An osmoregulated dipeptide in stressed Rhizobium meliloti.

One common mechanism of cellular adaptation to osmotic stress is the accumulation of organic solutes in the cytosol. We have used natural-abundance 13C nuclear magnetic resonance to identify all organic solutes that accumulate to significant levels in Rhizobium meliloti. Our studies led to the discovery of a new dipeptide, N-acetylglutaminylglutamine amide (NAGGN), which is accumulated during osmotic stress. Only rarely have peptides been shown to function in bacteria, and furthermore, this is the first example of a peptide playing a role in osmoregulation. Evidence for the biological role of NAGGN in osmotic-stress protection is presented.     

Glutamate catabolism in Rhizobium meliloti

The pathway by which glutamate is degraded as a carbon source has not previously been elucidated, but enzymatic analysis of Rhizobium meliloti CMF1 indicated that both glutamate dehydrogenase (GDH) and gamma-aminobutyrate (GABA) bypass activities were present in free living cells. However, when similar studies were performed on R. meliloti CMF1 bacteroids, isolated from alfalfa nodules, only GABA bypass activities were detectable. Both GDH and GABA bypass activities were influenced by the carbon source provided, with maximum activities being detected when glutamate was present as sole carbon and nitrogen source. Addition of a second carbon source, such as succinate, to the growth medium did not influence GDH activity but substantially decreased levels of the first enzyme of the GABA bypass, glutamate decarboxylase (GDC). Cyclic adenosine 3′5′-monophosphate (cAMP) failed to increase GDC activities in R. meliloti CMF1 cells grown in the presence of an additional carbon source. It is proposed that the GABA bypass is a major mechanism of glutamate carbon degradation in R. meliloti CMF1, a system whose enzymatic activities are influenced by the nature of the carbon source present in the growth environment.     

General transduction in Rhizobium meliloti

General transduction by phage phi M12 in Rhizobium meliloti SU47 and its derivatives is described. Cotransduction and selection for Tn5 insertions which are closely linked to specific loci were demonstrated. A derivative of SU47 carrying the recA::Tn5 allele of R. meliloti 102F34 could be transduced for plasmid R68.45 but not for chromosomally located alleles. Phage phi M12 is morphologically similar to Escherichia coli phage T4, and restriction endonuclease analysis indicated that the phage DNA was ca. 160 kilobases in size.     

Ammonium assimilation in Rhizobium meliloti

We have characterized a mutant of Rhizobium meliloti strain 2011 which cannot use ammonium as a nitrogen source. This mutant, RTm2620, was found to have significantly altered glutamate synthase activity. Both the mutant and the wild-type strains had glutamate dehydrogenase activity, which, although stimulated in the presence of glutamate and ammonium, was apparently insufficient to allow ammonium assimmilation. We conclude that the glutamine synthetase-glutamate synthase pathway may be the normal mode of ammonium assimilation by this strain in the free-living state. Independent revertants of Rm2620 were isolated and fell into two classes. Class I revertants regained partial glutamate synthase activity and had the same levels of glutamate dehydrogenase activity as Rm2620. Class II revertants retained the altered glutamate synthase activity but acquired a very high level of assimilatory glutamate dehydrogenase activity. Both classes were found to be altered in their symbiotic properties, although the original Rm2620 mutant was normal in this regard.     

Chemotaxis by Rhizobium meliloti

Summary Chemotaxis by Rhizobium meliloti strain Ve 26 has been studied and conditions required for chemotaxis have been defined, using the Adler capillary assay technique. Several sugars and amino-acids were shown to be attractants with varying effectiveness for this organism: sugars are weak attractants (except gluconate) and amino-acids are good attractants (except unpolar amino-acids).     

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.     

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.     

Ornithine cyclodeaminase activity in Rhizobium meliloti

Abstract Deamination of L-ornithine to L-proline by ornithine cyclodeaminase is an unusual enzyme reaction that has been shown to occur in only a few bacteria. Rhizobium meliloti strains GR4, 2011 and 41 are able to use ornithine as the sole carbon and nitrogen source. The main pathway of ornithine utilization in strain GR4 depends on ornithine cyclodeaminase activity. In addition, this enzymatic activity has been found to be dependent on NAD⁺ and L-arginine similar to Agrobacterium ornithine cyclodeaminases. The ornithine cyclodeaminase activity is also expressed in R. meliloti strains 2011 and 41 growing with L-ornithine.     

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.     

Rhizobium meliloti carries two megaplasmids

In Rhizobium meliloti strain 41 the existence of a second megaplasmid (pRme41c) with a molecular weight similar to the sym megaplasmid pRme41b was demonstrated. Derivatives of the wild-type strain carrying pRme41b or pRme41c tagged with Tn5 allowed the examination of the transfer ability of both megaplasmids. The introduction of megaplasmids into the wild-type R. meliloti was not detected, probably because of the action of plasmid genes coding for entry exclusion of the same type of plasmid. However, transmissibility of both megaplasmids was observed in matings with Nod- or Fix- pRme41b deletion mutant recipients and with Agrobacterium tumefaciens at frequencies of 10(-6) - 10(-8). Introduction of the megaplasmids into the R. meliloti recipients resulted in the loss of the same plasmid. On the other hand, pRme41b and pRme41c were compatible. From the extent of deletions in various Nod- and Fix- mutants a DNA region carrying genes probably involved in "surface exclusion" on pRme41b was located. This DNA region is about 50 kb distant from the nod genes and exhibits strong homology with a DNA segment of pRme41c. Symbiotic genes on pRme41c were not identified.     

Regulation of urease activity in Rhizobium meliloti

Abstract Using an ureC-lacZ fusion, the expression of urease structural genes of the soil bacterium Rhizobium meliloti strain AK631 was studied in response to different nitrogen sources and nickel contents in the growth medium. The expression of urease genes is repressed by ammonia and is not inducible by urea. Urease activity depends on the nickel concentration of the medium. Nickel uptake is repressed in medium containing ammonia and is not affected by the genes located in the urease operon investigated.     

Localization of symbiotic mutations in Rhizobium meliloti

A total of 5 Nod- and 57 Fix- symbiotic mutants of Rhizobium meliloti strain 41 have been isolated after either nitrosoguanidine or Tn5 transposition mutagenesis. Chromosomal locations of mutations in 1 Nod- and 11 Fix- derivatives were ascertained by transferring the chromosome (mobilized by plasmid R68.45), in eight fragments, into symbiotically effective recipients and testing the recombinants for symbiotic phenotype. Alternatively, the kanamycin resistance marker of Tn5 was mapped. In five mutants the fix alleles were localized on different chromosomal regions, but six other fix mutations and one nod mutation tested did not map onto the chromosome. It was shown that the chromosome-mobilizing ability (Cma+) of R68.45 was not involved in the mobilization of genes located extrachromosomally. Moreover, Cma- derivatives of R68.45 could mobilize regions of the indigenous plasmid pRme41b but not chromosomal genes. Thus, mobilization of a marker by Cma- R68.45 indicates its extrachromosomal location. With a 32P-labeled DNA fragment carrying Tn5 as a hybridization probe, it was shown that in five extrachromosomally located Tn5-induced fix mutants and one nod mutant Tn5 was localized on plasmid pRme41b. This is in agreement with the genetic mapping data.     

Genetic analyses of Rhizobium meliloti exopolysaccharides

We have recently obtained strong genetic evidence that the acidic Calcofluor-binding exopolysaccharide (EPS I) of Rhizobium meliloti Rm1021 is required for nodule invasion and possibly for later events in nodule development. Thirteen loci on the second megaplasmid have been identified that are required for, or affect, the synthesis of EPS I. Mutations in certain of these loci completely abolish the production of EPS I and result in mutants that form empty Fix- nodules. exoH mutants fail to succinylate their EPS I and form empty Fix- nodules. We have identified two unlinked regulatory loci, exoR and exoS, whose products play negative roles in the regulation of expression of the exo genes. We have recently discovered that R. meliloti has a latent capacity to synthesize a second exopolysaccharide (EPS II) that can substitute for the role(s) of EPS I in nodulation of alfalfa but not of other hosts. Possible roles for Rhizobium exopolysaccharides in nodulation are discussed.