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J Bacteriol. Sep 1988; 170(9): 4249–4256.
PMCID: PMC211434

Rhizobium meliloti mutants that overproduce the R. meliloti acidic calcofluor-binding exopolysaccharide.

Abstract

The acidic Calcofluor-binding exopolysaccharide of Rhizobium meliloti Rm1021 plays one or more critical roles in nodule invasion and possibly in nodule development. Two loci, exoR and exoS, that affect the regulation of synthesis of this exopolysaccharide were identified by screening for derivatives of strain Rm1021 that formed mucoid colonies that fluoresced extremely brightly under UV light when grown on medium containing Calcofluor. The exopolysaccharide produced in large quantities by the exoR95::Tn5 and exoS96::Tn5 strains was indistinguishable from that produced by the parental strain Rm1021, and its synthesis required the function of at least the exoA, exoB, and exoF genes. Both the exoR and exoS loci were located on the chromosome, and the exo96::Tn5 mutation was 84% linked to the trp-33 mutation by phi M12 transduction. Synthesis of the Calcofluor-binding exopolysaccharide by strain Rm1021 was greatly stimulated by starvation for ammonia. In contrast, the exoR95::Tn5 mutant produced high levels of exopolysaccharide regardless of the presence or absence of ammonia in the medium. The exoS96::Tn5 mutant produced elevated amounts of exopolysaccharide in the presence of ammonia, but higher amounts were observed after starvation for ammonia. The presence of either mutation increased the level of expression of exoF::TnphoA and exoP::TnphoA fusions (TnphoA is Tn5 IS50L::phoA). Analyses of results obtained when alfalfa seedlings were inoculated with the exoR95::Tn5 strain indicated that the mutant strain could not invade nodules. However, pseudorevertants that retained the original exoR95::Tn5 mutation but acquired unlinked suppressors so that they produced an approximately normal amount of exopolysaccharide were able to invade nodules and fix nitrogens. The exoS95::Tn5 strain formed Fix+ nodules, although some minor variability was observed.

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Selected References

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  • Allen P, Hart CA, Saunders JR. Isolation from Klebsiella and characterization of two rcs genes that activate colanic acid capsular biosynthesis in Escherichia coli. J Gen Microbiol. 1987 Feb;133(2):331–340. [PubMed]
  • Birnboim HC. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 1983;100:243–255. [PubMed]
  • Borthakur D, Johnston AW. Sequence of psi, a gene on the symbiotic plasmid of Rhizobium phaseoli which inhibits exopolysaccharide synthesis and nodulation and demonstration that its transcription is inhibited by psr, another gene on the symbiotic plasmid. Mol Gen Genet. 1987 Apr;207(1):149–154. [PubMed]
  • Brickman E, Beckwith J. Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and phi80 transducing phages. J Mol Biol. 1975 Aug 5;96(2):307–316. [PubMed]
  • De Vos GF, Walker GC, Signer ER. Genetic manipulations in Rhizobium meliloti utilizing two new transposon Tn5 derivatives. Mol Gen Genet. 1986 Sep;204(3):485–491. [PubMed]
  • Eckhardt T. A rapid method for the identification of plasmid desoxyribonucleic acid in bacteria. Plasmid. 1978 Sep;1(4):584–588. [PubMed]
  • Finan TM, Hartweig E, LeMieux K, Bergman K, Walker GC, Signer ER. General transduction in Rhizobium meliloti. J Bacteriol. 1984 Jul;159(1):120–124. [PMC free article] [PubMed]
  • Finan TM, Hirsch AM, Leigh JA, Johansen E, Kuldau GA, Deegan S, Walker GC, Signer ER. Symbiotic mutants of Rhizobium meliloti that uncouple plant from bacterial differentiation. Cell. 1985 Apr;40(4):869–877. [PubMed]
  • Finan TM, Kunkel B, De Vos GF, Signer ER. Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J Bacteriol. 1986 Jul;167(1):66–72. [PMC free article] [PubMed]
  • Friedman AM, Long SR, Brown SE, Buikema WJ, Ausubel FM. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. [PubMed]
  • Gottesman S, Trisler P, Torres-Cabassa A. Regulation of capsular polysaccharide synthesis in Escherichia coli K-12: characterization of three regulatory genes. J Bacteriol. 1985 Jun;162(3):1111–1119. [PMC free article] [PubMed]
  • Harding NE, Cleary JM, Cabañas DK, Rosen IG, Kang KS. Genetic and physical analyses of a cluster of genes essential for xanthan gum biosynthesis in Xanthomonas campestris. J Bacteriol. 1987 Jun;169(6):2854–2861. [PMC free article] [PubMed]
  • Ielpi L, Couso R, Dankert M. Lipid-linked intermediates in the biosynthesis of xanthan gum. FEBS Lett. 1981 Aug 3;130(2):253–256. [PubMed]
  • Klein S, Walker GC, Signer ER. All nod genes of Rhizobium meliloti are involved in alfalfa nodulation by exo mutants. J Bacteriol. 1988 Feb;170(2):1003–1006. [PMC free article] [PubMed]
  • Leigh JA, Signer ER, Walker GC. Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6231–6235. [PMC free article] [PubMed]
  • Leigh JA, Reed JW, Hanks JF, Hirsch AM, Walker GC. Rhizobium meliloti mutants that fail to succinylate their calcofluor-binding exopolysaccharide are defective in nodule invasion. Cell. 1987 Nov 20;51(4):579–587. [PubMed]
  • Long S, McCune S, Walker GC. Symbiotic loci of Rhizobium meliloti identified by random TnphoA mutagenesis. J Bacteriol. 1988 Sep;170(9):4257–4265. [PMC free article] [PubMed]
  • Long S, Reed JW, Himawan J, Walker GC. Genetic analysis of a cluster of genes required for synthesis of the calcofluor-binding exopolysaccharide of Rhizobium meliloti. J Bacteriol. 1988 Sep;170(9):4239–4248. [PMC free article] [PubMed]
  • Meade HM, Signer ER. Genetic mapping of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1977 May;74(5):2076–2078. [PMC free article] [PubMed]
  • Ruvkun GB, Ausubel FM. A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1;289(5793):85–88. [PubMed]
  • Smit G, Kijne JW, Lugtenberg BJ. Involvement of both cellulose fibrils and a Ca2+-dependent adhesin in the attachment of Rhizobium leguminosarum to pea root hair tips. J Bacteriol. 1987 Sep;169(9):4294–4301. [PMC free article] [PubMed]
  • Sutherland IW. Biosynthesis and composition of gram-negative bacterial extracellular and wall polysaccharides. Annu Rev Microbiol. 1985;39:243–270. [PubMed]
  • Thorne L, Tansey L, Pollock TJ. Clustering of mutations blocking synthesis of xanthan gum by Xanthomonas campestris. J Bacteriol. 1987 Aug;169(8):3593–3600. [PMC free article] [PubMed]
  • Tolmasky ME, Staneloni RJ, Leloir LF. Lipid-bound saccharides in Rhizobium meliloti. J Biol Chem. 1982 Jun 25;257(12):6751–6757. [PubMed]
  • Tolmasky ME, Staneloni RJ, Ugalde RA, Leloir LF. Lipid-bound sugars in Rhizobium meliloti. Arch Biochem Biophys. 1980 Aug;203(1):358–364. [PubMed]
  • Torres-Cabassa AS, Gottesman S. Capsule synthesis in Escherichia coli K-12 is regulated by proteolysis. J Bacteriol. 1987 Mar;169(3):981–989. [PMC free article] [PubMed]
  • Torres-Cabassa A, Gottesman S, Frederick RD, Dolph PJ, Coplin DL. Control of extracellular polysaccharide synthesis in Erwinia stewartii and Escherichia coli K-12: a common regulatory function. J Bacteriol. 1987 Oct;169(10):4525–4531. [PMC free article] [PubMed]
  • Troy FA, Frerman FE, Heath EC. The biosynthesis of capsular polysaccharide in Aerobacter aerogenes. J Biol Chem. 1971 Jan 10;246(1):118–133. [PubMed]

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