Extracellular polysaccharide composition, ex planta nitrogenase activity, and DNA homology in Rhizobium japonicum

Abstract

The composition of the major acidic extracellular polysaccharide (EPS) of 25 strains of Rhizobium japonicum was determined. Eight strains synthesized an acidic EPS containing rhamnose and 4-0methylglucuronic acid and were closely related according to DNA homology. These same strains also expressed high levels of ex planta nitrogenase activity. Sixteen strains produced an acidic EPS containing glucose, mannose, galacturonic acid, and galactose and were also related by DNA homology. These strains developed little or no nitrogenase activity under the experimental conditions employed.Previous results from this laboratory demonstrated a negative correlation between the amount of extracellular polysaccharide (EPS) produced and ex planta nitrogenase activity with many strains of Rhizobium japonicum (1). In addition, it was suggested that the differences in EPS production may not only be quantitative, but qualitative as well. Two different types of a major acidic EPS from R. japonicum have been characterized. One type has a tetrasaccharide repeating unit with a backbone of three rhamnosyl residues and a side chain containing one residue of 4-0-methylglucuronic acid (12). The second type has a pentasaccharide repeating unit with a backbone of two residues of glucose and one each of mannose and galacturonic acid and a side chain containing one residue of galactose or 4-0-methylgalactose (26). Compositional studies of the EPS from numerous strains of R. japonicum suggest that, with the exception of some strains isolated in the Far East (15, 16), most strains produce an EPS having a composition that is consistent with one of these two structures (11,12,25,26).Numerous workers have presented evidence suggesting that a reconsideration of Rhizobium classification is needed (see, e.g., references 14, 17, and 18). Recently, Hollis et al. (20) suggested that strains classified as R. japonicum could be separated into at least three DNA homology groups based on DNA-DNA hybridization studies. Two of the groups were so widely divergent that the authors suggested that separate species status may be appropriate. Using many of the same strains that Hollis et al. (20) studied, we have found that EPS composition and ex planta nitrogenase activity are related to the DNA homology group.Microorganisms. R. japonicum strains were obtained from and Rhizobium lupini ATCC 10319).Stock cultures were maintained on solid medium as previously described (1). The cells were routinely grown on * Corresponding author. t Contribution no. 824 from the Charles F. Kettering Research Laboratory.defined liquid medium containing 7.4 mM K2HPO4 (pH 6.5), 0.8 mM MgSO4, 50 FLM CaCl2, 0.1 mM FeSo4, 0.5% sodium gluconate, 0.1% glutamate, and trace metals (1).Composition of EPS. EPS was prepared by centrifuging 50 ml of log-phase cultures at 27,000 x g for 30 min. The clear supernatants were lyophylized and redissolved in 6 ml of water. Four milliliters of this concentrate was loaded onto a column (2.5 by 40 cm) containing Fractogel TSK HW-55F (Pierce Chemical Co., Roc...

Slow-growing Rhizobium japonicum comprises two highly divergent symbiotic types

“…Two derivatives of this strain may exist, since we observed considerably higher intrinsic antibiotic resistances for USDA 110 than are reported for the UC Davis strain 110.SU (17). It is therefore indicated that ex planta nitrogenase activity is generally associated with the sTIl genotype, but some sTI strains can be derepressed on a-ketoglutonate or malate medium (20,38). We note that these sTII strains are appropriate candidates for molecular genetic studies of the regulation of nitrogen fixation in R. japonicum.…”

“…It is reported that in gluconate-glutamate media, strains THA6, USDA 115, USDA 58, USDA 140, and USDA 110 produce no nitrogenase activity ex planta, whereas strains 61A76 and USDA 76 produce high activity ex planta (20). USDA 76 is a plant passage reisolate of USDA 74 (25).…”

“…A number of studies have indicated that there are considerable physiological and symbiotic differences among slowgrowing strains currently classified as R. japonicum (9,20,24,32). We asked whether this heterogeneity extends to the genome of these bacteria, particularly to the symbiotic genes involved in nodulation and nitrogen fixation.…”

Production of Extracellular Polysaccharide by aRhizobium Species from Root Nodules of the Leguminous TreeDalbergia lanceolaria

“…Hollingsworth et al [16] observed the presence of glucose, galactose and mannose in EPS, which were secreted by the Rhizobium strains MI-50A, M6±7B, and IRC 253. Some strains of Bradyrhizobium japonicum (Rhizobium as identified by the authors) contained EPS having rhamnose and 4-O-methylglucuronic acid [17]. Glycosyl components of the lipopolysaccharide (LPS) of Rhizobium trifolii 4S were found to be L-rhamnose, L-acetyl D-glucosamine and N-acetyl-D-mannosamine in a 3:1:1 molar proportion [18].…”

Division of Rhizobitoxine-Producing and Hydrogen-Uptake Positive Strains of <italic>Bradyrhizobium japonicum</italic> by <italic>nifDKE</italic> Sequence Divergence

“…Hup + strains were confined exclusively to the latter group of strains that were characterized by type A EPS and the absence of production of rhizobitoxine. Huber et al (1984) demonstrated a strict correlation between EPS composition and DNA homology group, which was based on DNA-DNA hybridization studies by Hollis et al (1981). DNA homology groups I and la are composed of B. japonicum strains that produce the type A EPS, while DNA homology group II is composed of the strains that produce the type B EPS.…”