Numerical Taxonomic Analysis of Some Strains of Rhizobium spp. That Uses a Qualitative Coding of Immunodiffusion Reactions.

Antigenic relationships among seven strains of Bradyrhizobium japonicum were examined by immunodiffusion reactions, in which cells of each strain were reacted against each of the seven corresponding antisera. Similar analyses were performed with Rhizobium trifolii (28 strains), Rhizobium meliloti (9 strains), and rhizobia of the cowpea miscellany (13 strains). Antigens and antisera were reacted within each species only; serological interspecies cross-reactions were not performed. The results, scored qualitatively as reactions of identity, cross-reactions, or no reaction, were formed into datum matrices and used to analyze the relationships between strains by applying the association measure of Bray and Curtis (J. R. Bray and J. T. Curtis, Ecol. Monogr. 27:325-349, 1957) and the UPGMA clustering algorithm (P. H. A. Sneath and R. R. Sokal, Numerical Taxonomy, 1973). No two strains were regarded as being serologically identical unless each gave the same results as the other in each immunodiffusion reaction against every antiserum. Despite the high level of cross-reactions and reactions of identity (totalling 93% of all cell-antiserum combinations) among strains of R. trifolii and R. meliloti, no strains were identical by the criterion described above; however, the strains of these species clustered rapidly and fused at the 70% similarity level. The B. japonicum strains and the rhizobia of the cowpea miscellany were much less cross-reactive (67 and 86% of all combinations were negative, respectively), and they clustered more slowly. The strains of B. japonicum fused completely only at the 4% similarity level, whereas of the 13 cowpea-nodulating strains, 4 reacted as two pairs of identical strains and 6 remained unfused.

Evidence for genetic exchange and recombination of Rhizobium symbiotic plasmids in a soil population.

A soil population of 16 Rhizobium leguminosarum bv. trifolii isolates was characterized by using three Sym (for symbiotic) plasmid-specific DNA hybridization probes: (i) an R. leguminosarum bv. trifolii-specific, repeated-sequence probe; (ii) a nifHDK gene probe, and (iii) a nod gene probe. A predominant Sym plasmid family was identified among the isolates. Three other unrelated Sym plasmid families were also identified. The isolates were also classified either by using a chromosomal DNA hybridization probe or by serological relatedness to 25 different R. leguminosarum bv. trifolii antisera. With either method, it was possible to group the 16 soil isolates into identical or related families. However, the correlation between the two techniques was not high. Irrespective of the means used to classify the bacterial host strain, it was possible to identify the same Sym plasmids in unrelated strains, as well as unrelated Sym plasmids in identical host strains. These data indicate that, within this soil population, there has been genetic exchange of Sym plasmids, and in one instance the hybridization pattern indicates that in vivo recombination of two different Sym plasmids may have occurred. Symbiotic effectiveness tests on red, strawberry, and subterranean clovers clearly differentiated the isolates. In general, the pattern of response was similar within groupings on the basis of Sym plasmid and chromosomal profiles but different between such groups.

Identification of pyruvated monosaccharides in polysaccharides by gas-liquid chromatography mass spectrometry.

Twelve bacterial polysaccharides of known structure containing a representative range of pyruvated monosaccharides, were methanolysed, trimethylsilylated, and analysed by g.l.c. and g.l.c.-m.s. Except for 3,4-O-(1-carboxyethylidene)-L-rhamnose, which was unusually labile, the pyruvic acid substituents were largely retained during methanolysis and the Me3Si derivatives of the resulting pyruvated methyl glycosides gave distinctive g.l.c. peaks with characteristic mass spectra. The pyranose rings of 4,6-O-(1-carboxyethylidene)-D-glucose, 4,6-O-(1-carboxyethylidene)-D-mannose, 4,6-O-(1-carboxyethylidene)-D-galactose, and 3,4-O-(1-carboxyethylidene)-D-galactose survived the methanolysis, but that of 2,3-O-(1-carboxyethylidene)-D-glucuronic acid was cleaved to give the methyl ester of 2,3-O-(1-carboxyethylidene)-aldehydo-D-glucuronic acid dimethyl acetal. In the case of 2,3-O-(1-carboxyethylidene)-D-galactose, cleavage of the pyranose ring was less complete; under the conditions used in these experiments two-thirds of the pyranose rings were intact while one-third were cleaved to give the methyl ester of 2,3-O-(1-carboxyethylidene)-aldehydo-D-galactose dimethyl acetal. A very small amount of 3,4-O-(1-carboxyethylidene)-L-rhamnose from one polysaccharide retained its pyruvic acid substituent after gentle methanolysis to give the methyl ester of 3,4-O-(1-carboxyethylidene)-aldehydo-L-rhamnose dimethyl acetal. Susceptibility to cleavage of the pyranose ring during methanolysis appears to be a property of pyruvated monosaccharides with trans-fused 1,3-dioxolane rings.

Immunochemical determination of the configuration of a haptenic substituent.

Quantitative inhibition of specific immune precipitation, a rapid microanalytical technique requiring no expensive equipment, was used to determine the stereoconfiguration of pyruvyl groups attached as acetals to two hydroxyls of nonreducing lateral end groups of the capsular polysaccharides of Klebsiella serotypes K11 and K21. The R and S isomers of 4,6-O-pyruvyl-D-alpha-methylgalactoside were used as inhibitors with appropriate polysaccharide antigens and antisera to the two serotypes. The R isomer was a potent inhibitor of precipitation in both antisera, showing that the pyruvylgalactosyl residues in the polysaccharides of both K11 and K21 are in the R form, in which the methyl group of pyruvyl is equatorial to the plane of the acetal ring.

Further examination of presumptive Rhizobium trifolii mutants that nodulate Glycine max.

Two recent reports described the isolation of derivatives of a Rhizobium trifolii strain that had gained the ability to nodulate Glycine max and Vigna radiata and that had demonstrated altered patterns of carbon source utilization, free-living nitrogen fixation, and hydrogen uptake. More extensive characterization of these strains now supports the conclusion that these strains are R. japonicum and are not derived from the putative parent R. trifolii.

Nitrogen fixation in nitrate reductase-deficient mutants of cultured rhizobia.

Forty-eight mutants unable to reduce nitrate were isolated from "cowpea" Rhizobium sp. strain 32Hl and examined for nitrogenase activity in culture. All but two of the mutants had nitrogenase activity comparable with the parental sttain and two nitrogenase-defective strains showed alterations in their symbiotic properties. One strain was unable to nodulate either Macroptilium atropurpureum or Vigna uguiculata and, with the other, nodules appeared promptly, but effective nitrogen fixation was delayed. These results, and the relatively low proportion of nitrate reductase mutants with impaired nitrogenase activity, do not support the proposed commanality between nitrogenase and nitrate reductase in cowpea rhizobia. Inhibition studies of the effect of nitrate and its reduction products on the nitrogenase activity in cultured strains 32Hl and the nitrate reductase-deficient, Nif+ strains, indicated that nitrogenase activity was sensitive to nitrite rather than to nitrate.

Nitrogenase activity and respiration of cultures of Rhizobium spp. with special reference to concentrations of dissolved oxygen.

Studies of nitrogenase in cultures of the cowpea rhizobia (Rhizobium spp.) strains 32H1 and CB756 are reported. Preliminary experiments established that, even when agar cultures were grown in air, suspensions of bacteria prepared anaerobically from them were most active at low concentrations of free dissolved O2. Consequently, assays for activity used low concentrations of O2, stabilized by adding the nodule pigment leghaemoglobin. In continuous, glutamine-limited cultures of 32H1, nitrogenase activity appeared only when the concentration of dissolved O2 in the cultures approached 1 muM. Lowering the glutamine concentration in the medium supplied to the culture from 2 to 1 mM halved the cell yield and nitrogenase activity was also diminished. Omitting succinate from the medium caused the concentration of dissolved O2 to rise and nitrogenase activity was lost. Upon restoration of the succinate supply, the O2 concentration immediately fell and nitrogenase was restored. The activity doubled in about 8 h, whereas the doubling time of this culture was 14 h. Sonic extracts of 32H1 cells from continuous cultures with active nitrogenase contained components reacting with antiserum against nitrogenase Mo-Fe protein from soybean bacteroids. Continuous cultures grown at higher O2 concentration, with only a trace of active nitrogenase, contained less of these antigens and they were not detected in highly aerobic cultures. Nitrogenase activity of a continuous culture was repressed by NH+4; the apparent half-life was about 90 min. Cells of 32H1 from a continuous culture growing at between 30 and 100 muM dissolved O2 possessed a protective mechanism which permitted respiration to increase following exposure to a rapid increase in O2 concentration from low levels (O2 shock). This effect disappeared as the O2 concentration for growth was reduced towards 1 muM.

Antigenic analysis of Rhizobium japonicum by immunodiffusion.

Immunodiffusion reactions were studied with seven strains of Rhizobium japonicum and three strains of the cowpea miscellany by using antisera against eight of the strains. Most strains yielded only weak precipitin bands when untreated cell suspensions were used as antigens in the diffusions. Ultrasonic disruption or heat treatment of the cells led to stronger bands, and immersion in boiling water for 20 min was used as the standard procedure for preparing these bacteria for immunodiffusion analysis. Heat-labile antigens were detected in only a few strains; the major antigens of all of the strains appeared to be heat-stable. Many of the strains cross-reacted, sometimes in a nonreciprocal manner; unheated cell suspensions cross-reacted more widely but more weakly than the heated suspensions. Heat-treated crushed nodule preparations reacted well in immunodiffusions. The antigens of cultured cell and nodule extract (bacteroid) forms of three strains were compared. In one of these strains, an antigen present in the cultured cells was absent from the bacteroids. Unknown strains present in soybean root nodules were readily identified by immunodiffusion.

Immunochemistry of newly found substituents of polysaccharides of Rhizobium species.

Acetyl and pyruvic acid groups were detected and estimated in the extracellular polysaccharides of Rhizobium trifolii and R. meliloti. Pyruvic acid was found also in the specific polysaccharide of pneumococcal type 27, and is believed to explain the cross-reactivity between antiserum to pneumococcal type 27 and these and other pyruvate-containing bacterial polysaccharides. Removal of pyruvate from a Rhizobium polysaccharide rendered it inactive with its homologous antiserum and with antiserum to pneumococcal type 27, but it then reacted with several antiserums to other pneumococcal types with which the intact material was unreactive.