“…S. meliloti strains were grown in TY (9) or in Luria-Bertani medium. For succinoglycan production S. meliloti strains were grown at 30°C in glutamate-D-mannitol-salts (GMS) medium (pH 7.0) supplemented with 0.24 M sodium chloride, biotin, thiamine, and trace elements (55).…”
Section: Methodsmentioning
confidence: 99%
“…S. meliloti strains were grown at 30°C for 10 days in GMS medium as described by Zevenhuizen and van Neerven (55). Cells were removed by centrifugation (11,200 ϫ g, 1 h, 10°C), and the clear culture supernatants, containing the secreted EPS, were lyophilized.…”
It is thought that in the gram-negative soil bacterium Sinorhizobium meliloti the protein ExoP is involved in biosynthesis of the acidic exopolysaccharide succinoglycan (EPS I). The amounts and compositions of EPS I produced by mutants expressing ExoP proteins characterized by specific amino acid substitutions in the C-terminal cytoplasmic domain were analyzed. The cytoplasmic domain of the ExoP protein was shown to have ATPase activity. Mutations in the highly conserved Walker A ATP-binding motif prevented ATPase activity of the ExoP protein. Phenotypically, these mutations resulted in much lower levels of succinoglycan which consisted only of monomers of the octasaccharide repeating unit. The ExoP protein has similarities to proteins with autophosphorylating protein tyrosine kinase activity. We found that ExoP was phosphorylated on tyrosine and that site-directed mutagenesis of specific tyrosine residues in the cytoplasmic domain of ExoP resulted in an altered ratio of low-molecular-weight succinoglycan to high-molecular-weight succinoglycan.
“…S. meliloti strains were grown in TY (9) or in Luria-Bertani medium. For succinoglycan production S. meliloti strains were grown at 30°C in glutamate-D-mannitol-salts (GMS) medium (pH 7.0) supplemented with 0.24 M sodium chloride, biotin, thiamine, and trace elements (55).…”
Section: Methodsmentioning
confidence: 99%
“…S. meliloti strains were grown at 30°C for 10 days in GMS medium as described by Zevenhuizen and van Neerven (55). Cells were removed by centrifugation (11,200 ϫ g, 1 h, 10°C), and the clear culture supernatants, containing the secreted EPS, were lyophilized.…”
It is thought that in the gram-negative soil bacterium Sinorhizobium meliloti the protein ExoP is involved in biosynthesis of the acidic exopolysaccharide succinoglycan (EPS I). The amounts and compositions of EPS I produced by mutants expressing ExoP proteins characterized by specific amino acid substitutions in the C-terminal cytoplasmic domain were analyzed. The cytoplasmic domain of the ExoP protein was shown to have ATPase activity. Mutations in the highly conserved Walker A ATP-binding motif prevented ATPase activity of the ExoP protein. Phenotypically, these mutations resulted in much lower levels of succinoglycan which consisted only of monomers of the octasaccharide repeating unit. The ExoP protein has similarities to proteins with autophosphorylating protein tyrosine kinase activity. We found that ExoP was phosphorylated on tyrosine and that site-directed mutagenesis of specific tyrosine residues in the cytoplasmic domain of ExoP resulted in an altered ratio of low-molecular-weight succinoglycan to high-molecular-weight succinoglycan.
“…The chv and ndv genes share sequence homology and are functionally interchangable in heterologous complementation experiments (10). Both bacteria synthesize a neutral cellular and (in some cases) extracellular polysaccharide, cyclic 1,2-13-D-glucan (5,27,31), and the chvB locus is required for this synthesis in A. tumefaciens (23). A role for the glucan in either infectious process has not been conclusively established.…”
Mutants of Rhizobium meliloti have been isolated which are deficient in exopolysaccharide (EPS) production and effective nodulation of alfalfa (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). We isolated approximately 100 analogous EPS-deficient (Exo) mutants of the closely related plant pathogen Agrobacterium tumefaciens, including strains whose EPS deficiencies were specifically complemented by each of five cloned R. meliloti exo loci. We also cloned A. tumefaciens genes which complemented EPS defects in three of the R. meliloti Exo mutants. In two of these cases, symbiotic defects were also complemented. All of the A. tumefaciens Exo mutants formed normal crown gall tumors on four different plant hosts, except ExoC mutants, which were nontumorigenic and unable to attach to plant cells in vitro. Like their R. meliloti counterparts, A. tumefaciens Exo mutants were deficient in production of succinoglycan, the major acidic EPS species produced by both genera. A. tumefaciens ExoC mutants also produced extremely low levels of another major EPS, cyclic 1,2-beta-D-glucan. This deficiency has been noted previously in a different set of nontumorigenic, attachment-defective A. tumefaciens mutants.
“…(l--+2)-P-D-Glucan is a polysaccharide produced by strains of Agrobacterium and Rhizobium and found in both the culture filtrates 1 - 11 ) and the cells. 6 ,12,13) In 1964, Dedonder and Hassid 3 ) studied the in vitro synthesis of (l--+2)-P-D-glucans.…”
mentioning
confidence: 99%
“…In 1979, Sandermann and Dekker 14 ) reported that a particulate fraction of Acetobacter xy-!inum synthesized an alkali-soluble material that was probably (l--+2)-P-D-glucan from UDP-D-[14C]glucose, although this glucan has not been isolated from cultures of Acetobacter. Recently7,8,[10][11][12][13]15,16) the (l--+2)-P-oglucans produced by cultures of Agrobacterium and Rhizobium were shown to have a cyclic structure composed exclusively of P-(1--+2)-linked o-glucosyl residues, and to consist of a mixture of components with different degrees of polymerization (DPs) the distribution patterns of which were characteristic for different strains. The DPs of the cyclic (l--+2)-P-o-glucaI)s produced by Agrobacterium radiobacter IFO 12665bl were distributed between 17 and 24 (mainly 19 to 22), while that of cyclic (l--+2)-P-o-glucan from Rhizobium phaseo!i AHU 1133 was mainly 17 with a minor proportion of molecules of 18 to 20.…”
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