Molecular cloning and expression of a locus (<i>mdoA</i>) implicated in the biosynthesis of membrane‐derived oligosaccharides in <i>Escherichia coli</i>

Lacroix, Jean-Marie; Tempête, M.; Menichi, Bernadette; Bohin, Jean-Pierre

doi:10.1111/j.1365-2958.1989.tb00267.x

Cited by 27 publications

(35 citation statements)

References 25 publications

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“…Both the pyrC and mdoA genes have also been mapped to the 23.5-min region of the E. coli genome (30,60 (27,30,60). The thin lines in the pTS1, pKS12, and pSKS1 restriction maps indicate pBR322 sequences.…”

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Isolation and characterization of the Escherichia coli htrB gene, whose product is essential for bacterial viability above 33 degrees C in rich media

et al. 1991

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We have identified and studied the htrB gene of Escherichia coli. Insertional inactivation of the htrB gene leads to bacterial death at temperatures above 33°C. The mutant bacterial phenotype at nonpermissive temperatures includes an arrest of cell division followed by the formation of bulges or filaments. The htrB + gene has been cloned by complementation and shown to reside at 23.4 min on the E. coli genetic map, the relative order of the neighboring loci being mboA-htrB-pyrC. The htrB gene is transcribed in a counterclockwise fashion, relative to the E. coli genetic map, and its product has been identified as a membrane-associated protein of 35,000 Da. Growth experiments in minimal media indicate that the HtrB function becomes dispensable at low growth rates.

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Isolation and characterization of the Escherichia coli htrB gene, whose product is essential for bacterial viability above 33 degrees C in rich media

et al. 1991

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“…In this bacterium, the defect in OPG synthesis does not confer an easily selectable phenotype in laboratory conditions. Thus, the mdoGH locus was cloned using the linked selectable genetic marker pyrC (23).…”

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Osmoregulated Periplasmic Glucan Synthesis Is Required for Erwinia chrysanthemi Pathogenicity

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Hugouvieux‐Cotte‐Pattat

et al. 2001

J Bacteriol

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Erwinia chrysanthemi is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the gram-negative bacterial envelope. We cloned the opgGH operon of E. chrysanthemi, encoding proteins involved in the glucose backbone synthesis of OPGs, by complementation of the homologous locus mdoGH of Escherichia coli. OpgG and OpgH show a high level of similarity with MdoG and MdoH, respectively, and mutations in the opgG or opgH gene abolish OPG synthesis. The opg mutants exhibit a pleiotropic phenotype, including overproduction of exopolysaccharides, reduced motility, bile salt hypersensitivity, reduced protease, cellulase, and pectate lyase production, and complete loss of virulence. Coinoculation experiments support the conclusion that OPGs present in the periplasmic space of the bacteria are necessary for growth in the plant host.Osmoregulated periplasmic glucans (OPGs) are a family of oligosaccharides found in the periplasmic space of gram-negative bacteria. Their two common features are the presence of glucose as the sole constituent sugar and their increased level in media of low osmolarity (5).Members of the family Enterobacteriaceae and related bacteria synthesize a family of linear and branched OPGs that are variously substituted. The linear backbone is constituted by glucose units joined by ␤,1-2 linkages, and the branches are made of one glucose unit linked to the main chain by a ␤,1-6 linkage. In Escherichia coli, the backbone, containing 7 to 13 glucose units, is substituted with phosphoglycerol, phosphoethanolamine, and succinyl residues (19). In Erwinia chrysanthemi, the backbone contains 5 to 12 glucose units substituted with succinyl and acetyl residues (9), and in Pseudomonas syringae, the backbone, consisting of 6 to 13 glucose units, is not substituted (38). In E. coli, the OPG backbone is synthesized by the products of the mdoGH operon located in the vicinity of pyrC, a gene involved in the biosynthesis of uracil (6). In this bacterium, the defect in OPG synthesis does not confer an easily selectable phenotype in laboratory conditions. Thus, the mdoGH locus was cloned using the linked selectable genetic marker pyrC (23).Many factors are involved in the virulence of pathogenic bacteria, and OPGs appear to be among them. In P. syringae pv. syringae, the causal agent of brown spot disease of the common bean (Phaseolus vulgaris), the hrpM mutant, obtained after transposon mutagenesis, was isolated because it failed to incite disease on the host plant and to cause the hypersensitive reaction on a non-host plant such as tobacco (29). The hrpM mutant does not synthesize OPGs, and the hrpM locus complements the OPG synthesis defect of the mdoH200::Tn10 mutant of E. coli. The amino acid sequences of HrpM and MdoH are 75.5% identical and 87.5% similar (25). More recently, a transposon insertion in a gene similar to hrpM/mdoH was isolated because it severely reduces the virulence in Pseudomonas aeruginosa PA14, an opp...

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“…Solid media were obtained by adding agar (15 g l 21 ). When low-osmolarity medium was required, lowosmolarity medium (LOS) or LB without NaCl was used (Lacroix et al, 1989). Antibiotics in media were used at the following concentrations: ampicillin, 50 mg ml 21 ; kanamycin, 50 mg ml 21 ; and tetracycline, 25 mg ml 21 .…”

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“…). OPGs were extracted as described previously (Lacroix et al, 1989) and purified as follows. Pyridine extract was chromatographed on a Biogel P4 column (Bio-Rad).…”

Section: Methodsmentioning

confidence: 99%

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Biosynthesis of osmoregulated periplasmic glucans in Escherichia coli: the membrane-bound and the soluble periplasmic phosphoglycerol transferases are encoded by the same gene

et al. 2008

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In Escherichia coli, osmoregulated periplasmic glucans (OPGs) are highly substituted by phosphoglycerol, phosphoethanolamine and succinyl residues. A two-step model was proposed to account for phosphoglycerol substitution: first, the membrane-bound phosphoglycerol transferase I transfers residues from membrane phosphatidylglycerol to nascent OPG molecules; second, the periplasmic phosphoglycerol transferase II swaps residues from one OPG molecule to another. Gene opgB was reported to encode phosphoglycerol transferase I. In this study, we demonstrate that the periplasmic enzyme II is a soluble form of the membrane-bound enzyme I. In addition, timing of OPG substitution was investigated. OPG substitution by succinyl residues occurs rapidly, probably during the backbone polymerization, whereas phosphoglycerol addition is a very progressive process. Thus, both phosphoglycerol transferase activities appear biologically necessary for complete OPG substitution.

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Molecular cloning and expression of a locus (mdoA) implicated in the biosynthesis of membrane‐derived oligosaccharides in Escherichia coli

Cited by 27 publications

References 25 publications

Isolation and characterization of the Escherichia coli htrB gene, whose product is essential for bacterial viability above 33 degrees C in rich media

Isolation and characterization of the Escherichia coli htrB gene, whose product is essential for bacterial viability above 33 degrees C in rich media

Osmoregulated Periplasmic Glucan Synthesis Is Required for Erwinia chrysanthemi Pathogenicity

Biosynthesis of osmoregulated periplasmic glucans in Escherichia coli: the membrane-bound and the soluble periplasmic phosphoglycerol transferases are encoded by the same gene

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