Downloaded from www.microbiologyresearch.org by IP: 54.90.7.114 On: Mon, 23 May 2016 20:39:15 Microbiology (1997), 143, 1951-1958 Printed in Great Britain Regulation of exopolysaccharide production in Rhizobium leguminosarum biovar viciae WSM710 involves exoR Wayne G. Reeve, Michael J. Dilworth, Ravi P. Tiwari and Andrew R. Glenn Author for correspondence: Andrew R. Glenn. Tel: +61 9 360 2231. Fax: $61 9 360 6303. e-mail: arglenn@central.murdoch.edu.au Nitrogen Fixation Research Group, School of Biological and Environmental Sciences, Murdoch University, Murdoch, Western Australia 61 50, Australia A mildly acid-sensitive mutant of Rhizobium leguminosarum bv. viciae WSM710 (WR6-35) produced colonies which were more mucoid in phenotype than the wild-type. Strain WR6-35 contained a single copy of Tn5 and the observed mucoid phenotype, acid sensitivity and TnS-induced kanamycin resistance were 100 O / O co-transducible using phage RL38. WR6-35 produced threefold more exopolysaccharide (EPS) than the wild-type in minimal medium devoid of a nitrogen source. EPS produced by the mutant and the wild-type was identical as determined by proton NMR spectra. An EcoRl rhizobial fragment containing Tn5 and flanking rhizobial sequences was cloned from the mutant, restriction mapped and sequenced. There was extensive similarity between the ORF disrupted by TnS in R. leguminosarum bv. viciae WR6-35 and the exoR gene of Rhizobium (Sinorhizobium) meliloti Rml02l (71-3 O/O identity over 892 bp). At the protein level there was 70% identity and 9303% similarity over 267 amino acids with the ExoR protein of R. meliloti RmlO21. Hydrophilicity profiles of the two proteins from these two rhizobia are superimposable. This gene in R. leguminosarum bv. viciae was thus designated exoR. The data suggest that Tn5 has disrupted a regulatory gene encoding a protein that negatively modulates EPS biosynthesis in R. leguminosarum bv. viciae WSM7lO. Despite earlier suggestions that EPS production and acid tolerance might be positively correlated, disruption of exoR in either R. leguminosarum bv. viciae or R. meliloti and its associated overproduction of EPS does not result in a more acid-tolerant phenotype than the wild-type when cultures are screened on conventional laboratory agar. Keywords : exopolysaccharide, acid tolerance, regulator, soil acidity, Rhizobium INTRODUCTION Root nodule bacteria are able to infect, nodulate, and convert atmospheric N, into NH, in association with a specific legume host. The symbiotic associations be- tween the root nodule bacteria and legumes are of immense importance since they provide the largest input from biological nitrogen fixation into agricultural pro- duction. Bacterial exopolysaccharide (EPS) is thought to play a ............................................................................................................................... .... .............. Abbreviation : EPS, exopolysaccharide. The GenBank accession number for the sequence reported in this paper is L39937. critical role in the rhizobial-plant interaction (Leigh & Coplin, 1992). Rhizobium can secrete EPS consisting of either homopolymers (1,2-P-glucans or cellulose) or heteropolysaccharides. The latter are acidic polymers composed of linear arrangements of repeating units containing neutral sugars and uronic acids as well as no n- car bo h y d r a t e su bs tit u en t s such a s acetate , p y r u v a t e , hydroxybutyrate and succinate (Aman et al., 1981 ; Canter Cremers et al., 1991; Cunningham & Munns, 1984; McNeil et al., 1986). Mutants of Rhizobium leguminosarum defective in EPS production are unable to induce visible nodule de- velopment on pea roots (Diebold & Noel, 1989). In contrast, exo mutants of Rhizobium meliloti (recently renamed Sinorhizobium meliloti: De Lajudie et al., 0002-1309 0 1997 SGM 1951