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At least 19 recordsLinked to original sources

PubMed2006

The cloning, crystallization and preliminary X-ray analysis of XC2113, a YaeQ protein from Xanthomonas campestris.

Xanthomonas campestris is a Gram-negative bacterium that is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. It is therefore important to identify potential pathogenic factors involved in this plant disease. Here, the cloning, expression, crystallization and preliminary X-ray analysis of XC2113, a YaeQ protein possibly involved in the production of virulence factors in Xanthomonas campestris pathovar campestris, are reported. The XC2113 crystals diffracted well to a resolution of at least 1.28 A. They are orthorhombic and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 32.86, b = 62.69, c = 79.96 A.

Bacterial Proteins↗

PubMed2004

Biofilm formation and dispersal in Xanthomonas campestris.

Xanthomonas campestris pathovar campestris is the causal agent of black rot disease of cruciferous plants. A cell-cell signalling system encoded by genes within the rpf cluster is required for the full virulence of this plant pathogen. This system has recently been implicated in regulation of the formation and dispersal of Xanthomonas biofilms.

Aconitate Hydratase↗

PubMed1997

Evaluation of the role of recA protein in plant virulence with recA mutants of Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris NRRL B1459 recA mutants were isolated by recombination with an interrupted Rhizobium etli recA gene and selection of double recombinants. The mutants were impaired in homologous genetic recombination and in DNA repair as judged by their sensitivity to methyl-methane-sulfonate and to UV irradiation; these defects are complemented in trans by the R. etli recA gene. Virulence of X. campestris pv. campestris NRRL B1459 to cabbage is considerably diminished by the recA mutation. The recA mutation is not correlated with the frequency of occurrence of a genetic rearrangement that affects chemotaxis, plant virulence, and xanthan gum production.

Blotting, Southern↗

PubMed2002

Isolation and characterization of a porin-like outer membrane protein from Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris, a plant-associated pathogenic bacterium, is the causal agent of foliar spots and blights in crucifers. The major outer membrane protein, Omp37, of 37 kDa, has been identified, purified to homogeneity, and its characterization has also been carried out. Native Omp37 behaved as a trimer, as revealed by gel filtration and SDS-PAGE. FTIR measurements revealed a high beta-structure content. The pore-forming ability of the purified Omp37 was studied by the liposome swelling assay. Omp37, to our knowledge, is the first porin that has been isolated from Xanthomonas. This study clearly demonstrates that Omp37 is related to the family of trimeric bacterial porins.

Chromatography, Gel↗

PubMed1996

Catabolite-repressor-like protein regulates the expression of a gene under the control of the Escherichia coli lac promoter in the plant pathogen Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris, the causal agent of black-rot disease of cruciferous plants, and an important industrial microbe, was able to express the Escherichia coli beta-glucuronidase reporter gene (uidA) when fused to the E. coli lactose operon promoter on a wide-host-range plasmid vector. The gene fusion is expressed constitutively at high levels in both complex and defined media using a wide range of carbon sources, and is not repressible by glucose or inducible by the gratuitous lac inducer isopropyl beta-D-thiogalactoside. An X. campestris campestris strain with a lesion in the clp (catabolite-repressor-like protein) locus, and containing the placluidA fusion, was tested for beta-glucuronidase activity. We found that the expression of the placluidA fusion gene is dependent on the presence of catabolite-repressor-like protein, with an approximately 75% reduction of expression in the clp -deficient mutant.

Cloning, Molecular↗

PubMed1995

Nucleotide sequence and expression of UDP-glucose dehydrogenase gene required for the synthesis of xanthan gum in Xanthomonas campestris.

Xanthomonas campestris pv. campestris, producing large amounts of exopolysaccharide xanthan gum, has a mucoid phenotype. Strain SD7 was a non-mucoid mutant deficient in UDP-glucose dehydrogenase. A DNA fragment able to complement the mutation of SD7 was cloned from the parental wild-type strain Xc11. Sequence analysis of the region required for the complementation revealed an open reading frame which could encode a polypeptide of 445 amino acids with a calculated molecular weight of 48,432, a size similar to that of the product produced by maxicell. The amino acid sequence had significant homology to that of the GDP-mannose dehydrogenase from Pseudomonas aeruginosa.

Amino Acid Sequence↗

PubMed2005

Cloning, purification, crystallization and preliminary X-ray analysis of XC229, a conserved hypothetical protein from Xanthomonas campestris.

Xanthomonas campestris pv. campestris is a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. Its genome contains approximately 4500 genes, roughly one third of which have no known structure and/or function. However, some of these unknown genes are highly conserved among several different bacterial genuses. XC229 is one such protein containing 134 amino acids. It was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal diffracted to a resolution of at least 1.80 A. It is cubic and belongs to space group I2(x)3, with unit-cell parameters a = b = c = 106.8 A. It contains one or two molecules per asymmetric unit.

Bacterial Proteins↗

PubMed2005

Preparation, crystallization and preliminary X-ray analysis of XC2382, an ApaG protein of unknown structure from Xanthomonas campestris.

Xanthomonas campestris pv. campestris is the causative agent of black rot, one of the major worldwide diseases of cruciferous crops. Its genome encodes approximately 4500 proteins, roughly one third of which have unknown function. XC2382 is one such protein, with a MW of 14.2 kDa. Based on a bioinformatics study, it was annotated as an ApaG gene product that serves multiple functions. The ApaG protein has been overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of at least 2.30 A. They are tetragonal and belong to space group P4(1/3), with unit-cell parameters a = b = 57.6, c = 122.9 A. There are two, three or four molecules in the asymmetric unit.

Bacterial Proteins↗

PubMed1996

The gene encoding UDP-glucose pyrophosphorylase is required for the synthesis of xanthan gum in Xanthomonas campestris.

Xanthomonas campestris pv. campestris produces a large quantity of exopolysaccharide, xanthan gum, rendering the colonies mucoid. G76E was a non-mucoid mutant isolated from Xc17 by Tn5 mutagenesis. A 3.0-kb KpnI-EcoRI fragment from the Xc17 chromosome was able to restore mucoid phenotype to G76E. Sequence analysis of the region responsible for the restoration revealed an open reading frame, ORF324, able to encode a polypeptide of 35,232 Da which shows striking similarity to the UDP-glucose pyrophosphorylases from bacteria. The activity of UDP-glucose pyrophosphorylase reduced drastically in G76E was found to be regained in the presence of the cloned 3.0-kb KpnI-EcoRI fragment. In vitro expression of the gene in the S30 transcription/translation system produced a protein of ca. 35 kDa.

Amino Acid Sequence↗

PubMed2005

Preparation, crystallization and preliminary X-ray characterization of a conserved hypothetical protein XC1692 from Xanthomonas campestris.

Xanthomonas campestris pv. campestris strain 17 is a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. Its genome contains approximately 4500 genes, one third of which have no known structure and/or function yet are highly conserved among several different bacterial genuses. One of these gene products is XC1692 protein, containing 141 amino acids. It was overexpressed in Escherichia coli, purified and crystallized in a variety of forms using the hanging-drop vapour-diffusion method. The crystals diffract to at least 1.45 A resolution. They are hexagonal and belong to space group P6(3), with unit-cell parameters a = b = 56.9, c = 71.0 A. They contain one molecule per asymmetric unit.

Bacterial Proteins↗

PubMed2005

Cloning, purification crystallization and preliminary X-ray characterization of a conserved hypothetical protein XC6422 from Xanthomonas campestris.

Xanthomonas campestris pv. campestris is a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. Its genome contains approximately 4500 genes, roughly one third of which have no known structure and/or function. However, some genes of unknown function are highly conserved among several different bacterial genuses. XC6422 is one such conserved hypothetical protein and has been overexpressed in Escherichia coli, purified and crystallized in a variety of forms using the hanging-drop vapour-diffusion method. Crystals grew to approximately 2 x 1.5 x 0.4 mm in size after one week and diffracted to at least 1.6 A resolution. They belong to the monoclinic space group C2, with one molecule per asymmetric unit and unit-cell parameters a = 75.8, b = 79.3, c = 38.2 A, beta = 109.4 degrees. Determination of this structure may provide insights into the protein's function.

Bacterial Proteins↗

PubMed2003

[Identification and cloning of a novel gene involved in EPS biosynthesis of Xanthomonas campestris pv. campestris].

Xanthomonas campestris pv. campestris ( Xcc), causative agent of the black rot disease of cruciferous crops worldwide, produces large amount of extracellular polysaccharide( EPS), which has found wide applications in industry. In order to clone genes involved in EPS biosynthesis, Xcc wild-type strain 8004 was mutagenized with transposon Tn5gus A5, and a number of EPS-defective mutants were isolated. The Tn5gusA5 insertion sites in the mutants were analyzed by using thermal asymmetric interlaced PCR(TAIL-PCR), and the corresponding genes were identified by homology blast to the completely sequenced genome of Xcc 8004 strain. A novel gene, waxE, identified from the EPS-defective mutant 151D09, was found to be disrupted by the insertion of Tn5gusA5 in the open reading frame(ORF) with genome coordinates 4478998bp to 4479819bp.This gene showed 52% similarity to the kdtX gene of Serratia marcescens and 50% to the waaE of Klebsiella pneumoniae at amino acid level, with characteristics of glycostransferase 2 family domain. In order to identify the function of waxE gene, waxE gene deletion mutant of Xcc 8004 was constructed by gene replacement strategy in which waxE gene of genome was replaced by kanamycin resistant gene kan. The waxE gene deletion mutant strain, named Xcc 8570, was confirmed by both PCR and southern analysis. The growth rate of the deletion mutant 8570 in rich medium was not affected, but the EPS yield reduced by 35% as compared with the wildtype strain 8004. The deletion mutant could be completmented in trans with plasmid pLATC8976 harboring an intact waxE gene, and the EPS yield of the mutant was restored. The combined data showed that waxE gene involved in EPS biosynthesis in Xcc.

Amino Acid Sequence↗

PubMed2005

Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv. campestris.

Xanthomonas campestris pathovar campestris (Xcc) is the causative agent of crucifer black rot disease, which causes severe losses in agricultural yield world-wide. This bacterium is a model organism for studying plant-bacteria interactions. We sequenced the complete genome of Xcc 8004 (5,148,708 bp), which is highly conserved relative to that of Xcc ATCC 33913. Comparative genomics analysis indicated that, in addition to a significant genomic-scale rearrangement cross the replication axis between two IS1478 elements, loss and acquisition of blocks of genes, rather than point mutations, constitute the main genetic variation between the two Xcc strains. Screening of a high-density transposon insertional mutant library (16,512 clones) of Xcc 8004 against a host plant (Brassica oleraceae) identified 75 nonredundant, single-copy insertions in protein-coding sequences (CDSs) and intergenic regions. In addition to known virulence factors, full virulence was found to require several additional metabolic pathways and regulatory systems, such as fatty acid degradation, type IV secretion system, cell signaling, and amino acids and nucleotide metabolism. Among the identified pathogenicity-related genes, three of unknown function were found in Xcc 8004-specific chromosomal segments, revealing a direct correlation between genomic dynamics and Xcc virulence. The present combination of comparative and functional genomic analyses provides valuable information about the genetic basis of Xcc pathogenicity, which may offer novel insight toward the development of efficient methods for prevention of this important plant disease.

Bacterial Proteins↗

PubMed2003

Molecular genetic analyses of potential beta-galactosidase genes in Xanthomonas campestris.

Xanthomonas campestris pv. campestris, which displays no significant beta-1,4-D-galactopyranosidase activity, has three annotated beta-galactosidase genes in the sequenced genome, designated galA, galB and galC herein. GalA and GalB are similar to glycosyl hydrolase (GH) family 2 enzymes, including Escherichia coli LacZ. galA and galB cannot express detectable activity even after being cloned in-frame and driven by the vector's promoter. GalC is a GH35 enzyme homologous to the Xanthomonas axonopodis pv. manihotis Bga. The latter cleaves beta,1-3-linked galactose 1,000 times faster than beta,1-4-linked galactose and is not responsible for lactose utilization. In X. campestris pv. campestris cells, GalC is readily detectable by Western blotting, and the levels can be increased by cloning the gene under the control of the vector's promoter. Results of insertional mutation, transcriptional fusion assay and Western blotting indicated that galC, clustered with several GH genes, is cotranscribed with the upstream gene(s) and is expressed constitutively. Xc17L is a previously isolated mutant with elevated beta-galactosidase activity and a greatly improved ability to grow on lactose. Results of DNA sequencing of Xc17L galA, galB and galC, enzyme assays of galA, galB and galC mutants derived from Xc17L, and Western blotting of GalC in Xc17L indicated that the three beta-galactosidase genes do not encode the elevated beta-galactosidase activity in Xc17L. The presence of a fourth beta-galactosidase gene is proposed.

Amino Acid Motifs↗

PubMed1989

Genetic and biochemical analysis of protein export from Xanthomonas campestris.

Xanthomonas campestris pv. campestris, a Gram-negative phytopathogen, produces a number of extracellular enzymes which can degrade components of the host plant cell. Some non-pathogenic mutants, derived by chemical mutagenesis, were found to be defective in the export but not the synthesis of a number of these enzymes. The pathogenicity and export lesions in one such mutant, strain 8288, could be complemented by a cosmid clone pIJ3000 from the Xanthomonas library. Mutagenesis of pIJ3000 with the transposon Tn5 followed by recombination into the corresponding region of the chromosome has revealed a cluster of 6 to 8 genes whose function is required for enzyme export. Sequence analysis of part of the cluster has revealed two open reading frames that would encode proteins with extensive hydrophobic domains. Export-defective mutants retain the normally exported enzymes in the periplasmic space. These forms have the same molecular weight as the extracellular forms, suggesting that the signal sequence has been properly processed. The results are consistent with a mechanism of sequential translocation across cytoplasmic and outer membrane via the periplasm. The second translocation step may be mediated by the products of export genes. This may be a common export mechanism amongst Gram-negative bacteria but other mechanisms do exist, sometimes in parallel in the same cell, and these are briefly reviewed.

Bacterial Proteins↗

PubMed1987

Genetic and physical analyses of a cluster of genes essential for xanthan gum biosynthesis in Xanthomonas campestris.

Xanthomonas campestris produces copious amounts of a complex exopolysaccharide, xanthan gum. Nonmucoid mutants, defective in synthesis of xanthan polysaccharide, were isolated after nitrosoguanidine mutagenesis. To isolate genes essential for xanthan polysaccharide synthesis (xps), a genomic library of X. campestris DNA, partially digested with SalI and ligated into the broad-host-range cloning vector pRK293, was constructed in Escherichia coli. The pooled clone bank was conjugated en masse from E. coli into three nonmucoid mutants by using pRK2013, which provides plasmid transfer functions. Kanamycin-resistant exconjugants were then screened for the ability to form mucoid colonies. Analysis of plasmids from several mucoid exconjugants indicated that overlapping segments of DNA had been cloned. These plasmids were tested for complementation of eight additional nonmucoid mutants. A 22-kilobase (kb) region of DNA was defined physically by restriction enzyme analysis and genetically by ability to restore mucoid phenotype to 10 of the 11 nonmucoid mutants tested. This region was further defined by subcloning and by transposon mutagenesis with mini-Mu(Tetr), with subsequent analysis of genetic complementation of nonmucoid mutants. A region of 13.5 kb of DNA was determined to contain at least five complementation groups. The effect of plasmids containing cloned xps genes on xanthan gum synthesis was evaluated. One plasmid, pCHC3, containing a 12.4-kb insert and at least four linked xanthan biosynthetic genes, increased the production of xanthan gum by 10% and increased the extent of pyruvylation of the xanthan side chains by about 45%. This indicates that a gene affecting pyruvylation of xanthan gum is linked to this cluster of xps genes.

Chromosome Mapping↗

PubMed1984

Genetic Construction of Lactose-Utilizing Xanthomonas campestris.

Xanthomonas campestris, the producer of xanthan gum, possesses a beta-galactosidase of very low specific activity. Plasmid pGC9114 (RP1::Tn951), generated by the transposition of the lactose transposon Tn951 to RP1, was conjugally transferred into XN1, a nalidixic acid-resistant derivative of X. campestris NRRL B-1459S-4L. Transfer occurred on membrane filters and in broth. The beta-galactosidase gene of Tn951 was expressed in X. campestris. The specific activity of beta-galactosidase in transconjugants was over 200-fold higher than that in XN1, and transconjugants grew as well in lactose-based media as in glucose-based media. The lactose-utilizing transconjugants could potentially be used to produce xanthan gum from cheese whey.

Journal Article↗

PubMed1984

Differentiation between Xanthomonas campestris pv. oryzae, Xanthomonas campestris pv. oryzicola and the bacterial 'brown blotch' pathogen on rice by numerical analysis of phenotypic features and protein gel electrophoregrams.

Thirty-five Xanthomonas campestris pv. oryzae, fourteen X. campestris pv. oryzicola strains and six 'brown blotch' pathogens of rice, all of different geographical origin, were studied by numerical analysis of 133 phenotype features and gel electrophoregrams of soluble proteins, %G + C determinations and DNA:rRNA hybridizations. The following conclusions were drawn. (i) The Xanthomonas campestris pathovars oryzae and oryzicola display clearly distinct protein patterns on polyacrylamide gels and can be differentiated from each other by four phenotype tests. (ii) Both pathovars are indeed members of Xanthomonas which belongs to a separate rRNA branch of the second rRNA superfamily together with the rRNA branches of Pseudomonas fluorescens, Marinomonas, Azotobacter, Azomonas and Frateuria. (iii) 'Brown blotch' strains are considerably different from X. campestris pv. oryzae and oryzicola. They are not members of the genus Xanthomonas, but are more related to the generically misnamed. Flavobacterium capsulatum, Pseudomonas paucimobilis, Flavobacterium devorans and 'Pseudomonas azotocolligans' belonging in the fourth rRNA superfamily. (iv) No correlation was found between the virulence, pathogenic groups or geographical distribution of X. campestris pv. oryzae or oryzicola strains and any phenotypic or protein electrophoretic property or clustering.

Base Composition↗