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A Tn5-induced mutant strain (CTNUX5) of Rhizobium etli unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into an argC-homologous gene. Unlike its wild-type parent (strain CE3), the mutant strain CTNUX5 had an absolute dependency on arginine to grow. The argC gene was cloned from the wild-type strain CE3, and the resulting plasmid, pAR207, after transformation was shown to relieve the arginine auxotrophy of strain CTNUX5. Unlike strain CE3 or CTNUX5-pAR207, strain CTNUX5 showed undetectable levels of N-acetyl-gamma-glutamylphosphate reductase activity. Unless arginine was added to the growth medium, strain CTNUX5 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (nodulation factors) and to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris.

By means of a detailed ultrastructural analysis of nodules induced by Rhizobium etli on the roots of Phaseolus vulgaris, we observe that the development of host-invaded cells is not synchronous. An accumulation of mitochondria was found in freshly invaded host cells, containing only a few symbiosomes (SBs) that are released from highly branched intracellular ramification of the infection threads. Moreover, besides the fusion between the SB membrane with host secretory vesicles, we observe also a great number of fusions between the outer leaflets of adjoining SB membranes, thus resulting in structures that resemble the tight junction network (zona occludens with a five-layered structure) of epithelian cells. This process was found to be induced strongly and earlier both in the invaded host cells of ineffective nodules (elicited by Fix- mutant strains of R. etli) and in the older (senescence) invaded cells of effective nodules, whereas bacteroid division is seldom if ever observed. Our observations strongly suggest that multiple-occupancy SBs also arise by fusion of single-occupancy SBs and the physiological consequence of this process is discussed.

A mutant strain (CTNUX4) of Rhizobium etli carrying Tn5 unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a trpB (tryptophan synthase)-homologous gene. When tested on the roots of Phaseolus vulgaris, strain CTNUX4 was able to induce only small, slightly pink, ineffective (Fix-) nodules. However, under free-living conditions, strain CTNUX4 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) unless tryptophan was added to the growth medium. These data and histological observations indicate that the lack of tryptophan biosynthesis affects the symbiotic behavior of R. etli.

A mutant strain (CTNUX23) of Rhizobium etli carrying Tn5 unable to grow with sulfate as the sole sulfur source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a metZ (O-succinylhomoserine sulfhydrylase)-homologous gene. The CTNUX23 mutant strain had a growth dependency for methionine, although cystathionine or homocysteine, but not homoserine or O-succinylhomoserine, allowed growth of the mutant. RNase protection assays showed that the metZ-like gene had a basal level of expression in methionine- or cysteine-grown cells, which was induced when sulfate or thiosulfate was used. The metZ gene was cloned from the parent wild-type strain, CE3, and the resulting plasmid pAR204 relieved, after transformation, the methionine auxotrophy of both strains CTNUX23 of R. etli and PAO503(metZ) of Pseudomonas aeruginosa. Unlike strain CE3 or CTNUX23 (pAR204), strain CTNUX23 showed undetectable levels of O-succinylhomoserine sulfhydrylase activity. Strain CTNUX23 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) or to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris, unless methionine was added to the growth medium. These data and our previous results support the notion that cysteine or glutathione, but not methionine, is supplied by the root cells to bacteria growing inside the plant.