Structure of complex flagellar filaments in Rhizobium meliloti

The complex flagella of Rhizobium meliloti 2011 and MVII-1 were analyzed with regard to serology, fine structure, subunits, and amino acid composition. The serological identities of flagellar filaments of the two strains were demonstrated by double immunodiffusion with antiflagellin antiserum. The filaments had a diameter of 16 nm. Their morphology was dominated by the prominent undulations of an external three-start helix running at a 10-nm axial distance and at an angle of 32 degrees. Faint nearly axial striations indicated the presence of a tubular core of a different helical order. The complex filaments consisted of 40,000-dalton flagellin monomers. Typically, the amino acid composition was 3 to 4% higher in nonpolar residues and 5 to 7% lower in aspartic and glutamic acids (and their amides) than that of plain flagellar proteins. There were no immunochemical relationships among Pseudomonas rhodos, Rhizobium lupini, and R. meliloti complex flagella, suggesting that the latter represent a new class.     

Role of divalent cations in EOG generation

Underwater EOGs in response to 10^–4 M isoamyl acetatewere recorded in vivo in frogs. The olfactory mucosa was superfusedby sodium-free sucrose solutions, and the effect of the additionof millimolar concentrations of sodium, calcium, cobalt, bariumor cadmium was studied. All voltage responses were standardizedaccording to the solution resistivities. Barium and calciumefficiently restored the response; sodium and cobalt were lessefficient. Cobalt did not alter the restoring effect of calcium.Cadmium induced an irreversible blockage irrespective of thepresence of calcium. These results are discussed in terms ofpossible ionic mechanisms mediating olfactory responses.     

Generalized transduction in Rhizobium meliloti.

Generalized transduction of Rhizobium meliloti 1021 was carried out by bacteriophage N3. Genetic markers on the chromosome and the pSym megaplasmid were transduced, along with markers on several IncP plasmids. Cotransduction between transposon Tn5 insertions and integrated recombinant plasmid markers permitted correlation of cotransductional frequencies and known physical distances. Bacteriophage N3 was capable of infecting several commonly used strains of R. meliloti.     

Catabolite-repression-like phenomenon in Rhizobium meliloti.

We report a phenomenon similar to catabolite repression in Rhizobium meliloti. Succinate, which allows the highest observed rate of growth of R. meliloti, caused an immediate reduction of beta-galactosidase activity when added to cells growing in lactose. A Lac- mutant was unaltered in nodulation and nitrogen fixation capacities, but a pleiotropic mutant deficient in several catabolic properties was unable to produce effective nitrogen-fixing nodules.     

Beauvericin and divalent cations: crystal structure of the barium complex.

The molecular structure of the 2:2 complex of the cyclohexadepsipeptide antibiotic beauvericin with barium picrate has been determined by X-ray crystallography. The structure serves to confirm previous observations on the bimolecular behavior of beauvericin and of the ions transported by beauvercin. The intimate involvement of the anions in the coordination of the barium also explains observations that the cation specificity of beauvericin in membrane transport depends on the species of anions present.     

Role of divalent cations in the resumption of meiosis of rat oocytes.

Resumption of meiotic maturation was induced in follicle-enclosed rat-oocytes by treatment with the divalent cationophore A23187 (10(-5)M). However, the same effect was attained by incubation in Ca++-deficient medium, even in the presence of EDTA or EGTA (1mM). The stability of the first polar body was increased under Ca++-deficient conditions. Neither the ionophore nor Ca++-deficient medium interfered with the spontaneous maturation of isolated oocytes of the rat. The experiments with cultured follicles suggest that redistribution of divalent cations may participate in the physiological control of meiosis in mammalian oocytes.     

High-frequency transformation of Rhizobium meliloti.

Transformation of R factor RP4 and its derivative pRK290 from Escherichia coli to Rhizobium meliloti is reported. The efficiency of transformation was in the range of 10(-5) per viable cell. In addition, chromosomal DNA prepared from one R. meliloti strain resistant to streptomycin was transferred to the isoleucine-valine-requiring mutant susceptible to streptomycin.     

Role of divalent cations in the genesis of the frog electro-olfactogram

In frogs, underwater electro-olfactograms (EOGs) in response to 10(-5) M isoamyl acetate were recorded under restricted cationic environments. The olfactory mucosa was superfused by sodium-free sucrose solutions, and the effect of the addition of millimolar concentrations of calcium, cobalt, barium, or cadmium was studied. Only calcium and barium restored the response; sodium and cobalt were less efficient. Cobalt did not alter the restoring effect of calcium. Cadmium resulted in irreversible blocking irrespective of the presence of calcium. These results are discussed in terms of possible ionic mechanisms mediating olfactory responses.     

Cloning of the glutamine synthetase I gene from Rhizobium meliloti.

Glutamine synthetase is a major enzyme in the assimilation of ammonia by members of the genus Rhizobium. Two forms of glutamine synthetase are found in members of the genus Rhizobium, a heat-stable glutamine synthetase I (GSI) and a heat-labile GSII. As a step toward clarifying the role of these enzymes in symbiotic nitrogen fixation, we have cloned the structural gene for GSI from Rhizobium meliloti 104A14. A gene bank of R. meliloti was constructed by using the bacteriophage P4 cosmid pMK318. Cosmids that contain the structural gene for GSI were isolated by selecting for plasmids that permit ET8051, an Escherichia coli glutamine autotroph, to grow with ammonia as the sole nitrogen source. One of the cosmids, pJS36, contains an insert of 11.9 kilobases. ET8051(pJS36) grows slowly on minimal media. When a 3.7-kilobase HindIII fragment derived from this DNA is cloned into the HindIII site of pACYC177 and the plasmids are transformed into ET8051, rapid growth is observed when the insert is in one orientation (pJS44) but not the other (pJS45). Glutamine synthetase activity can be detected in ET8051(pJS44); most of this activity is heat stable. pJS36 hybridizes with the glnA structural gene from Escherichia coli. Insertion of a 2.7-kilobase Tetr determinant into a BglII site located within pJS44 abolishes all glutamine synthetase activity. This interrupted version of a glutamine synthetase gene was substituted for the normal R. meliloti sequence by homologous recombination in R. meliloti. Recombinants lose GSI activity, but retain GSII activity and grow well with ammonia as the sole nitrogen source. These mutants are unaffected in nodulation and nitrogen fixation.     

Role of galactosyltransferase activity in phage sensitivity and nodulation competitiveness of Rhizobium meliloti.

A stock culture of Rhizobium meliloti 102F51 contains colonies of two distinct phenotypes (Handelsman et al., J. Bacteriol. 157:703-707, 1984); one colony type is agglutinated by high dilutions of the alfalfa agglutinin, is sensitive to phage F20, and is resistant to phage 16B, and the other is agglutinated only by low dilutions of the alfalfa agglutinin, is resistant to phage F20, and is sensitive to phage 16B. Cells of the latter phenotype have an inner-membrane-bound galactosyltransferase activity that transfers galactose from UDP-galactose to a water-insoluble anionic polymer. This enzymatic activity is absent in cells of the first phenotype. All of the phage 16B-resistant mutants selected from a sensitive strain were agglutinated by high dilutions of the alfalfa agglutinin, were sensitive to phage F20, and lacked galactosyltransferase activity. The galactose-containing polymer prepared in vitro was immunologically cross-reactive with the cell surface.     

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

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.     

Succinate dehydrogenase mutant of Rhizobium meliloti.

A succinate dehydrogenase mutant strain of Rhizobium meliloti was isolated after nitrosoguanidine mutagenesis. It failed to grow on succinate, glutamate, acetate, pyruvate, or arabinose but grew on glucose, sucrose, fructose, and other carbohydrates. The mutant strain showed delayed nodulation of lucerne plants, and the nodules were white and ineffective. A spontaneous revertant strain of normal growth phenotype induced red and effective nodules.     

alpha-Ketoglutarate dehydrogenase mutant of Rhizobium meliloti.

A mutant of Rhizobium meliloti selected as unable to grow on L-arabinose also failed to grow on acetate or pyruvate. It grew, but slower than the parental strain, on many other carbon sources. Assay showed it to lack alpha-ketoglutarate dehydrogenase (kgd) activity, and revertants of normal growth phenotype contained the activity again. Other enzymes of the tricarboxylic acid cycle and of the glyoxylate cycle were present in both mutant and parent strains. Enzymes of pyruvate metabolism were also assayed. L-Arabinose degradation in R. meliloti was found to differ from the known pathway in R. japonicum, since the former strain lacked 2-keto-o-deoxy-L-arabonate aldolase but contained alpha-ketoglutarate semialdehyde dehydrogenase; thus, it is likely that R. meliloti has the L-arabinose pathway leading to alpha-ketoglutarate rather than the one to glycolaldehyde and pyruvate. This finding accounts for the L-arabinose negativity of the mutant. Resting cells of the mutant were able to metabolize the three substrates which did not allow growth.     

Phosphoglucose isomerase mutant of Rhizobium meliloti.

A mutant strain of complex phenotype was selected in Rhizobium meliloti after nitrosoguanidine mutagenesis. It failed to grow on mannitol, sorbitol, fructose, mannose, ribose, arabitol, or xylose, but grew on glucose, maltose, gluconate, L-arabinose, and many other carbohydrates. Assay showed the enzyme lesion to be in phosphoglucose isomerase (pgi), and revertants, which were of normal growth phenotype, contained the enzyme again. Nonpermissive substrates such as fructose and xylose prevented growth on permissive ones such as L-arabinose, and in such situations there was high accumulation of fructose 6-phosphate. The mutant strain had about 20% as much exopolysaccharide as the parent. Nitrogen fixation by whole plants was low and delayed when the mutant strain was the inoculant.     

Expression of Rhizobium meliloti nod genes in Rhizobium and Agrobacterium backgrounds.

Rhizobium meliloti nod genes are required for the infection of alfalfa. Induction of the nodC gene depends on a chemical signal from alfalfa and on nodD gene expression. By using a nodC-lacZ fusion, we have shown that the induction of the R. meliloti nodC gene and the expression of nodD occur at almost normal levels in other Rhizobium backgrounds and in Agrobacterium tumefaciens, but not in Escherichia coli. Xanthomonas campestris, or Pseudomonas savastanoi. Our results suggest that bacterial genes in addition to nodDABC are required for nod gene response to plant cells. We have found that inducing activity is present in other plant species besides alfalfa. Acetosyringone, the A. tumefaciens vir gene inducer, does not induce nodC.     

Interference between Rhizobium meliloti and Rhizobium trifolii nodulation genes: genetic basis of R. meliloti dominance.

Transfer of an IncP plasmid carrying the Rhizobium meliloti nodFE, nodG, and nodH genes to Rhizobium trifolii enabled R. trifolii to nodulate alfalfa (Medicago sativa), the normal host of R. meliloti. Using transposon Tn5-linked mutations and in vitro-constructed deletions of the R. meliloti nodFE, nodG, and nodH genes, we showed that R. meliloti nodH was required for R. trifolii to elicit both root hair curling and nodule initiation on alfalfa and that nodH, nodFE, and nodG were required for R. trifolii to elicit infection threads in alfalfa root hairs. Interestingly, the transfer of the R. meliloti nodFE, nodG, and nodH genes to R. trifolii prevented R. trifolii from infecting and nodulating its normal host, white clover (Trifolium repens). Experiments with the mutated R. meliloti nodH, nodF, nodE, and nodG genes demonstrated that nodH, nodF, nodE, and possibly nodG have an additive effect in blocking infection and nodulation of clover.