| 1. |
Liu CC,
Wang HR,
Chou HC,
Chang WT,
Tu J,
( 1992 ) Analysis of the genes and gene products of Xanthomonas transposable elements ISXc5 and ISXc4. PMID : 1327971 : DOI : 10.1016/0378-1119(92)90015-h Abstract >>
A series of deletion mutants have been constructed for the gene analyses of transposable elements, ISXc5 and ISXc4, derived from Xanthomonas. At least two element-encoded polypeptides of 48 kDa and 40 kDa have been identified in the minicell-producing Escherichia coli strain, TC410. A study of the element transposition and cointegrate resolution revealed that the 48-kDa and 40-kDa polypeptide are both involved in translocation of the elements, the 48-kDa product being involved in transposition of the elements and the 40-kDa product being involved in cointegrate resolution.
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2. |
Hwang I,
Lim SM,
Shaw PD,
( 1992 ) Cloning and characterization of pathogenicity genes from Xanthomonas campestris pv. glycines. PMID : 1312532 : DOI : 10.1128/jb.174.6.1923-1931.1992 PMC : PMC205798 Abstract >>
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.
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3. |
Kholodii G,
Bogdanova E,
( 2002 ) Tn5044-conferred mercury resistance depends on temperature: the complexity of the character of thermosensitivity. PMID : 12403178 : Abstract >>
Escherichia coli K12 containing the transposon Tn5044 mer operon (merR, T, P, C, and A genes) is resistant to mercuric chloride at 30 degrees C but sensitive to this compound at 37-41.5 degrees C. We have studied the mechanism underlying the temperature-sensitive nature of this mercury resistance phenotype, and found that the expression of the Tn5044 merA gene coding for mercuric reductase (MerA) is severely inhibited at non-permissive temperatures. Additionally, MerA showed a considerably reduced functional activity in vivo at non-permissive temperatures. However, the temperature-sensitive character of the functioning of this enzyme in cell extracts, where it interacted with one of the low-molecular weight SH compounds rather than with the transport protein MerT (as is the case in vivo), was not apparent. These data suggest that the temperature-sensitive mercury resistance phenotype should stay under control at two stages: when the merA gene is expressed and when its product interacts with MerT to accept the mercuric ion.
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4. |
Heu S,
Oh J,
Kang Y,
Ryu S,
Cho SK,
Cho Y,
Cho M,
( 2001 ) gly gene cloning and expression and purification of glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines 8ra. PMID : 11526012 : DOI : 10.1128/aem.67.9.4105-4110.2001 PMC : PMC93136 Abstract >>
Glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines, inhibits the growth of X. campestris pv. vesicatoria. We have cloned and expressed the genes encoding glycinecin A in Escherichia coli. Recombinant glycinecin A was purified from cell extracts by ammonium sulfate precipitation followed by chromatography on Q-Sepharose, Mono Q (ion exchange), and size exclusion columns. Purified glycinecin A is composed of two polypeptides, is active over a wide pH range (6 to 9), and is stable at temperatures up to 60 degrees C. Glycinecin A is a heterodimer consisting of 39- and 14-kDa subunits, as revealed through size exclusion chromatography and cross-linking analysis. Two genes, glyA and glyB, encoding the 39- and 14-kDa subunits, respectively, were identified based on the N-terminal sequences of the subunits. From the nucleotide sequences of glyA and glyB, we conclude that both genes are translated as bacteriocin precursors that include N-terminal leader sequences. When expressed in E. coli, recombinant glycinecin A was found primarily in cell extracts. In contrast, most glycinecin A from Xanthomonas was found in the culture media. E. coli transformed with either glyA or glyB separately did not show the bacteriocin activity.
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5. |
Gosset G,
Bonner CA,
Jensen RA,
( 2001 ) Microbial origin of plant-type 2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate synthases, exemplified by the chorismate- and tryptophan-regulated enzyme from Xanthomonas campestris. PMID : 11395471 : DOI : 10.1128/JB.183.13.4061-4070.2001 PMC : PMC95290 Abstract >>
Enzymes performing the initial reaction of aromatic amino acid biosynthesis, 2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthases, exist as two distinct homology classes. The three classic Escherichia coli paralogs are AroA(I) proteins, but many members of the Bacteria possess the AroA(II) class of enzyme, sometimes in combination with AroA(I) proteins. AroA(II) DAHP synthases until now have been shown to be specifically dedicated to secondary metabolism (e.g., formation of ansamycin antibiotics or phenazine pigment). In contrast, here we show that the Xanthomonas campestris AroA(II) protein functions as the sole DAHP synthase supporting aromatic amino acid biosynthesis. X. campestris AroA(II) was cloned in E. coli by functional complementation, and genes corresponding to two possible translation starts were expressed. We developed a 1-day partial purification method (>99%) for the unstable protein. The recombinant AroA(II) protein was found to be subject to an allosteric pattern of sequential feedback inhibition in which chorismate is the prime allosteric effector. L-Tryptophan was found to be a minor feedback inhibitor. An N-terminal region of 111 amino acids may be located in the periplasm since a probable inner membrane-spanning region is predicted. Unlike chloroplast-localized AroA(II) of higher plants, X. campestris AroA(II) was not hysteretically activated by dithiols. Compared to plant AroA(II) proteins, differences in divalent metal activation were also observed. Phylogenetic tree analysis shows that AroA(II) originated within the Bacteria domain, and it seems probable that higher-plant plastids acquired AroA(II) from a gram-negative bacterium via endosymbiosis. The X. campestris AroA(II) protein is suggested to exemplify a case of analog displacement whereby an ancestral aroA(I) species was discarded, with the aroA(II) replacement providing an alternative pattern of allosteric control. Three subgroups of AroA(II) proteins can be recognized: a large, central group containing the plant enzymes and that from X. campestris, one defined by a three-residue deletion near the conserved KPRS motif, and one possessing a larger deletion further downstream.
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6. |
Rowe-Magnus DA,
Guerout AM,
Ploncard P,
Dychinco B,
Davies J,
Mazel D,
( 2001 ) The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons. PMID : 11209061 : DOI : 10.1073/pnas.98.2.652 PMC : PMC14643 Abstract >>
Integrons are genetic elements that acquire and exchange exogenous DNA, known as gene cassettes, by a site-specific recombination mechanism. Characterized gene cassettes consist of a target recombination sequence (attC site) usually associated with a single open reading frame coding for an antibiotic resistance determinant. The affiliation of multiresistant integrons (MRIs), which contain various combinations of antibiotic resistance gene cassettes, with transferable elements underlies the rapid evolution of multidrug resistance among diverse Gram-negative bacteria. Yet the origin of MRIs remains unknown. Recently, a chromosomal super-integron (SI) harboring hundreds of cassettes was identified in the Vibrio cholerae genome. Here, we demonstrate that the activity of its associated integrase is identical to that of the MRI integrase, IntI1. We have also identified equivalent integron superstructures in nine distinct genera throughout the gamma-proteobacterial radiation. Phylogenetic analysis revealed that the evolutionary history of the system paralleled that of the radiation, indicating that integrons are ancient structures. The attC sites of the 63 antibiotic-resistance gene cassettes identified thus far in MRIs are highly variable. Strikingly, one-fifth of these were virtually identical to the highly related yet species-specific attC sites of the SIs described here. Furthermore, antimicrobial resistance homologues were identified among the thousands of genes entrapped by these SIs. Because the gene cassettes of SIs are substrates for MRIs, these data identify SIs as the source of contemporary MRIs and their cassettes. However, our demonstration of the metabolic functions, beyond antibiotic resistance and virulence, of three distinct SI gene cassettes indicates that integrons function as a general gene-capture system for bacterial innovation.
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7. |
Slater H,
Alvarez-Morales A,
Barber CE,
Daniels MJ,
Dow JM,
( 2000 ) A two-component system involving an HD-GYP domain protein links cell-cell signalling to pathogenicity gene expression in Xanthomonas campestris. PMID : 11123673 : DOI : 10.1046/j.1365-2958.2000.02196.x Abstract >>
The synthesis of extracellular enzymes and extracellular polysaccharide (EPS) in Xanthomonas campestris pv. campestris (Xcc) is regulated by a cluster of genes called rpf (for regulation of pathogenicity factors). Two of the genes, rpfF and rpfB, have previously been implicated in the synthesis of a diffusible regulatory molecule, DSF. Here, we describe a screen of transposon insertion mutants of Xcc that identified two DSF-overproducing strains. In each mutant, the gene disrupted is rpfC, which encodes a hybrid two-component regulatory protein in which the sensor and regulator domains are fused and which contains an additional C-terminal phosphorelay (HPt) domain. We show that rpfC is in an operon with rpfH and rpfG. The predicted protein RpfG has a regulatory input domain attached to a specialized version of an HD domain, previously suggested to function in signal transduction. The predicted protein RpfH is structurally related to the sensory input domain of RpfC. We show that RpfC and RpfG act positively to regulate the synthesis of extracellular enzymes and EPS, but that RpfC acts negatively to regulate the synthesis of DSF. We propose that RpfGHC is a signal transduction system that couples the synthesis of pathogenicity factors to sensing of environmental signals that may include DSF itself.
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8. |
Gorlenko Z,
Rybochkin V,
Mindlin S,
Yurieva O,
Kholodii G,
( 2000 ) Tn5044, a novel Tn3 family transposon coding for temperature-sensitive mercury resistance. PMID : 10875286 : Abstract >>
We report the discovery and characterization of the mercury resistance transposon, Tn5044, from a Xanthomonas strain from the Kamchatka peninsula. In addition to the standard set of merRTPCAD genes, the mer operon of Tn5044 contains a gene named sigY that encodes the RNA polymerase sigma factor-like protein. Mercury resistance determined by Tn5044 is expressed at low (30 degrees C) but not at elevated temperatures (37 degrees C). None of the mer operon genes downstream of merA is responsible for the temperature-sensitive mercury resistance. The transposition module of Tn5044 is closely related to those of Tn1412 isolated from medical sources and to Tn5563 and ISXc5 from environmental sources. However, Tn5044 differs from these transposons in that it has unusually long terminal inverted repeats. Sequence analysis of the transposase (tnpA) genes places Tn5044 and its close relatives into the Tn3 subgroup of the Tn3 family. However, the orientation of their resolvase and transposase genes is unusual for the Tn3 family: tnpR is proximal to the end of the transposon, while divergently transcribed tnpA is oriented inwardly. The region between tnpA and tnpR genes is unusually large and contains two short conserved open reading frames. In addition to the complete set of sequence motifs common to true resolvases, the resolvase of Tn5044 and its close relatives possesses a C-terminal extension showing no homology to known proteins. Despite this peculiarity, Tn5044 resolvase can resolve cointegrates formed during Tn5044 transposition controlled by tnpA. Genetic data suggest that the extension is essential for TnpR functioning.
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9. |
Sundin GW,
( 2000 ) Examination of base pair variants of the strA-strB streptomycin resistance genes from bacterial pathogens of humans, animals and plants. PMID : 11062214 : DOI : 10.1093/jac/46.5.848 Abstract >>
N/A
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10. |
Abdian PL,
Lellouch AC,
Gautier C,
Ielpi L,
Geremia RA,
( 2000 ) Identification of essential amino acids in the bacterial alpha -mannosyltransferase aceA. PMID : 11001941 : DOI : 10.1074/jbc.M007496200 Abstract >>
The alpha-mannosyltransferase AceA from Acetobacter xylinum belongs to the CaZY family 4 of retaining glycosyltransferases. We have identified a series of either highly conserved or invariant residues that are found in all family 4 enzymes as well as other retaining glycosyltransferases. These residues included Glu-287 and Glu-295, which comprise an EX(7)E motif and have been proposed to be involved in catalysis. Alanine replacements of each conserved residue were constructed by site-directed mutagenesis. The mannosyltransferase activity of each mutant was examined by both an in vitro transferase assay using recombinant mutant AceA expressed in Escherichia coli and by an in vivo rescue assay by expressing the mutant AceA in a Xanthomonas campestris gumH(-) strain. We found that only mutants K211A and E287A lost all detectable activity both in vitro and in vivo, whereas E295A retained residual activity in the more sensitive in vivo assay. H127A and S162A each retained reduced but significant activities both in vitro and in vivo. Secondary structure predictions of AceA and subsequent comparison with the crystal structures of the T4 beta-glucosyltransferase and MurG suggest that AceA Lys-211 and Glu-295 are involved in nucleotide sugar donor binding, leaving Glu-287 of the EX(7)E as a potential catalytic residue.
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11. |
Kholodii G,
Mindlin S,
Minakhina S,
( 1999 ) Tn5053 family transposons are res site hunters sensing plasmidal res sites occupied by cognate resolvases. PMID : 10476039 : DOI : 10.1046/j.1365-2958.1999.01548.x Abstract >>
DNA sequence database search revealed that most of Tn5053/Tn402 family transposons inserted into natural plasmids were located in putative res regions upstream of genes encoding various resolvase-like proteins. Some of these resolvase genes belonged to Tn3 family transposons and were closely related to the tnpR genes of Tn1721 and a recently detected Tn5044. Using recombinant plasmids containing fragments of Tn1721 or Tn5044 as targets in transposition experiments, we have demonstrated that Tn5053 displays striking insertional preference for the res regions of these transposons: more than 70% of Tn5053 insertion events occur in clusters inside the target res regions, while most remaining insertion events occur no further than 200 base pairs away from both sides of the res regions. We demonstrate that Tn5053 insertions (both into and outside a res region of the target plasmid) require the presence of a functional cognate resolvase gene either in cis or in trans. To our knowledge, this is the first case when a site-specific recombination system outside a transposon has been shown to be involved in transposition.
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12. |
Ryan RP,
Ryan DJ,
Sun YC,
Li FM,
Wang Y,
Dowling DN,
( 2007 ) An acquired efflux system is responsible for copper resistance in Xanthomonas strain IG-8 isolated from China. PMID : 17263848 : DOI : 10.1111/j.1574-6968.2006.00592.x Abstract >>
The genus Xanthomonas contains plant pathogens exhibiting innate resistance to a range of antimicrobial agents. In other genera, multidrug resistance is mediated by a synergy between a low-permeability outer membrane and expression of a number of multidrug efflux systems. This report describes the isolation of a novel gene cluster xmeRSA from Xanthomonas strain IG-8 that mediates copper chloride resistance. Subsequent analysis of these genes showed that they were responsible for the high level of multiple resistance in this strain and were homologues of the sme system of Stenotrophomonas maltophilia. Knock-out mutants of this gene cluster indicate that these genes are required for the copper resistance phenotype of strain IG-8. Expression analysis using lacZ fusions indicates that the genes are regulated by copper and other antimicrobials. Bioinformatic analysis suggests that these genes were acquired by horizontal gene transfer.
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13. |
de Crécy-Lagard V,
Lejeune P,
Bouvet OM,
Danchin A,
( 1991 ) Identification of two fructose transport and phosphorylation pathways in Xanthomonas campestris pv. campestris. PMID : 1650911 : DOI : 10.1007/bf00273939 Abstract >>
Fructose was shown to be phosphorylated by a specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) in Xanthomonas campestris pv. campestris. Transposon mutagenesis of X. campestris was performed and two mutants affected in growth on fructose were isolated. Both mutants were deficient in PTS activity. Comparison of the rate of uptake and phosphorylation of fructose in the wild-type and in the mutant strains revealed the presence of a second fructose permeation and phosphorylation pathway in this bacterium: an unidentified permease coupled to an ATP-dependent fructokinase. One of the two mutants was also deficient in fructokinase activity. Chromosomal DNA fragments containing the regions flanking the transposon insertion site were cloned from both mutant strains. Their physical study revealed that the insertion sites were separated by 1.4 kb, allowing the reconstruction of a wild-type DNA fragment which complemented one of the two mutants. The region flanking the transposon insertion site was sequenced in one of the mutants, showing that the transposon had interrupted the gene encoding the fructose EII. The mutant strains also failed to utilize mannose, sucrose and mannitol, suggesting the existence of a branch point between the metabolism of fructose and of these latter carbohydrates.
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14. |
Gillings MR,
Holley MP,
Stokes HW,
Holmes AJ,
( 2005 ) Integrons in Xanthomonas: a source of species genome diversity. PMID : 15755815 : DOI : 10.1073/pnas.0406620102 PMC : PMC555480 Abstract >>
Integrons are best known for assembling antibiotic resistance genes in clinical bacteria. They capture genes by using integrase-mediated site-specific recombination of mobile gene cassettes. Integrons also occur in the chromosomes of many bacteria, notably beta- and gamma-Proteobacteria. In a survey of Xanthomonas, integrons were found in all 32 strains representing 12 pathovars of two species. Their chromosomal location was downstream from the acid dehydratase gene, ilvD, suggesting that an integron was present at this site in the ancestral xanthomonad. There was considerable sequence and structural diversity among the extant integrons. The majority of integrase genes were predicted to be inactivated by frameshifts, stop codons, or large deletions, suggesting that the associated gene cassettes can no longer be mobilized. In support, groups of strains with the same deletions or stop codons/frameshifts in their integrase gene usually contained identical arrays of gene cassettes. In general, strains within individual pathovars had identical cassettes, and these exhibited no similarity to cassettes detected in other pathovars. The variety and characteristics of contemporary gene cassettes suggests that the ancestral integron had access to a diverse pool of these mobile elements, and that their genes originated outside the Xanthomonas genome. Subsequent inactivation of the integrase gene in particular lineages has largely fixed the gene cassette arrays in particular pathovars during their differentiation and specialization into ecological niches. The acquisition of diverse gene cassettes by different lineages within Xanthomonas has contributed to the species-genome diversity of the genus. The role of gene cassettes in survival on plant surfaces is currently unknown.
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15. |
Hu NT,
Hung MN,
Huang AM,
Tsai HF,
Yang BY,
Chow TY,
Tseng YH,
( 1992 ) Molecular cloning, characterization and nucleotide sequence of the gene for secreted alpha-amylase from Xanthomonas campestris pv. campestris. PMID : 1527504 : DOI : 10.1099/00221287-138-8-1647 Abstract >>
alpha-Amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) of apparent molecular mass 45 kDa was secreted by Xanthomonas campestris pv. campestris grown in medium containing starch or maltose. We isolated its structural gene from a recombinant lambda library and located it on a 2.7 kb DNA fragment. Nucleotide sequencing of the fragment revealed a potential ORF encoding a protein of 475 amino acid residues, including a potential signal sequence of 35 amino acids. The signal processing site was confirmed by N-terminal amino acid sequence analysis of the exported alpha-amylase. The deduced amino acid sequence of the mature protein is very similar to that of the alpha-amylase of Aeromonas hydrophila. It also contains all four amino acid sequences highly conserved in the alpha-amylases from a wide range of organisms. Expression of the amy gene in Escherichia coli was poor from its own promoter, but was enhanced by the upstream promoter on the vector. The alpha-amylase synthesized in E. coli was located in the periplasm.
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16. |
Weng SF,
Lin JW,
Chen CH,
Chen YY,
Tseng YH,
Tseng YH,
( 2004 ) Constitutive expression of a chromosomal class A (BJM group 2) beta-lactamase in Xanthomonas campestris. PMID : 14693541 : DOI : 10.1128/aac.48.1.209-215.2004 PMC : PMC310161 Abstract >>
Sequencing of the upstream region of the beta-lactamase gene from Xanthomonas campestris pv. campestris 11 (bla(XCC-1)) revealed the cognate ampR1 gene (289 amino acids, 31 kDa). It runs divergently from bla(XCC-1) with a 100-bp intergenic region (IG) containing partially overlapped promoters with structural features typical of the bla-ampR IG. The deduced AmpR1 protein shows significant identity in amino acid sequence and conserved motifs with AmpR proteins of other species, e.g., of Pseudomonas aeruginosa (58.2% amino acid identity). Results of insertional mutation, complementation tests, and beta-lactamase assays suggested that expression of bla(XCC-1) was constitutive and dependent on AmpR1. Four bla genes and two ampR genes are present in the fully sequenced X. campestris pv. campestris ATCC 33913 genome, with XCC3039 and XCC3040 considered the analogues of bla(XCC-1) and ampR1, respectively. An ampR1 homologue was detected by Southern hybridization in the ampicillin-resistant Xanthomonas strains, which appear to express beta-lactamase constitutively. Although the significance remains to be studied, constitutive expression of beta-lactamase by a widespread bacterial genus raises environmental concerns regarding the dissemination of resistance genes.
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17. |
Schneider KL,
Marrero G,
Alvarez AM,
Presting GG,
( 2011 ) Classification of plant associated bacteria using RIF, a computationally derived DNA marker. PMID : 21533033 : DOI : 10.1371/journal.pone.0018496 PMC : PMC3080875 Abstract >>
A DNA marker that distinguishes plant associated bacteria at the species level and below was derived by comparing six sequenced genomes of Xanthomonas, a genus that contains many important phytopathogens. This DNA marker comprises a portion of the dnaA replication initiation factor (RIF). Unlike the rRNA genes, dnaA is a single copy gene in the vast majority of sequenced bacterial genomes, and amplification of RIF requires genus-specific primers. In silico analysis revealed that RIF has equal or greater ability to differentiate closely related species of Xanthomonas than the widely used ribosomal intergenic spacer region (ITS). Furthermore, in a set of 263 Xanthomonas, Ralstonia and Clavibacter strains, the RIF marker was directly sequenced in both directions with a success rate approximately 16% higher than that for ITS. RIF frameworks for Xanthomonas, Ralstonia and Clavibacter were constructed using 682 reference strains representing different species, subspecies, pathovars, races, hosts and geographic regions, and contain a total of 109 different RIF sequences. RIF sequences showed subspecific groupings but did not place strains of X. campestris or X. axonopodis into currently named pathovars nor R. solanacearum strains into their respective races, confirming previous conclusions that pathovar and race designations do not necessarily reflect genetic relationships. The RIF marker also was sequenced for 24 reference strains from three genera in the Enterobacteriaceae: Pectobacterium, Pantoea and Dickeya. RIF sequences of 70 previously uncharacterized strains of Ralstonia, Clavibacter, Pectobacterium and Dickeya matched, or were similar to, those of known reference strains, illustrating the utility of the frameworks to classify bacteria below the species level and rapidly match unknown isolates to reference strains. The RIF sequence frameworks are available at the online RIF database, RIFdb, and can be queried for diagnostic purposes with RIF sequences obtained from unknown strains in both chromatogram and FASTA format.
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18. |
Kearney B,
Staskawicz BJ,
( 1990 ) Characterization of IS476 and its role in bacterial spot disease of tomato and pepper. PMID : 2152895 : DOI : 10.1128/jb.172.1.143-148.1990 PMC : PMC208411 Abstract >>
IS476 is an endogenous insertion sequence present in copper-tolerant strains of Xanthomonas campestris pv. vesicatoria. Sequence analysis has revealed that the element is 1,225 base pairs in length, has 26-base-pair inverted repeats, and causes a 4-base-pair target site duplication upon insertion into the avirulence gene avrBs1. Comparison of the full-length sequence with sequences in the National Biomedical Research Foundation and National Institutes of Health data bases showed that one of the predicted IS476 proteins is partially homologous to the putative transposase of IS3 from Escherichia coli, and the inverted repeats of IS476 have significant homology to the inverted repeats of the IS51 insertion sequence of Pseudomonas syringae pv. savastanoi. A transposition assay based on the insertional inactivation of the sacRB locus of Bacillus subtilis was used to demonstrate that one of the three copies of IS476 residing on the 200-kilobase copper plasmid pXVCU1 is capable of transposition in several strains of Xanthomonas campestris. The position of IS476 insertion in several avrBs1 mutants was established and was shown to influence both induction of hypersensitivity and bacterial growth in planta.
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19. |
Chakrabarty PK,
Duan YP,
Gabriel DW,
( 1997 ) Cloning and Characterization of a Member of the Xanthomonas avr/pth Gene Family That Evades All Commercially Utilized Cotton R Genes in the United States. PMID : 18945013 : DOI : 10.1094/PHYTO.1997.87.11.1160 Abstract >>
ABSTRACT The highly virulent African strains of Xanthomonas campestris pv. malvacearum are quarantined pathogens in the United States and can evade or overcome all commercially utilized resistance (R) genes in cotton grown in the United States including the entire set of host differential lines used to distinguish 19 races of the pathogen. Nevertheless, the African strains carry multiple DNA fragments that strongly hybridize with members of the Xanthomonas avirulence (avr)/pathogenicity (pth) gene family. Since all previously tested members of the gene family confer avirulence against one or more R genes in cotton, strains carrying multiple members might not be expected to evade so many different R genes. The hybridizing DNA fragments were cloned from African strain XcmN and found to confer water-soaking ability to a nearly asymptomatic mutant strain of the pathogen. Restriction mapping, Southern hybridization, and DNA sequencing of the cloned fragments from XcmN were used to identify two water-soaking genes, pthN and pthN2, as new members of the Xanthomonas avr/pth gene family. The complete DNA sequence of pthN was obtained, and it is >94% identical with all other sequenced members of the gene family. Gene fusions of pthN with avrb6 (another family member) and other experiments revealed that the ability of African strain XcmN to water-soak cotton and avoid recognition by commercially used cotton R genes is determined by the specific repeats of multiple functional members of the Xanthomonas avr/pth gene family.
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20. |
Parkinson N,
Aritua V,
Heeney J,
Cowie C,
Bew J,
Stead D,
( 2007 ) Phylogenetic analysis of Xanthomonas species by comparison of partial gyrase B gene sequences. PMID : 18048743 : DOI : 10.1099/ijs.0.65220-0 Abstract >>
The genus Xanthomonas currently comprises 27 species with validly published names that are important crop and horticultural pathogens. We have constructed a phylogram from alignment of gyrase B (gyrB) sequences for all xanthomonad species, both to indicate inter-species relatedness and as an aid for rapid and accurate species-level identification. The phylogeny indicated a monophyletic group, with X. albilineans and X. sacchari as the most ancestral species. Three species, X. hyacinthi, X. translucens and X. theicola, formed an early-branching group. Three clades were supported by high bootstrap values: group 1 comprised X. cucurbitae, X. cassavae and X. codiaei; group 2 comprised X. arboricola, X. campestris, X. populi, X. hortorum, X. gardneri and X. cynarae; group 3 contained the remaining species, within which two further clades, supported by a 100 % bootstrap value, were identified. Group 3A comprised X. axonopodis, X. euvesicatoria, X. perforans and X. melonis, together with X. alfalfae, X. citri and X. fuscans, whose names were recently validly published. Group 3B contained the monocot pathogens X. vasicola and X. oryzae. Two recently identified species, X. cynarae and X. gardneri, were poorly discriminated and were related closely to X. hortorum. Three species, X. perforans, X. euvesicatoria and X. alfalfae, had identical gyrB sequences. Partial sequencing of a further five genes from these species found only minor sequence differences that confirmed their close relatedness. Although branch lengths between species varied, indicating different degrees of genetic distinctiveness, the majority (n=21) were well-differentiated, indicating the utility of the method as an identification tool, and we now use this method for routine diagnosis of xanthomonad species.
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21. |
Osbourn AE,
Clarke BR,
Stevens BJ,
Daniels MJ,
( 1990 ) Use of oligonucleotide probes to identify members of two-component regulatory systems in Xanthomonas campestris pathovar campestris. PMID : 2233675 : DOI : 10.1007/bf00283036 Abstract >>
Two-component regulatory systems comprising a sensor and a regulator protein, both with highly conserved amino acid domains, and commonly genetically linked, have been described in a range of bacterial species and are involved in sensing environmental stimuli. We used two oligonucleotide probes matching the postulated coding regions for domains of sensor and regulator proteins respectively in Xanthomonas campestris pathovar campestris (Xcc) to identify possible two-component regulatory systems in Xcc. Two different fragments of Xcc DNA with homology to both of these probes were cloned. The DNA sequence of part of one of these fragments encompassed a potential open reading frame (ORF), the predicted amino acid sequence of which had extensive homology with regulator proteins of two-component regulatory systems. Analysis of the predicted amino acid sequence for the 3' end of an adjacent ORF revealed a very high level of homology with the C-terminal end of sensor proteins. Strains of Xcc with Tn5-induced mutations in the regulator gene were affected in extracellular polysaccharide production, and also in resistance to salt and chloramphenicol. No effects of mutation in the second clone were observed.
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22. |
( 1997 ) The Xanthomonas campestris gumD gene required for synthesis of xanthan gum is involved in normal pigmentation and virulence in causing black rot. PMID : 9144435 : DOI : 10.1006/bbrc.1997.6365 Abstract >>
A cloned 4.1-kb EcoRI fragment from Xanthomonas campestris pv. campestris was previously shown to complement the non-mucoid mutant P22 and increase xanthan gum production after being transformed into the wild-type strain Xc17. The gene responsible for these effects was identified, sequenced, and shown to be the gumD gene which has previously been proposed to encode glucose transferase activity, an enzyme required for adding the first glucose residue to the isoprenoid glycosyl carrier lipid during xanthan synthesis. A gumD mutant, isolated from Xc17 by gene replacement, was shown to possess altered pigment xanthomonadin profiles and exhibit reduced virulence in causing black rot in broccoli. This study appears to be the first to demonstrate that interruption of a gene required for xanthan synthesis can lead to reduced virulence of X. campestris.
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( 1997 ) Isolation and analysis of the Xanthomonas alkyl hydroperoxide reductase gene and the peroxide sensor regulator genes ahpC and ahpF-oxyR-orfX. PMID : 9190810 : DOI : 10.1128/jb.179.12.3944-3949.1997 PMC : PMC179203 Abstract >>
From Xanthomonas campestris pv. phaseoli, we have isolated by two independent methods genes involved in peroxide detoxification (ahpC and ahpF), a gene involved in peroxide sensing and transcription regulation (oxyR), and a gene of unknown function (orfX). Amino acid sequence analysis of AhpC, AhpF, and OxyR showed high identity with bacterial homologs. OrfX was a small cysteine-rich protein with no significant homology to known proteins. The genes ahpC, ahpF, oxyR, and orfX were arranged in a head-to-tail fashion. This unique arrangement was conserved in all of the Xanthomonas strains tested. The functionalities of both the ahpC and oxyR genes were demonstrated. In X. campestris pv. phaseoli, increased expression of ahpC alone conferred partial protection against growth retardation and killing by organic hydroperoxides but not by H2O2 or superoxide generators. These genes are likely to have important physiological roles in protection against peroxide toxicity in Xanthomonas.
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24. |
( 1997 ) A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal molecule. PMID : 9179849 : DOI : 10.1046/j.1365-2958.1997.3721736.x Abstract >>
Mutations in the seven clustered rpf genes cause downregulated synthesis of extracellular enzymes and reduced virulence of Xanthomonas campestris pathovar campestris (Xcc). The phenotype of mutants in one of the genes, rpfF, can be restored by a diffusible extracellular factor (DSF) produced by all Xcc strains tested, apart from rpfF and rpfB mutants. DSF accumulates in early stationary phase (when synthesis of enzymes is maximal), but levels decline subsequently. Addition of DSF to exponentially-growing wild-type bacteria does not cause precocious enzyme synthesis. rpfB and rpfF are expressed throughout growth, but the rate increases in early stationary phase. RpfB is predicted to be a long-chain fatty acyl CoA ligase, and RpfF shows some relatedness to enoyl CoA hydratases. The properties of DSF suggest that it may be a fatty-acid derivative, and certain lipid preparations possess DSF activity at higher concentrations. These include lipid extracts and acid-hydrolysed lipoplysaccharide and lipid A from Xcc, and purified dodecanoic and hydroxydodecanoic acid. DSF production is confined to certain xanthomonads. We propose a model for the DSF system, which represents a novel mechanism for regulating virulence factor synthesis in response to physiological or environmental changes.
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25. |
( 1996 ) The rpoN gene of Xanthomonas campestris pv. vesicatoria is not required for pathogenicity. PMID : 8969534 : Abstract >>
We have cloned the rpoN region of the pepper and tomato pathogen Xanthomonas campestris pv. vesicatoria to analyze its role in pathogenicity and hrp gene activation. Open reading frame 3 (ORF3) from this region is predicted to encode a protein that is up to 45% identical and 65% similar to previously described RpoN proteins and was shown to be essential for the utilization of nitrate as sole nitrogen source. The X. campestris pv. vesicatoria rpoN gene is, however, not required for pathogenicity and hrp gene expression.
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26. |
( 1996 ) The gene encoding UDP-glucose pyrophosphorylase is required for the synthesis of xanthan gum in Xanthomonas campestris. PMID : 8831665 : DOI : 10.1006/bbrc.1996.1403 Abstract >>
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.
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27. |
( 1997 ) Characterization of the fimA gene encoding bundle-forming fimbriae of the plant pathogen Xanthomonas campestris pv. vesicatoria. PMID : 9023213 : DOI : 10.1128/jb.179.4.1280-1290.1997 PMC : PMC178827 Abstract >>
The fimA gene of Xanthomonas campestris pv. vesicatoria was identified and characterized. A 20-mer degenerate oligonucleotide complementary to the N-terminal amino acid sequence of the purified 15.5-kDa fimbrillin was used to locate fimA on a 2.6-kb SalI fragment of the X. campestris pv. vesicatoria 3240 genome. The nucleotide sequence of a 1.4-kb fragment containing the fimA region revealed two open reading frames predicting highly homologous proteins FimA and FimB. FimA, which was composed of 136 amino acids and had a calculated molecular weight of 14,302, showed high sequence identity to the type IV fimbrillin precursors. fimB predicted a protein product of 135 amino acids and a molecular weight of 13,854. The open reading frame for fimB contained near the 5' end a palindromic sequence with a terminator loop potential, and the expression level of fimB in vitro and in Xanthomonas was considerably lower than that of fimA. We detected an efficiently transcribed fimA-specific mRNA of 600 bases as well as two weakly expressed, longer mRNA species that reacted with both fimA and fimB. A homolog of fimA but not of fimB was detected by Southern hybridization in strains of X. campestris pv. vesicatoria, campestris, begoniae, translucens, and graminis. A fimA::omega mutant of strain 3240 was not significantly reduced in virulence or adhesiveness to tomato leaves. However, the fimA mutant was dramatically reduced in cell aggregation in laboratory cultures and on infected tomato leaves. The fimA mutant strain also exhibited decreased tolerance to UV light.
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28. |
( 1995 ) The type IV pre-pilin leader peptidase of Xanthomonas campestris pv. campestris is functional without conserved cysteine residues. PMID : 8817497 : DOI : 10.1111/j.1365-2958.1995.mmi_18040769.x Abstract >>
Type IV pre-pilin leader peptidase was demonstrated to be required for protein secretion, in addition to its involvement in biogenesis of type IV pIII. The type IV pre-pilin leader peptidase gene of Xanthomonas campestris pv. campestris was located on a 3 kb Accl 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.
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29. |
( 1993 ) The opsX locus of Xanthomonas campestris affects host range and biosynthesis of lipopolysaccharide and extracellular polysaccharide. PMID : 8376331 : DOI : 10.1128/jb.175.18.5839-5850.1993 PMC : PMC206663 Abstract >>
Xanthomonas campestris pv. citrumelo strain 3048 is the causal agent of citrus bacterial leaf spot disease and has a wide host range that includes rutaceous and leguminous plants. A spontaneous prototrophic mutant of strain 3048 (strain M28) that had lost virulence on citrus but retained virulence on bean plants was recovered. Growth studies in planta showed that M28 cells died rapidly in citrus leaves but grew normally in bean leaves. In addition to the loss of citrus-specific virulence, M28 displayed the following mutant phenotypes in culture: decreased growth rate, reduction of the amount of exopolysaccharide (to ca. 25% of the amount in 3048), loss of capsules, and significant alterations of the two 3048 lipopolysaccharide (LPS) bands visualized by silver stain on polyacrylamide gels, consistent with a defect(s) in LPS assembly. A 38-kb DNA fragment from a 3048 total DNA library that complemented the mutant phenotypes of M28 was identified. The 38-kb fragment did not hybridize to two similarly sized fragments carrying different hrp (hypersensitive response and pathogenicity) genes cloned from 3048. Subcloning, DNA sequence analyses, and gene disruption experiments were used to identify a single gene, opsX (for outer-membrane polysaccharide), responsible for the mutant phenotypes of M28. At least one other gene downstream from opsX also affected the same phenotypes and may be part of a gene cluster. We report here the DNA sequence and transcriptional start site of opsX. A search of protein sequence data bases with the predicted 31.3-kDa OpsX sequence found strong similarity to Lsi-1 of Neisseria gonorrhoeae and RfaQ of Escherichia coli (both are involved in LPS core assembly). The host-specific virulence function of opsX appears to involve biosynthesis of the extracellular polysaccharide and a complete LPS. Both may be needed in normal amounts for protection from citrus, but not bean, defense compounds.
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30. |
Lin CS,
Lin NT,
Yang BY,
Weng SF,
Tseng YH,
( 1995 ) Nucleotide sequence and expression of UDP-glucose dehydrogenase gene required for the synthesis of xanthan gum in Xanthomonas campestris. PMID : 7857269 : DOI : 10.1006/bbrc.1995.1176 Abstract >>
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.
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31. |
Oku T,
Alvarez AM,
Kado CI,
( 1995 ) Conservation of the hypersensitivity-pathogenicity regulatory gene hrpX of Xanthomonas campestris and X. oryzae. PMID : 7626786 : Abstract >>
The hrpX gene is essential for pathogenicity of Xanthomonas species. Loss of hrpX by mutation results in the loss of pathogenicity and a gain in the ability of Xanthomonas to cause the hypersensitive response in their respective host plants, suggesting that hrpX confers a means to evade this host defense response. The function of HrpX protein was predicted by sequencing of hrpXc and hrpXo from X. campestris pv. campestris and X. oryzae, respectively. The predicted amino acid sequences of the protein encoded by these respective genes revealed similarities (45.96%) to the HrpB protein of Burkholderia solanacearum, which has sequence identity to the transcriptional activator VirF of Yersinia enterocolitica and AraC of Escherichia coli. Thus, HrpX may regulate Xanthomonas virulence genes since a putative DNA binding domain present in the carboxyl terminal half of HrpX is highly conserved among HrpB, VirF and AraC and since over-expression of the carboxyl terminal half of HrpX in E. coli is lethal.
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32. |
Sundin GW,
Bender CL,
( 1995 ) Expression of the strA-strB streptomycin resistance genes in Pseudomonas syringae and Xanthomonas campestris and characterization of IS6100 in X. campestris. PMID : 7487022 : PMC : PMC167566 Abstract >>
Expression of the strA-strB streptomycin resistance (SMr) genes was examined in Pseudomonas syringae pv. syringae and Xanthomonas campestris pv. vesicatoria. The strA-strB genes in P. syringae and X. campestris were encoded on elements closely related to Tn5393 from Erwinia amylovora and designated Tn5393a and Tn5393b, respectively. The putative recombination site (res) and resolvase-repressor (tnpR) genes of Tn5393 from E. amylovora, P syringae, and X. campestris were identical; however, IS6100 mapped within tnpR in X. campestris, and IS1133 was previously located downstream of tnpR in E. amylovora (C.-S Chiou and A. L. Jones, J. Bacteriol. 175:732-740, 1993). Transcriptional fusions (strA-strB::uidA) indicated that a strong promoter sequence was located within res in Tn5393a. Expression from this promoter sequence was reduced when the tnpR gene was present in cis position relative to the promoter. In X. campestris pv. vesicatoria, analysis of promoter activity with transcriptional fusions indicated that IS6100 increased the expression of strA-strB. Analysis of codon usage patterns and percent G+C in the third codon position indicated that IS6100 could have originated in a gram-negative bacterium. The data obtained in the present study help explain differences observed in the levels of SMr expressed by three genera which share common genes for resistance. Furthermore, the widespread dissemination of Tn5393 and derivatives in phytopathogenic prokaryotes confirms the importance of these bacteria as reservoirs of antibiotic resistance in the environment.
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33. |
Becker A,
Niehaus K,
Pühler A,
( 1995 ) Low-molecular-weight succinoglycan is predominantly produced by Rhizobium meliloti strains carrying a mutated ExoP protein characterized by a periplasmic N-terminal domain and a missing C-terminal domain. PMID : 7565082 : DOI : 10.1111/j.1365-2958.1995.tb02292.x Abstract >>
The membrane topology of the Rhizobium meliloti 2011 ExoP protein involved in polymerization and export of succinoglycan was analysed by translational fusions of lacZ and phoA reporter genes to the exoP gene. Based on this analysis, the ExoP protein could be divided into an N-terminal domain mainly located in the periplasmic space and a C-terminal domain located in the cytoplasm. Whereas the C-terminal domain of ExoP is characterized by a potential nucleotide-binding motif, the N-terminal ExoP domain contains the sequence motif 'PX2PX4SPKX11GXMXG', which is also present in proteins involved in the determination of O-antigen chain length. R. meliloti strains carrying mutated exoP* genes, exclusively encoding the N-terminal ExoP domain, produced a reduced amount of succinoglycan. This reduction could be suppressed by a mutation in the regulatory gene exoR. The ratio of low-molecular-weight to high-molecular-weight succinoglycan was significantly increased in the exoP* mutant strain. In the exoP*/exoR mutant strain only low-molecular-weight succinoglycan could be detected. Based on sequence homologies and similar hydropathic profiles, the N-terminal domain of ExoP was proposed to be a member of a protein family thought to be involved in polysaccharide chain-length determination.
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34. |
Hong HJ,
Kim TH,
Song WS,
Ko HJ,
Lee GS,
Kang SG,
Kim PH,
Yoon SI,
( 2018 ) Crystal structure of FlgL and its implications for flagellar assembly. PMID : 30250171 : DOI : 10.1038/s41598-018-32460-9 PMC : PMC6155364 Abstract >>
Bacteria move toward attractants and away from repellants by rotating their flagellum. The bacterial flagellum assembles through the ordered organization of more than 30 different proteins. Among the diverse flagellar proteins, FlgL forms the junction between the hook and the filament in the flagellum together with FlgK and provides a structural base where flagellin, a filament-forming protein, is inserted for the initiation of filament elongation. However, the functional and structural information available for FlgL is highly limited. To provide structural insights into the cross-linkage between the FlgL junction and the flagellin filament, we determined the crystal structures of FlgL from gram-positive Bacillus cereus (bcFlgL) and gram-negative Xanthomonas campestris (xcFlgL). bcFlgL contains one domain (D1), whereas xcFlgL adopts a two-domain structure that consists of the D1 and D2 domains. The constant D1 domain of FlgL adopts a rod structure that is generated by four longitudinal segments. This four-segment structure is recapitulated in filament and junction proteins but not in hook and rod proteins, allowing us to propose a junction-filament assembly mechanism based on a quasi-homotypic interaction. The D2 domain of xcFlgL resembles that of another junction protein, FlgK, suggesting the structural and functional relatedness of FlgL and FlgK.
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35. |
( 2012 ) A low-temperature-active alkaline pectate lyase from Xanthomonas campestris ACCC 10048 with high activity over a wide pH range. PMID : 22983714 : DOI : 10.1007/s12010-012-9872-8 Abstract >>
Alkaline pectate lyases are favorable for the textile industry. Here, we report the gene cloning and expression of a low-temperature-active alkaline pectate lyase (PL D) from Xanthomonas campestris ACCC 10048. Deduced PL D consists of a putative 27-residue signal peptide and a catalytic domain of 320 residues belonging to family PF09492. Recombinant PL D (r-PL D) produced in Escherichia coli was purified to electrophoretic homogeneity with a single step of Ni(2+)-NTA affinity chromatography and showed an apparent molecular weight of ~38 kDa. The pH and temperature optima of r-PL D were found to be 9.0 �XC and 30 �XC, respectively. Compared with its microbial counterparts, r-PL D had higher activity over a wide pH range (>45 % of the maximum activity at pH 3.0-12.0) and at lower temperatures (>35 % of activity even at 0 �XC). The K(m) and V(max) values of r-PL D for polygalacturonic acid were 4.9 gl(-1) and 30.1 �gmolmin(-1) mg(-1), respectively. Compared with the commercial compound pectinase from Novozymes, r-PL D showed similar efficacy in reducing the intrinsic viscosity of polygalacturonic acid (35.1 % vs. 36.5 %) and in bioscouring of jute (10.25 % vs. 10.82 %). Thus, r-PL D is a valuable additive candidate for the textile industry.
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36. |
( 1997 ) The aroQ and pheA domains of the bifunctional P-protein from Xanthomonas campestris in a context of genomic comparison. PMID : 9689222 : DOI : 10.1089/omi.1.1997.2.141 Abstract >>
The gene (denoted aroQp.pheA) encoding the bifunctional P-protein (chorismate mutase-P/prephenate dehydratase) from Xanthomonas campestris was cloned. aroQp.pheA is essential for L-phenylalanine biosynthesis. DNA sequencing of the smallest subclone capable of functional complementation of an Escherichia coli phenylalanine auxotroph revealed a putative open reading frame (ORF) of 1200 bp that would encode a 43,438-Da protein. AroQp.PheA exhibited 51% amino acid identity with a Pseudomonas stutzeri homologoue and greater than 30% identities with AroQp.PheA proteins from Haemophilus influenzae, Neisseria gonorrhoeae, and a number of enteric bacteria. AroQp.PheA from X. campestris, when expressed in E. coli, possesses a 40-residue amino-terminal extension that is lysine-rich and that is absent in all of the AroQp.PheA homologues known at present. About 95% of AroQp.PheA was particulate and readily sedimented by low-speed centrifugation. Soluble preparations of cloned AroQp.PheA exhibited a native molecular mass of 81,000 Da, indicating that the active enzyme species is a homodimer. These preparations were unstable after purification of about 40-fold, even in the presence of glycerol, which was an effective protectant before fractionation. When AroQp.PheA was overproduced by a T7 translation vector, unusual inclusion bodies having a macromolecular structure consisting of protein fibrils were observed by electron microscopy. Insoluble protein collected at low-speed centrifugation possessed high catalytic activity. The single band obtained via SDS-PAGE was used to confirm the translational start via N-terminal amino acid sequencing. A perspective on the evolutionary relationships of monofunctional AroQ and PheA proteins and the AroQp.PheA family of proteins is presented. A serC gene located immediately upstream of X. campestris aroQp.pheA appears to reflect a conserved gene organization, and both may belong to a single transcriptional unit.
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37. |
( 1998 ) The rpfA gene of Xanthomonas campestris pathovar campestris, which is involved in the regulation of pathogenicity factor production, encodes an aconitase. PMID : 9663682 : DOI : 10.1046/j.1365-2958.1998.00852.x Abstract >>
Xanthomonas campestris pv campestris (Xcc) is a plant pathogenic bacterium that controls the production of pathogenicity factors in part by a cluster of genes designated rpf (regulation of pathogenicity factors). Sequence analysis of one of these genes (rpfA) revealed an open reading frame with amino acid sequence similarity to aconitases from other bacteria. Aconitase activity was lower in cellular extracts of an rpfA::Tn5 mutant than in those from the wild type. A zymogram of aconitase activity after native gel electrophoresis showed the presence of two distinct aconitases in Xcc; the major aconitase was absent in the rpfA::Tn5 mutant. This mutant also had reduced levels of extracellular enzymes and extracellular polysaccharide (EPS). Supplying rpfA in trans to the rpfA::Tn5 mutant restored both the major aconitase activity and the synthesis of these pathogenicity factors. The transcription of the genes for two extracellular enzymes (prtA, encoding a serine protease, and engXCA, encoding endoglucanase) was reduced in the rpfA mutant background. Because some eukaryotic aconitases are also involved in iron regulation, we explored a possible connection between rpfA and iron metabolism. Intracellular iron levels in the mutants were lower than in the wild type as assessed by sensitivity to the iron-activated antibiotic, streptonigrin. Wild-type bacteria grown in iron-deficient conditions had a similar sensitivity to streptonigrin as the aconitase mutant. Overall, these results suggest that a prokaryotic aconitase can also act as a regulator of gene expression and that the regulation is possibly related to changes in intracellular iron levels.
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38. |
( 1998 ) Xanthomonas campestris pv. campestris gum mutants: effects on xanthan biosynthesis and plant virulence. PMID : 9537354 : PMC : PMC107069 Abstract >>
Xanthan is an industrially important exopolysaccharide produced by the phytopathogenic, gram-negative bacterium Xanthomonas campestris pv. campestris. It is composed of polymerized pentasaccharide repeating units which are assembled by the sequential addition of glucose-1-phosphate, glucose, mannose, glucuronic acid, and mannose on a polyprenol phosphate carrier (L. Ielpi, R. O. Couso, and M. A. Dankert, J. Bacteriol. 175:2490-2500, 1993). A cluster of 12 genes in a region designated xpsI or gum has been suggested to encode proteins involved in the synthesis and polymerization of the lipid intermediate. However, no experimental evidence supporting this suggestion has been published. In this work, from the biochemical analysis of a defined set of X. campestris gum mutants, we report experimental data for assigning functions to the products of the gum genes. We also show that the first step in the assembly of the lipid-linked intermediate is severely affected by the combination of certain gum and non-gum mutations. In addition, we provide evidence that the C-terminal domain of the gumD gene product is sufficient for its glucosyl-1-phosphate transferase activity. Finally, we found that alterations in the later stages of xanthan biosynthesis reduce the aggressiveness of X. campestris against the plant.
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