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By screening with the fluorescent stain Calcofluor, we have isolated 26 independent transposon Tn5 insertion mutants of Rhizobium meliloti that are deficient in the production of a known extracellular polysaccharide (Exo-). The mutants belonged to six distinct genetic groups based on the ability of their Exo- phenotype to be complemented by different recombinant plasmids from a R. meliloti clone bank. With few exceptions, all of the mutants formed ineffective (non-nitrogen-fixing) nodules on alfalfa. For all but one group, the complementing plasmids restored effective nodulation. These results establish a firm and extensive correlation between the ability of Rhizobium to produce a particular polysaccharide and symbiotic proficiency. The ineffective nodules appeared to contain no bacteroids and to form without shepherds' crooks or infection threads; this symbiotic phenotype matches that described for a set of independently isolated mutants that belong phenotypically and genetically to the group B exopolysaccharide mutants described previously [Finan et al. (1985) Cell 40, 869-877]. Apparently the exopolysaccharide, although not required for nodule formation, is involved in wild-type nodule invasion.

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.

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.

Monoclonal antibodies were produced to the surface of the symbiotic nitrogen-fixing bacterium Rhizobium meliloti. Bacterial lysis in the presence of complement or cycles of agglutination and growth were used to select mutants no longer recognized by the antibodies. The mutants were used to produce new antibodies with different specificities. Several mutants had altered sensitivity to one or more bacteriophages. R. meliloti strains from different sources had distinct patterns of sensitivity to monoclonal antibodies and phages, which together can be used for discriminative typing.

Two recent reports described the isolation of derivatives of a Rhizobium trifoliistrain that had gained the ability to nodulate Glycine maxand Vigna radiataand 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. japonicumand are not derived from the putative parent R. trifolii.

Mutants of Rhizobium meliloti selected primarily for bacteriophage resistance fall into 13 groups. Mutants in the four best-characterized groups (class A, lpsB, lpsC, and class D), which map to the rhizobial chromosome, appear to affect lipopolysaccharide (LPS) as judged by the reactivity with monoclonal antibodies and behavior on sodium dodecyl sulfate-polyacrylamide gels of extracted LPS. Mutations in all 13 groups, in an otherwise wild-type genetic background, are Fix+ on alfalfa. This suggests that LPS does not play a major role in symbiosis. Mutations in lpsB, however, are Fix- in one particular genetic background, evidently because of the cumulative effect of several independent background mutations. In addition, an auxotrophic mutation evidently equivalent to Escherichia coli carAB is Fix- on alfalfa.

Nodulation of alfalfa by exoB mutants of Rhizobium meliloti occurred without root hair curling or infection thread formation. nod exoB double mutants had the same nodulation deficiency as single nod mutants. Therefore, all the known nod genes are involved in nodule induction by exoB mutants, which apparently occurs via intercellular invasion.

Transposon Tn5 encodes streptomycin resistance in addition to kanamycin-neomycin resistance. This resistance was not detectable in Escherichia coli but was efficiently expressed in Rhizobium meliloti and certain other strains. By analysis of cloned Tn5 restriction endonuclease fragments, the streptomycin resistance (str) gene was located in the right-hand side of the central region as the transposon is conventionally drawn. Transcription of str appeared to originate at pL, the promoter for the neo gene (neomycin phosphotransferase type II). Expression of streptomycin resistance in E. coli was obtained after cloning of the neo-str region downstream of a strong E. coli promoter. A construct in which PL was deleted also showed differential expression of streptomycin resistance.

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.

The occurrence in Azospirillum brasilense of genes that code for exopolysaccharide (EPS) synthesis was investigated through complementation studies of Rhizobium meliloti Exo- mutants. These mutants are deficient in the synthesis of the major acidic EPS of Rhizobium species and form empty, non-nitrogen-fixing root nodules on alfalfa (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). We demonstrated that the exoC mutation of R. meliloti could be corrected for EPS production by several cosmid clones of a clone bank of A. brasilense ATCC 29145. However, the EPS produced differed in structure from the wild-type R. meliloti EPS, and the symbiotic deficiency of the exoC mutation was not reversed by any of these cosmid clones. The exoB mutation could be corrected not only for EPS production but also for the ability to form nitrogen-fixing nodules on alfalfa by one particular cosmid clone of A. brasilense. Tn5 insertions in the cloned DNA were isolated and used to construct Azospirillum mutants with mutations in the corresponding loci by marker exchange. It was found that these mutants failed to produce the wild-type high-molecular-weight EPS, but instead produced EPSs of lower molecular weight.

Using physical and genetic data, we have demonstrated that Rhizobium meliloti SU47 has a symbiotic megaplasmid, pRmeSU47b, in addition to the previously described nod-nif megaplasmid pRmeSU47a. This plasmid includes four loci involved in exopolysaccharide (exo) synthesis as well as two loci involved in thiamine biosynthesis. Mutations at the exo loci have previously been shown to result in the formation of nodules which lack infection threads (Inf-) and fail to fix nitrogen (Fix-). Thus, both megaplasmids contain genes involved in the formation of nitrogen-fixing root nodules. Mutations at two other exo loci were not located on either megaplasmid. To mobilize the megaplasmids, the oriT of plasmid RK2 was inserted into them. On alfalfa, Agrobacterium tumefaciens strains containing pRmeSU47a induced marked root hair curling with no infection threads and Fix- nodules, as reported by others. This plant phenotype was not observed to change with A. tumefaciens strains containing both pRmeSU47a and pRmeSU47b megaplasmids, and strains containing pRmeSU47b alone failed to curl root hairs or form nodules.

A high proportion of Rhizobium phaseoli mutants that survived infection with phage F1 were found to be nodulation deficient. Two that were examined in detail had internal defects in addition to the expected surface defects. One internal defect was in the enzyme phosphoglucose isomerase. The use of phages to select appropriate mutants should apply generally in any system in which surface components are involved.