The
            <i>mdoC</i>
            Gene of
            <i>Escherichia coli</i>
            Encodes a Membrane Protein That Is Required for Succinylation of Osmoregulated Periplasmic Glucans

Lacroix, Jean-Marie; Lanfroy, Eric; Cogez, Virginie; Lequette, Yannick; Bohin, Anne; Bohin, Jean-Pierre

doi:10.1128/jb.181.12.3626-3631.1999

Cited by 44 publications

(33 citation statements)

References 28 publications

(29 reference statements)

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“…Using a procedure very similar to that developed for the isolation of an E. coli mutant, mdoC, deficient in the succinyl substitution of OPGs [32], a mutant (NFB4000) with nonacidic OPG (see below) was obtained from strain WS8. OPGs extracted from clones obtained after transposon Tn5TpMCS mutagenesis were tested by thin layer chromatographic analysis [23].…”

Section: R E S U L T S Isolation and Characterization Of The Osmoregumentioning

confidence: 99%

Osmoregulated periplasmic glucans of the free‐living photosynthetic bacterium Rhodobacter sphaeroides

Talaga¹,

Cogez²,

Wieruszeski³

et al. 2002

European Journal of Biochemistry

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The osmoregulated periplasmic glucans (OPGs) produced by Rhodobacter sphaeroides, a free-living organism, were isolated by trichloracetic acid treatment and gel permeation chromatography. Compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry and nuclear magnetic resonance. R. sphaeroides predominantly synthesizes a cyclic glucan containing 18 glucose residues that can be substituted by one to seven succinyl esters residues at the C 6 position of some of the glucose residues, and by one or two acetyl residues. The glucans were subjected to a mild alkaline treatment in order to remove the succinyl and acetyl substituents, analyzed by MALDI mass spectrometry and purified by high-performance anion-exchange chromatography. Methylation analysis revealed that this glucan is linked by 17 1,2 glycosidic bonds and one 1,6 glycosidic bond. Homonuclear and 1 H/ 13 C heteronuclear NMR experiments revealed the presence of a single a-1,6 glycosidic linkage, whereas all other glucose residues are b-1,2 linked. The different anomeric proton signals allowed a complete sequence-specific assignment of the glucan. The structural characteristics of this glucan are very similar to the previously described OPGs of Ralstonia solanacearum and Xanthomonas campestris, except for its different size and the presence of substituents. Therefore, similar OPGs are synthesized by phytopathogenic as well as free-living bacteria, suggesting these compounds are intrinsic components of the Gram-negative bacterial envelope.Keywords: periplasm; osmoregulation; cyclic glucans.Osmoregulated periplasmic glucans (OPGs) appear to be general constituents of the envelope of Gram-negative bacteria [1]. Their abundance in the periplasmic space is the greatest when the medium osmolarity is very low. Under these conditions, OPGs can represent between 5 and 10% of the cellular dry weight. These compounds play an important role in the interaction with specific plant hosts; in Sinorhizobium meliloti [2] and Bradyrhizobium japonicum [3] they are essential for nitrogen fixation; and in Agrobacterium tumefaciens [4], Pseudomonas syringae [5,6], and Erwinia chrysanthemi [7] for the development of plant disease. In this latter case, experiments in which OPG deficient mutants were coinoculated with wild-type bacteria have established that OPGs must be present in the periplasmic space of the bacteria to enable growth in the plant host [7]. However, beyond this functional homology, and the fact that glucose is the sole monosaccharide present, OPGs from various origins display an unexpected structural diversity. This variation occurs at two levels: the glucose backbone organization, and the absence or the presence of various substituents.Four families of OPGs can now be distinguished (a) OPGs of Escherichia coli, P. syringae and Erwinia chrysanthemi appears to range from six to 13 glucose residues [8,9]. The structure is highly branched, the backbone consisting of b-1,2 linked glucose units to which the...

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Section: R E S U L T S Isolation and Characterization Of The Osmoregumentioning

confidence: 99%

Osmoregulated periplasmic glucans of the free‐living photosynthetic bacterium Rhodobacter sphaeroides

Talaga¹,

Cogez²,

Wieruszeski³

et al. 2002

European Journal of Biochemistry

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“…In E. coli, localization of the succinylation was not determined but the mdoC gene, which is necessary for succinylation of OPGs, encodes a protein with 10 predicted transmembrane segments. Consequently, it was proposed that this protein could catalyze the transfer of succinyl residues from the cytoplasmic side of the membrane to the nascent glucan backbones on the periplasmic side of the membrane [19]. If this is true, one can postulate the existence of a protein complex where the enzymes necessary for backbone synthesis and the enzymes necessary for backbone modi¢cation could work coordinately (Fig.…”

Section: Glucan Substitutionmentioning

confidence: 99%

Osmoregulated periplasmic glucans in Proteobacteria

Bohin

2000

FEMS Microbiology Letters

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Large amounts of osmoregulated periplasmic glucans (OPGs) are found in the periplasmic space of Proteobacteria. Four families of OPGs are described on the basis of structural features of the polyglucose backbone. Depending on the species considered, OPGs can be modified to various extent by a variety of substituents. Genes governing the backbone synthesis are identified in a limited number of species. They belong to three unrelated families. OPG synthesis is subject to osmoregulation and feedback control. Osmoregulation can occur at the level of gene expression and/or at the level of enzyme activity. Mutants defective in OPG synthesis have a highly pleiotropic phenotype, indicative of an overall alteration of their envelope properties. Mutants of this kind were obtained as attenuated or avirulent derivatives of plant or animals pathogen. Thus, OPGs appear to be important intrinsic components of the Gram‐negative bacterial envelope, which can be essential in extreme conditions found in nature, and especially when bacteria must interact with an eukaryotic host.

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“…Each Tn 5 TpMCS mutant generated from WS8 was screened for production of OPGs as described previously: 4 mL of an overnight culture in Luria–Bertani without NaCl were treated to give a 15‐µL extract in water [14]. Samples of 5 µL were analyzed by chromatography on aluminium silica gel 60 plates (Merk) in ethanol/butanol/water (5 : 5 : 4) solvent.…”

Section: Methodsmentioning

confidence: 99%

“…Our initial purpose was to obtain OPG defective mutants by screening a transposon insertion library with a thin layer chromatographic assay [14]. A single mutant clone was detected that produced neutral OPGs.…”

mentioning

confidence: 99%

The opgGIH and opgC genes of Rhodobacter sphaeroides form an operon that controls backbone synthesis and succinylation of osmoregulated periplasmic glucans

Cogez¹,

Gak²,

Puskás³

et al. 2002

European Journal of Biochemistry

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Osmoregulated periplasmic glucans (OPGs) of Rhodobacter sphaeroides are anionic cyclic molecules that accumulate in large amounts in the periplasmic space in response to low osmolarity of the medium. Their anionic character is provided by the substitution of the glucosidic backbone by succinyl residues. A wild-type strain was subject to transposon mutagenesis, and putative mutant clones were screened for changes in OPGs by thin layer chromatography. One mutant deficient in succinyl substitution of the OPGs was obtained and the gene inactivated in this mutant was characterized and named opgC. opgC is located downstream of three ORFs, opgGIH, two of which are similar to the Escherichia coli operon, mdoGH, governing OPG backbone synthesis. Inactivation of opgG, opgI or opgH abolished OPG production and complementation analysis indicated that the three genes are necessary for backbone synthesis. In contrast, inactivation of a gene similar to ndvB, encoding the OPG-glucosyl transferase in Sinorhizobium meliloti, had no consequence on OPG synthesis in Rhodobacter sphaeroides. Cassette insertions in opgH had a polar effect on glucan substitution, indicating that opgC is in the same transcription unit. Expression of opgIHC in E. coli mdoB/mdoC and mdoH mutants allowed the production of slightly anionic and abnormally long linear glucans.

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The mdoC Gene of Escherichia coli Encodes a Membrane Protein That Is Required for Succinylation of Osmoregulated Periplasmic Glucans

Cited by 44 publications

References 28 publications

Osmoregulated periplasmic glucans of the free‐living photosynthetic bacterium Rhodobacter sphaeroides

Osmoregulated periplasmic glucans of the free‐living photosynthetic bacterium Rhodobacter sphaeroides

Osmoregulated periplasmic glucans in Proteobacteria

The opgGIH and opgC genes of Rhodobacter sphaeroides form an operon that controls backbone synthesis and succinylation of osmoregulated periplasmic glucans

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