basis of similarities in nucleotide sequence and function with the Escherichia coli dif site required for chromosome dimer resolution and whose deletion causes filamentation of the cells. The oriC and dif sites were located at 12:00 and 6:00, respectively, on the circular X. campestris pv. campestris 17 chromosome map, similar to the locations found for E. coli sites. Computer searches revealed the presence of both the dif site and XerC/XerD recombinase homologues in 16 of the 42 fully sequenced eubacterial genomes, but eight of the dif sites are located far away from the 6:00 point instead of being placed opposite the cognate oriC. The differences in the relative position suggest that mechanisms different from that of E. coli may participate in the control of chromosome replication.     

oriC Region and Replication Termination Site, dif, of the Xanthomonas campestris pv. campestris 17 Chromosome

A 13-kb DNA fragment containing oriC and the flanking genes thdF, orf900, yidC, rnpA, rpmH, oriC, dnaA, dnaN, recF, and gyrB was cloned from the gram-negative plant pathogen Xanthomonas campestris pv. campestris 17. These genes are conserved in order with other eubacterial oriC genes and code for proteins that share high degrees of identity with their homologues, except for orf900, which has a homologue only in Xylella fastidiosa. The dnaA/dnaN intergenic region (273 bp) identified to be the minimal oriC region responsible for autonomous replication has 10 pure AT clusters of four to seven bases and only three consensus DnaA boxes. These findings are in disagreement with the notion that typical oriCs contain four or more DnaA boxes located upstream of the dnaA gene. The X. campestris pv. campestris 17 attB site required for site-specific integration of cloned fragments from filamentous phage Lf replicative form DNA was identified to be a dif site on the basis of similarities in nucleotide sequence and function with the Escherichia coli dif site required for chromosome dimer resolution and whose deletion causes filamentation of the cells. The oriC and dif sites were located at 12:00 and 6:00, respectively, on the circular X. campestris pv. campestris 17 chromosome map, similar to the locations found for E. coli sites. Computer searches revealed the presence of both the dif site and XerC/XerD recombinase homologues in 16 of the 42 fully sequenced eubacterial genomes, but eight of the dif sites are located far away from the 6:00 point instead of being placed opposite the cognate oriC. The differences in the relative position suggest that mechanisms different from that of E. coli may participate in the control of chromosome replication.     

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.     

Homing in the field cricket,Gryllus campestris

Homing ability of the cricket Gryllus campestriswas experimentally studied in the field after passive and active displacements. Whatever the weather conditions and the nature of the displacement, crickets (18/18) home directly when they are located between 5 and 15 cm from their burrow by performing real-time orientation. When moved by the experimenter between 15 and 55 cm away from home, some crickets (26/43) can retrieve their burrow by systematic searching only after winding inward trips but they get lost (0/18) beyond 55 cm. After self-generated movements between 5 and 85 cm away from home, crickets (33/35) return to their burrow under blue sky, but they cannot home as well under overcast sky (22/34 from 5 to 65 cm and 0/5 beyond). The volatile memory orientation system allowing path integration is based on the polarized skylight of the sun.     

XerR, a negative regulator of XccR in Xanthomonas campestris pv. campestris, relieves its repressor function in planta

We previously reported that XccR, a LuxR-type regulator of Xanthomonas campestris pv. campestris (Xcc), activates the downstream proline iminopeptidase virulence gene (pip) in response to certain host plant factor(s). In this report, we further show that the expression of the xccR gene was repressed in the culture medium by an NtrC-type response regulator, which we named XerR (XccR expression-related, repressor), and that this repression was relieved when the bacteria were grown in planta. Such a regulatory mechanism is reinforced by the observations that XerR directly bound to the xccR promoter in vitro, and that mutations at the phosphorylation-related residues of XerR resulted in the loss of its repressor function. Furthermore, the expression level of xccR increased even in XerR-overexpressing Xcc cells when they were vacuum infiltrated into cabbage plants. We also preliminarily characterized the host factor(s) involved in the above mentioned interactions between Xcc and the host plant, showing that a plant material(s) with molecular weight(s) less than 1 kDa abolished the binding of XerR to the xccR promoter, while the same material enhanced the binding of XccR to the luxXc box in the pip promoter. Taken together, our results implicate XerR in a new layer of the regulatory mechanism controlling the expression of the virulence-related xccR/pip locus and provide clues to the identification of plant signal molecules that interact with XerR and XccR to enhance the virulence of Xcc.     

hxc2, an Arabidopsis mutant with an altered hypersensitive response to Xanthomonas campestris pv. campestris

A chemical mutagenized population of Arabidopsis Col-0-gl plants was screened for an altered hypersensitive response (HR) after spray inoculation with an HR-inducing isolate of Xanthomonas campestris pv. campestris (strain 147). Three classes of mutant were identified: those exhibiting an HR- phenotype or partial loss of HR; hyper-responsive mutants showing necrotic lesions rapidly leading to the collapse of leaves; and susceptible mutants. One mutant belonging to the susceptible class, hxc-2, was extensively characterized. The compatible phenotype observed several days after initiation of the interaction was confirmed by measurement of in planta bacterial growth and use of bacterial strains constitutively expressing the GUS reporter gene. In the same way, accumulation of autofluorescent compounds, salicylic acid production and defence gene expression in the mutant were found to be similar to that displayed by the susceptible ecotype. Inoculation of hxc-2 with different avirulent bacteria suggests that the mutation is specific for the interaction with the Xcc 147 strain, although the mutation has been shown to affect a single dominant locus, different from the resistance locus defined by genetic analysis of resistance to Xcc 147. Genetic mapping of the mutation indicated that it is located on chromosome III, defining a previously unknown resistance function in response to X. c. campestris.     

十字花科黑腐病菌hrpG基因原核表达

在十字花科黑腐病菌(Xcc)中,hrp基因对寄主的致病性和非寄主的超敏反应中起核心作用,而hrpG对整个hrp基因簇起调控作用.HrpG为OmpR家族的双组分系统感受调控蛋白,含有两个结构域,分别是N端Response_reg和C端Trans reg_C.本研究利用表达载体pQE-30 Xa,成功构建了HrpG的表达重组子,在E.coli M15 [pREP4]中进行诱导表达.通过调节诱导温度、IPTG浓度和诱导时间最终确定在温度为20℃,IPTG浓度为0.8 mmol/L,诱导表达4 h.hrpG基因在宿主细胞E.coli M15获得高效可溶性表达.目前尚未有可溶性HrpG蛋白获得成功表达的报导,本研究中获得HrpG蛋白在大肠杆菌获得大量可溶性的表达,将为in vitro研究HrpG的生理活性,特异的结合位点和调控功能研究打下良好基础.     

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.     

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.     

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.     

The selection of lactose-utiljzing,polysaccharide-producing strains ofXanthomonas campestris

Summary A procedure was developed to discriminate between singleXanthomonas campestris colony isolates which differed in growth form on solid media and in viscosity production in liquid media. The method was used to assess degree of variability between colonies and between successive generations of isolates chosen for industrial application.     

Pan Proteome of Xanthomonas campestris pv. campestris Isolates Contrasting in Virulence

Fully sequenced genomes of Xanthomonas campestris pv. campestris (Xcc) strains are reported. However, intra‐pathovar differences are still intriguing and far from clear. In this work, the contrasting virulence between two isolates of Xcc ‐ Xcc51 (more virulent) and XccY21 (less virulent) is evaluated by determining their pan proteome profiles. The bacteria are grown in NYG and XVM1 (optimal for induction of hrp regulon) broths and collected at the max‐exponential growth phase. Shotgun proteomics reveals a total of 329 proteins when Xcc isolates are grown in XVM1. A comparison of both profiles reveals 47 proteins with significant abundance fluctuations, out of which, 39 show an increased abundance in Xcc51 and are mainly involved in virulence/adaptation mechanisms, genetic information processing, and membrane receptor/iron transport systems, such as BfeA, BtuB, Cap, Clp, Dcp, FyuA, GroEs, HpaG, Tig, and OmpP6. Several differential proteins are further analyzed by qRT‐PCR, which reveals a similar expression pattern to the protein abundance. The data shed light on the complex Xcc pathogenicity mechanisms and point out a set of proteins related to the higher virulence of Xcc51. This information is essential for the development of more efficient strategies aiming at the control of black rot disease.     

Qualitative and comparative proteomic analysis of Xanthomonas campestris pv. campestris 17

The bacterium Xanthomonas campestris pathovar campestris (XCC) 17 is a local isolate that causes crucifer black rot disease in Taiwan. In this study, its proteome was separated using 2-DE and the well-resolved proteins were excised, trypsin digested, and analyzed by MS. Over 400 protein spots were analyzed and 281 proteins were identified by searching the MS or MS/MS spectra against the proteome database of the closely related XCC ATCC 33913. Functional categorization of the identified proteins matched 141 (50%) proteins to 81 metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In addition, we performed a comparative proteome analysis of the pathogenic strain 17 and an avirulent strain 11A to reveal the virulence-related proteins. We detected 22 up-regulated proteins in strain 17 including the degrading enzymes EngXCA, HtrA, and PepA, which had been shown to have a role in pathogenesis in other bacteria, and an anti-host defense protein, Ohr. Thus, further functional studies of these up-regulated proteins with respect to their roles in XCC pathogenicity are suggested.     

The galU gene of Xanthomonas campestris pv. campestris is involved in bacterial attachment,cell motility,polysaccharide synthesis,virulence, and tolerance to various stresses

Uridine triphosphate (UTP)-glucose-1-phosphate uridylyltransferase (GalU; EC 2.7.7.9) is an enzyme that catalyzes the formation of uridine diphosphate (UDP)-glucose from UTP and glucose-1-phosphate. GalU is involved in virulence in a number of animal-pathogenic bacteria since its product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharide and exopolysaccharide. However, its function in Xanthomonas campestris pv. campestris, the phytopathogen that causes black rot in cruciferous plants, is unclear. Here, we characterized a galU mutant of X. campestris pv. campestris and showed that the X. campestris pv. campestris galU mutant resulted in a reduction in virulence on the host cabbage. We also demonstrated that galU is involved in bacterial attachment, cell motility, and polysaccharide synthesis. Furthermore, the galU mutant showed increased sensitivity to various stress conditions including copper sulfate, hydrogen peroxide, and sodium dodecyl sulfate. In addition, mutation of galU impairs the expression of the flagellin gene fliC as well as the attachment-related genes xadA, fhaC, and yapH. In conclusion, our results indicate involvement of galU in the virulence factor production and pathogenicity in X. campestris pv. campestris, and a role for galU in stress tolerance of this crucifer pathogen.