Location and cloning of the ketal pyruvate transferase gene of Xanthomonas campestris

Marzocca, Marina Patricia; Harding, Nancy E.; Petroni, Alejandro; Cleary, Joseph M.; Ielpi, Luis

doi:10.1128/jb.173.23.7519-7524.1991

Cited by 42 publications

(24 citation statements)

References 30 publications

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“…Preparation of EDTA-treated cells and the assay for in vitro synthesis of xanthan gum were done as previously described (Ielpi et al, 1981a), but using UDP-['4C]GlcA as radiolabelled sugar, and unlabelled UDP-Glc and GDP-Man as sugar donors (Marzocca et al, 1991). Under these conditions, in incubations done with EDTA-treated wild-type cells, the main product extracted in the lipid fraction is ['4C-GlcA]ps-P-P-lipid, but small amounts of a [14C-GalAIlipid-bound galacturonide, which is also formed, may be present (Baldessari et al, 1990).…”

Section: Methodsmentioning

confidence: 99%

Identification, Genetic and Biochemical Analysis of Genes involved in Synthesis of Sugar Nucleotide Precursors of Xanthan Gum

Harding¹,

Raffo²,

Raimondi³

et al. 1993

Journal of General Microbiology

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A genetic and biochemical analysis of Xanthomonas campestris chromosomal functions required for xanthan polysaccharide synthesis (xps) was undertaken. Seven xps DNA regions were isolated after conjugation of chemically induced non-mucoid mutants with a genomic library of X. campestris DNA. No overlapping segments between regions were detected, based on physical mapping, indicating the unlinked character of these regions. Clones complementing several different mutants belonging to the same region contained overlapping segments of X. campestris chromosomal DNA. Complementation and biochemical analysis, and DNA mapping were used to identify and characterize xpsZZZ, ZV and VZ DNA regions. Mutants in these three regions were able to synthesize both lipid intermediates and xanthan gum in vitro when sugar nucleotides were provided as substrates. HPLC analysis of the intracellular sugar nucleotide content showed that the XpsIII group comprises two different classes of mutants : XpsIIIA, defective in UDP-glucose, UDP-glucuronic acid and GDP-mannose, and XpsIIIB, defective in GDP-mannose. XpsIV mutants were defective in UDP-glucose and UDP-glucuronic acid, and XpsVI mutants were defective only in UDP-glucuronic acid. Analysis of enzyme activities involved in the synthesis of UDP-glucose, GDP-mannose and UDP-glucuronic acid indicated that the xpsZZZA region affects the activity of the phosphoglucomutase/phosphomannomutase enzyme, and the xpsZZZB region affects the mannoisomerase/ phosphomannoisomerase activities. The xpsZV mutations affect the activity of the UDPG-pyrophosphorylase enzyme, and the xps VZ mutations affect the activity of the UDPG-dehydrogenase enzyme.

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Section: Methodsmentioning

confidence: 99%

Identification, Genetic and Biochemical Analysis of Genes involved in Synthesis of Sugar Nucleotide Precursors of Xanthan Gum

Harding¹,

Raffo²,

Raimondi³

et al. 1993

Journal of General Microbiology

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“…It has been suggested that the EPS may play a role in the ability of Xanthomonas spp. to infect plants (58) have induced mutations affecting its EPS (2,6,26,28,33,38,60,65) and no definitive role of its EPS or cell surface molecules in virulence has been determined (2,9,17,29,36,44,48,59,61). Defects in the LPS of Rhizobium, Erwinia, and Pseudomonas species can lead to partial or complete loss of virulence (30,43,51).…”

Section: M28mentioning

confidence: 99%

“…The opsXY locus affected the EPS of 3048 but was not observed to complement any of the 90 non-or low-mucoid mutants of X. campestris tested in the Kelco, Inc., collection. The mutational lesions in these 90 mutants were mapped to eight different regions of the chromosome and are involved in the synthesis of the pentasaccharide (26), synthesis of the sugar nucleotide precursors (38), or to unknown functions of xanthan biosynthesis (25a). These results indicated that the opsXY locus was not primarily involved in EPS biosynthesis.…”

Section: Gca Cgc Gcg CM Tgg Gac Gac Gat Ggc Cgc Cct Gta Ctg Atg Atc Tmentioning

confidence: 99%

The opsX locus of Xanthomonas campestris affects host range and biosynthesis of lipopolysaccharide and extracellular polysaccharide

et al. 1993

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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 ofEscherichia 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.Bacterial cell-surface molecules compose the glycocalyx (8) and play critical roles in the survival of free-living and parasitic prokaryotes. In gram-negative bacteria, the outermost molecules include extracellular polysaccharide (EPS), which is secreted to form an outer layer in the form of capsules or loose slime, and lipopolysaccharide (LPS), a component of the outer membrane. The ability of bacteria to colonize a specific niche may depend upon the EPS layer, which serves a variety of roles, including protecting pathogens from desiccation, toxic metals, and host defense responses (6,7,31,42). The outer membrane LPS acts as a permeability barrier to various toxic molecules such as hydrophobic antibiotics, detergents, and hydrophobic dyes (39,42,45). Since they have many charged substituents, the polysaccharides of the LPS are believed to be involved in the barrier function, including protecting both animal and plant pathogens from toxic hydrophobic host molecules (42,43,45).With some plant-associated microbes, cell surface molecules have been shown to be crucial for determining the success or failure of parasiti...

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“…(19,20), Rhizobium sp. (21,22), and Bacillus anthracis (23), among other species, it was of interest to determine whether S. pombe has a genome-encoded functional analog of these proteins.…”

mentioning

confidence: 99%

Five Genes Involved in Biosynthesis of the Pyruvylated Galβ1,3-Epitope in Schizosaccharomyces pombe N-Linked Glycans

Andreishcheva

Kunkel

Gemmill

et al. 2004

Journal of Biological Chemistry

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The N-linked galactomannans of Schizosaccharomyces pombe have pyruvylated Gal␤1,3-(PvGal) caps on a portion of the Gal␣1,2-residues in their outer chains (Gemmill, T. R., and Trimble, R. B. (1998) Glycobiology 8, 1087-1095). PvGal biosynthesis was investigated by ethyl methanesulfonate mutagenesis of S. pombe, followed by the isolation of cells devoid of negatively charged N-glycans by Q-Sepharose exclusion and failure to bind human serum amyloid P component, which acts as a lectin for terminal PvGal residues. Mutant glycans were characterized by lectin binding, saccharide composition, exoglycosidase sensitivity, and NMR spectroscopy. Restoration of the cell surface negative charge by complementation with an S. pombe genomic library led to the identification of five genes involved in PvGal biosynthesis, which we designated pvg1-pvg5. Pvg1p may be a pyruvyltransferase, since NMR of pvg1 ؊ mutant N-glycans revealed the absence of only the pyruvyl moiety. Pvg2p-Pvg5p are crucial for attachment of the Gal␤1,3-residue that becomes pyruvylated. Pvg3p is predicted to be a member of the ␤1,3-galactosyltransferase family, and Pvg3p-green fluorescent protein labeling was consistent with Golgi localization. Predicted Pvg1p and Pvg3p functions imply that Gal␤1,3-is added to the galactomannans and is then pyruvylated in situ, rather than by an en bloc addition of PvGal␤1,3-caps to the outer chain. Pvg4p-green fluorescent protein targeted to the nucleus, and its sequence contains a MADSbox DNA-binding and dimerization domain; however, it does not appear to solely control transcription of the other identified genes. Pvg2p and/or Pvg5p may contribute to an enzyme complex. Whereas a functional role for the PvGal epitope in S. pombe remains unclear, it is nonessential for either cell growth or mating under laboratory conditions.

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Location and cloning of the ketal pyruvate transferase gene of Xanthomonas campestris

Cited by 42 publications

References 30 publications

Identification, Genetic and Biochemical Analysis of Genes involved in Synthesis of Sugar Nucleotide Precursors of Xanthan Gum

Identification, Genetic and Biochemical Analysis of Genes involved in Synthesis of Sugar Nucleotide Precursors of Xanthan Gum

The opsX locus of Xanthomonas campestris affects host range and biosynthesis of lipopolysaccharide and extracellular polysaccharide

Five Genes Involved in Biosynthesis of the Pyruvylated Galβ1,3-Epitope in Schizosaccharomyces pombe N-Linked Glycans

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