We have physically and genetically characterized 20 symbiotic and 20 auxotrophic mutants of Rhizobium meliloti, the nitrogen-fixing symbiont of alfalfa (Medicago sativa), isolated by transposon Tn5 mutagenesis. A "suicide plasmid" mutagenesis procedure was used to generate TN-5-induced mutants, and both auxotrophic and symbiotic mutants were found at a frequency of 0.3% among strains containing random TN5 insertions. Two classes of symbiotic mutants were isolated: 4 of the 20 formed no nodules at all (Nod-), and 16 formed nodules which failed to fix nitrogen (Fix-). We used a combination of physical and genetic criteria to determine that in most cases the auxotrophic and symbiotic phenotypes could be correlated with the insertion of a single Tn5 elements. Once the Tn5 element was inserted into the R. meliloti genome, the frequency of its transposition to a new site was approximately 10-8 and the frequency of precise excision was less than 10-9. In approximately 25% of the mutant strains, phage Mu DNA sequences, which originated from the suicide plasmid used to generate the Tn5 transpositions, were also found in the R. meliloti genome contiguous with Tn5. These later strains exhibited anomalous conjugation properties, and therefore we could not correlate the symbiotic phenotype with a Tn5 insertion. In general, we found that both physical and genetic tests were required to fully characterize transposon-induced mutations.

The fixA, fixB, fixC, and flxX genes of Rhizobium meliloti 1021 constitute an operon and are required for nitrogen fixation in alfalfa nodules. DNA homologous to the R. meliloti fixABC genes is present in all other Rhizobium and Bradyrhizobium species examined, butJlxABC-homologous sequences were found in only one free-living diazotroph, Azotobacter vinelandii. To determine whether the fixABCX genes share sequence homology with any of the 17 KlebsieUla pneumoniae nif genes, we determined the entire nucleotide sequence of the fixA, fixB, fixC, and fixX genes and defined four open reading frames that code for polypeptides of molecular weights 31,146, 37,786, 47,288, and 10,937, respectively. Neither DNA nor amino acid sequence homology to the R. melilotifixA, -B, -C, and -X genes was found in the K. pneumoniae nifoperon. The fixX gene contains a cluster of cysteine residues characteristic of ferredoxins and is highly homologous to an Azotobacter ferredoxin which has been shown to donate electrons to nitrogenase. ThefixABC operon contains a promoter region that is highly homologous to other nifA-activated promoters. We also found a duplication of the 5' end of the fixABCX operon; a 250-bp region located 520 bp upstream of theJixABCX promoter bears more than 65% homology to the 5' end of the transcribed region, including the first 32 codons of fixA.Several genes have been identified as essential for symbiotic nitrogen fixation by the bacterium Rhizobium meliloti.These genes are characterized as nif and fix genes. By definition, the nif genes of R. meliloti are those which bear structural or functional homology to the well-characterized nifgenes of the free-living, nitrogen-fixing species Klebsiella pneumoniae. Thefix genes, on the other hand, are essential for nitrogen fixation by virtue of the Fix-phenotype of nodules elicited by strains which contain mutations in these genes (1, 4, 15, 29, 31) but have not yet been assigned a biochemical function.In R. meliloti three fix genes, fixA, fixB, and fixC, were previously identified which are closely linked to a cluster of nif genes located on a large endogenous plasmid, the socalled Sym plasmid (29,31) (Fig. 1). The fixABC genes are located in a single operon (6, 31) and are transcriptionally activated coordinately with the nitrogenase structural genes nifH, -D, and -K by the nifA gene product (38).In the work reported here, we examined the fine structure of the fixABC genes and attempted to determine their evo-

A HindIII (17.0 kb) and an EcoR1 restriction fragment (6.9 kb) of Klebsiella pneumoniae nif DNA were cloned on two small amplifiable plasmids, pCM1 and pSA30 respectively. These plasmids between them carry 14 of the 15 known Klebsiella nif genes. The operon for the three structural genes for nitrogenase, nifpHDK, is carried on pSA30: four and five of the remaining six operons are on pCRA37 and pCM1 respectively. All of the nif genes were assigned to endonuclease restriction fragments of DNA using the Southern blotting technique (Southern, 1975) with total DNA of nif insertion mutants and radioactive plasmid DNA which contained cloned nif DNA sequences. Their locations were consistent with the genetic map of nif genes. The estimated size of the nif gene cluster was 24 kb.

We have cloned and characterized three distinct Rhizobium meliloti loci involved in glutamine biosynthesis (ginA, glnlI, and glnT

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