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

Hotte, B; Ratharnold, I; Pühler, Alfred; Simon, Reinhard

doi:10.1128/jb.172.5.2804-2807.1990

Cited by 64 publications

(37 citation statements)

References 28 publications

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“…In V. cholerae, Deleya marina and X. campestris pv. campestris, rough appearance has been linked to EPS production (Hotte et al, 1990;Shea et al, 1991;Wai et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis

et al. 2009

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The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis Azospirillum brasilense is a plant root-colonizing bacterium that exerts beneficial effects on the growth of many agricultural crops. Extracellular polysaccharides of the bacterium play an important role in its interactions with plant roots. The pRhico plasmid of A. brasilense Sp7, also named p90, carries several genes involved in synthesis and export of cell surface polysaccharides. We generated two Sp7 mutants impaired in two pRhico-located genes, noeJ and noeL, encoding mannose-6-phosphate isomerase and GDP-mannose 4,6-dehydratase, respectively. Our results demonstrate that in A. brasilense Sp7, noeJ and noeL are involved in lipopolysaccharide and exopolysaccharide synthesis. noeJ and noeL mutant strains were significantly altered in their outer membrane and cytoplasmic/periplasmic protein profiles relative to the wild-type strain. Moreover, both noeJ and noeL mutations significantly affected the bacterial responses to several stresses and antimicrobial compounds. Disruption of noeL, but not noeJ, affected the ability of the A. brasilense Sp7 to form biofilms. The pleiotropic alterations observed in the mutants could be due, at least partially, to their altered lipopolysaccharides and exopolysaccharides relative to the wild-type.

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“…In V. cholerae, Deleya marina and X. campestris pv. campestris, rough appearance has been linked to EPS production (Hotte et al, 1990;Shea et al, 1991;Wai et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis

et al. 2009

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“…X. campestris IS element B100-2 is most probably the cause of an exopolysaccharide defect in at least two X. campestris pv. campestris exopolysaccharide mutants (18).…”

Section: Discussionmentioning

confidence: 99%

Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors

et al. 1991

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On the basis of an RSF1010-derived broad-host-range vector, three different systems which enable positive detection and isolation of insertion sequence (IS) elements from gram-negative bacteria were constructed. Vectors pSUP104-pheS, pSUP104-rpsL, and pSUP104-sac were used successfully in a number of Rhizobium strains and in Xanthomonas campestris. More than 20 different IS elements were isolated and characterized. The 16 IS elements from Rhizobium meliloti were further used to characterize various R. meliloti strains by hybridization. The resulting hybridization patterns were different for every strain and gave a dear and definite IS fingerprint of each strain. These IS fingerprints can be used to identify and characterize R. meliot strains rapidly and unequivocally, as they proved to be relatively stable. Some of the IS elements were found to be identical when the IS fingerprints from a given strain were compared. This method of IS fingerprinting can also establish whether IS elements are the same, related, or different.Insertion sequence (IS) elements are defined as relatively small mobile genetic entities which, unlike drug resistance transposons, do not contain selectable genes (5). This definition makes it clear that direct selection for the presence of an IS element is usually not possible. IS elements in procaryotes were first identified as causative agents of spontaneous mutations in Escherichia coli (21, 36). Since then, various classes of transposable elements have been found to be natural constituents of many bacterial chromosomes, plasmids, and bacteriophages. As far as gram-negative bacteria are concerned, most IS elements have been isolated from enteric bacteria (mainly E. coli; for reviews and lists of isolated elements, see references 14 and 20). However, there have been a substantial number of reports on IS elements identified in other gram-negative bacterial species, such as Agrobacterium tumefaciens (3,15,24,42), Xanthomonas campestris (22, 23), and various Rhizobium (9,28,32) and Pseudomonas (6,13,35,41,45) strains.In general, these elements have been found more or less by chance because of the capacity to inactivate or activate particular genes. However, there have been successful attempts to screen for transposable elements by means of direct selection procedures (8,15,30,35).In this report, we present the construction and use of three broad-host-range vectors designed to enable systematic searches for the presence of transposable elements in gramnegative bacteria. Advantage was taken of two direct selection systems initially constructed to simplify cloning experiments with E. coli (7, 17), as well as a system described by Gay and coworkers (15). The common principle of these systems is based upon insertional inactivation of a vectorborne dominant sensitivity marker. With pSUP104-pheS and pSUP104-rpsL, this leads to drug resistance. Inactivation of pSUP104-sac allows its host to grow on medium containing 5% sucrose. Both of these events are positively selectable phenotypes in the hosts. (A...

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

Cited by 64 publications

References 28 publications

The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis

The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis

Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors

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

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