Bacteriocinogeny of Rhizobium trifolii.

Rhizobium trifolii strains differing in cell and colony morphology, streptomycin resistance, phage sensitivity pattern and infectivity to clover plants produced bacteriocins sensitive to proteases. Elimination of bacteriocin production ability wtih SDS and rifampicin treatment indicates that this feature is plasmid controlled. Elimination of bacteriocinogenic plasmid did not influence other features of R. trifolii.     

Polyol metabolism by Rhizobium trifolii.

In Rhizobium trifolii 7000, the polyols myo-inositol, xylitol, ribitol, D-arabitol, D-mannitol, D-sorbital, and dulcitol are metabolized by inducible nicotinamide adenine dinucleotide-dependent polyol dehydrogenases. Five different polyol dehydrogenases were recognized: inositol dehydrogenase, specific for inositil; ribitol dehydrogenase, specific for ribitol; D-arabitol dehydrogenase, which oxidized D-arabitol, D-mannitol, and D-sorbitol; xylitol dehydrogenase, which oxidized xylitol and D-sorbitol; and dulcitol dehydrogenase, which oxidized dulcitol, ribitol, xylitol, and sorbitol. Apart from inositil and xylitol, all of the polyols induced more than one polyol dehydrogenase and polyol transport system, but the heterologous polyol dehydrogenases and polyol transport systems were not coordinately induced by a particular polyol. With the exception of xylitol, all of the polyols tested served as growth substrates. A mutant of trifolii 7000, which was constitutive for dulcitol dehydrogenase, could also grow on xylitol.     

Transformation of an R-factor from Pseudomonas aeruginosa into Rhizobium trifolii

Summary The transfer by bacterial transformation of an R-factor giving resistance to kanamycin, ampicillin and chloramphenicol from Pseudomonas aeruginosa into Rhizobium trifolii, strain T1 is described.     

Genetic transformation in Rhizobium trifolii.

Markers controlling the synthesis of amino acids and organic bases as well as streptomycin resistance and sensitivity to acriflavine were transformed in Rhizobium trifolii. The results indicate that the str marker was transformed independently of leu, his, ade and trp markers. Co-transformation of leu and utra markers ranged from 3 to 7%, whereas that of thi and acr was 10%.     

Plasmid deoxyribonucleic acid in Rhizobium trifolii.

In deoxyribonucleic acid of Rhizobium trifolii centrifuged in cesium chloride-ethidium bromide equilibrium was found a sattelite peak containing covalently closed circular deoxyribonucleic acid. The plasmid had a molecular weight of about 64 x 10(6) shown by sedimentation in sucrose gradients and electron microscopy.     

Conserved Nodulation Genes in Rhizobium meliloti and Rhizobium trifolii

Plasmids which contained wild-type or mutated Rhizobium meliloti nodulation (nod) genes were introduced into Nod⁻R. trifolii mutants ANU453 and ANU851 and tested for their ability to nodulate clover. Cloned wild-type and mutated R. meliloti nod gene segments restored ANU851 to Nod⁺, with the exception of nodD mutants. Similarly, wild-type and mutant R. meliloti nod genes complemented ANU453 to Nod⁺, except for nodCII mutants. Thus, ANU851 identifies the equivalent of the R. meliloti nodD genes, and ANU453 specifies the equivalent of the R. meliloti nodCII genes. In addition, cloned wild-type R. trifolii nod genes were introduced into seven R. meliloti Nod⁻ mutants. All seven mutants were restored to Nod⁺ on alfalfa. Our results indicate that these genes represent common nodulation functions and argue for an allelic relationship between nod genes in R. meliloti and R. trifolii.     

Genetic mapping of the chromosome of Rhizobium trifolii.

Cultures of the wild strain and auxotrophic mutants of Rhizobium trifolii T37 synchronized by means of phenylethanol have been mutagenized with nitrosoguanidine. Fifteen genetic markers were characterized in respect of their order and the time of replication based on the peaks of mutations of the genes. The time of R. trifolii chromosome replication was estimated using inhibitors of the initiation of DNA replication: rifampicin, chloramphenicol and phenylethanol. The replicative map of R. trifolii chromosome has been constructed. Taking into account the replicative map, linkages of the genes, and the bidirectional model of the Rhizobium chromosome replication, a circular genetic map of the chromosome of R. trifolii T37 was elaborated.     

Properties of some bacteriocins produced by Rhizobium trifolii.

Bacteriocins produced by six strains of Rhizobium trifolii were found to be of the relatively low molecular weight, non-phage type. The molecular weights ranged from approximately 1-8 X 105 to 2-0 X 105. All were of protein composition, as indicated by buoyant density (1-32 to 1-34 g/cm3) in CsC1 and by sensitivity to proteolytic enzymes. They were resistant to RNAase but sensitive to DNAase. The six bacteriocins could further be separated into two subgroups on the basis of sensitivity to extremes of pH, binding to filter membranes, activity spectrum on sensitive strains of R. trifolii, and possibly mode of action on sensitive bacteria. Bacteriocin production occurred spontaneously during the early-to mid-exponential phase of bacterial growth in broth culture.     

Plasmids and stability of symbiotic properties of Rhizobium trifolii.

A conjugal plasmid which encodes both peak nodulation genes and nitrogenase genes, and which is labeled with the transposon Tn5, was transferred to a wild-type Rhizobium trifolii strain to examine the stability and expression of the host range and fixation (Fix+) phenotypes. Transconjugates were isolated which were shown to initially form nitrogen-fixing nodules (Nod+ Fix+) on both clovers and peas. These hybrid strains were then repeatedly passaged through either pea or clover nodules or onto a solid agar medium to determine whether these broadened-host-range characteristics were stably maintained. An instability was noted in the capacity of some of these hybrids to form nitrogen-fixing nodules on all of the host plants used. The broadened nodulation ability was, however, more readily maintained. In some cases, the changes in the Nod+ Fix+ phenotype could be attributed to demonstrable changes in the plasmid profile of the hybrid strains, whereas in other cases no demonstrable plasmid alterations could be detected.     

Regulation of phenolic catabolism in Rhizobium leguminosarum biovar trifolii.

In members of the family Rhizobiaceae, many phenolic compounds are degraded by the protocatechuate branch of the beta-ketoadipate pathway. In this paper we describe a novel pattern of induction of protocatechuate (pca) genes in Rhizobium leguminosarum biovar trifolii. Isolation of pca mutant strains revealed that 4-hydroxybenzoate, quinate, and 4-coumarate are degraded via the protocatechuate pathway. At least three inducers govern catabolism of 4-hydroxybenzoate to succinyl coenzyme A and acetyl coenzyme A. The enzyme that catalyzes the initial step is induced by its substrate, whereas the catabolite beta-carboxy-cis,cis-muconate induces enzymes for the upper protocatechuate pathway, and beta-ketoadipate elicits expression of the enzyme for a subsequent step, beta-ketoadipate succinyl-coenzyme A transferase. Elucidation of the induction pattern relied in part on complementation of mutant Rhizobium strains by known subclones of Acinetobacter genes expressed off the lac promoter in a broad-host-range vector.