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Rhizobium sp. strain NGR234 produces a flavonoid-inducible rhamnose-rich lipopolysaccharide (LPS) that is important for the nodulation of legumes. Many of the genes encoding the rhamnan part of the molecule lie between 87 degrees and 110 degrees of pNGR234a, the symbiotic plasmid of NGR234. Computational methods suggest that 5 of the 12 open reading frames (ORFs) within this arc are involved in synthesis (and subsequent polymerization) of L-rhamnose. Two others probably play roles in the transport of carbohydrates. To evaluate the function of these ORFs, we mutated a number of them and tested the ability of the mutants to nodulate a variety of legumes. At the same time, changes in the production of surface polysaccharides (particularly the rhamnan O antigen) were examined. Deletion of rmlB to wbgA and mutation in fixF abolished rhamnan synthesis. Mutation of y4gM (a member of the ATP-binding cassette transporter family) did not abolish production of the rhamnose-rich LPS but, unexpectedly, the mutant displayed a symbiotic phenotype very similar to that of strains unable to produce the rhamnan O antigen (NGRDeltarmlB-wbgA and NGROmegafixF). At least two flavonoid-inducible regulatory pathways are involved in synthesis of the rhamnan O antigen. Mutation of either pathway reduces rhamnan production. Coordination of rhamnan synthesis with rhizobial release from infection threads is thus part of the symbiotic interaction.

To understand the genetic mechanism of host specificity in the interaction between rhizobia and their hosts, it is important to identify genes that influence both early and late steps in symbiotic development. This paper focuses on the little-understood genetics of host-specific nitrogen fixation. A deletion mutant of Bradyrhizobium japonicum, strain NAD163, was found to induce effective, nitrogen-fixing nodules on soybean and siratro plants but produced ineffective nodules on cowpea plants. Additional transposon and deletion mutants defined a small region that conferred this phenotype, and this region was sequenced to identify two putative open reading frames (ORFs). Data indicate that only one of these ORFs is detectable in bacteroids. This ORF was termed hsfA, with a predicted protein product of 11 kDa. The transcriptional start site of hsfA was determined and found to coincide with a predicted RpoN-dependent promoter. Microscopic studies of nodules induced by the wild type and hsfA mutants on cowpea and soybean plants indicate that the cowpea mutant nodules are slow to develop. The data indicate that hsfA appears to play a crucial role in bacteroid development on cowpea but does not appear to be essential for nitrogen fixation on the other hosts tested.

Purine auxotrophs of various Rhizobium species are symbiotically defective, usually unable to initiate or complete the infection process. Earlier studies demonstrated that, in the Rhizobium etli-bean symbiosis, infection by purine auxotrophs is partially restored by supplementation of the plant medium with 5-amino-imidazole-4-carboxamide (AICA) riboside, the unphosphorylated form of the purine biosynthetic intermediate AICAR. The addition of purine to the root environment does not have this effect. In this study, purine auxotrophs of Rhizobium fredii HH303 and Rhizobium leguminosarum 128C56 (bv. viciae) were examined. Nutritional and genetic characterization indicated that each mutant was blocked in purine biosynthesis prior to the production of AICAR. R. fredii HH303 and R. leguminosarum 128C56 appeared to be deficient in AICA riboside transport and/or conversion into AICAR, and the auxotrophs derived from them grew very poorly with AICA riboside as a purine source. All of the auxotrophs elicited poorly developed, uninfected nodules on their appropriate hosts. On peas, addition of AICA riboside or purine to the root environment led to enhanced nodulation; however, infection threads were observed only in the presence of AICA riboside. On soybeans, only AICA riboside was effective in enhancing nodulation and promoting infection. Although AICA riboside supplementation of the auxotrophs led to infection thread development on both hosts, the numbers of bacteria recovered from the nodules were still 2 or more orders of magnitude lower than in fully developed nodules populated by wild-type bacteria. The ability to AICA riboside to promote infection by purine auxotrophs, despite serving as a very poor purine source for these strains, supports the hypothesis that AICAR plays a role in infection other than merely promoting bacterial growth.

The tropical legume Sesbania rostrata can be nodulated by Azorhizobium caulinodans on both its stem and its root system. Here we investigate in detail the process of root nodulation and show that nodules develop exclusively at the base of secondary roots. Intercellular infection leads to the formation of infection pockets, which then give rise to infection threads. Concomitantly with infection, cortical cells of the secondary roots dedifferentiate, forming a meristem which has an "open-basket" configuration and which surrounds the initial infection site. Bacteria are released from the tips of infection threads into plant cells via "infection droplets," each containing several bacteria. Initially, nodule differentiation is comparable to that of indeterminate nodules, with the youngest meristematic cells being located at the periphery and the nitrogen-fixing cells being located at the nodule center. Because of the peculiar form of the meristem, Sesbania root nodules develop uniformly around a central axis. Nitrogen fixation is detected as early as 3 days following inoculation, while the nodule meristem is still active. Two weeks after inoculation, meristematic activity ceases, and nodules then show the typical histology of determinate nodules. Thus, root nodule organogenesis in S. rostrata appears to be intermediate between indeterminate and determinate types.

The interaction between Rhizobium lipopolysaccharide (LPS) and white clover roots was examined. The Limulus lysate assay indicated that Rhizobium leguminosarum bv. trifolii (hereafter called R. trifolii) released LPS into the external root environment of slide cultures. Immunofluorescence and immunoelectron microscopy showed that purified LPS from R. trifolii 0403 bound rapidly to root hair tips and infiltrated across the root hair wall. Infection thread formation in root hairs was promoted by preinoculation treatment of roots with R. trifolii LPS at a low dose (up to 5 micrograms per plant) but inhibited at a higher dose. This biological activity of LPS was restricted to the region of the root present at the time of exposure to LPS, higher with LPS from cells in the early stationary phase than in the mid-exponential phase, incubation time dependent, incapable of reversing inhibition of infection by NO3- or NH4+, and conserved among serologically distinct LPSs from several wild-type R. trifolii strains (0403, 2S-2, and ANU843). In contrast, infections were not increased by preinoculation treatment of roots with LPSs from R. leguminosarum bv. viciae strain 300, R. meliloti 102F28, or members of the family Enterobacteriaceae. Most infection threads developed successfully in root hairs pretreated with R. trifolii LPS, whereas many infections aborted near their origins and accumulated brown deposits if pretreated with LPS from R. meliloti 102F28. LPS from R. leguminosarum 300 also caused most infection threads to abort. Other specific responses of root hairs to infection-stimulating LPS from R. trifolii included acceleration of cytoplasmic streaming and production of novel proteins. Combined gas chromatography-mass spectroscopy and proton nuclear magnetic resonance analyses indicated that biologically active LPS from R. trifolii 0403 in the early stationary phase had less fucose but more 2-O-methylfucose, quinovosamine, 3,6-dideoxy-3-(methylamino)galactose, and noncarbohydrate substituents (O-methyl, N-methyl, and acetyl groups) on glycosyl components than did inactive LPS in the mid-exponential phase. We conclude that LPS-root hair interactions trigger metabolic events that have a significant impact on successful development of infection threads in this Rhizobium-legume symbiosis.

Two Rhizobium phaseoli mutants affected in cytochrome expression were obtained by Tn5-mob mutagenesis of the wild-type strain (CE3). Mutant strain CFN031 expressed sevenfold less cytochrome o in culture, expressed cytochrome aa3 under microaerophilic culture conditions, in contrast to strain CE3, and was affected in its vegetative growth properties and proliferation inside plant host cells. Mutant CFN037 expressed cytochrome aa3 under microaerophilic culture conditions, while bacteroid development and nitrogen fixation occurred earlier than in strain CE3.

Iron acquisition by symbiotic Rhizobium spp. is essential for nitrogen fixation in the legume root nodule symbiosis. Rhizobium leguminosarum 116, an ineffective mutant strain with a defect in iron acquisition, was isolated after nitrosoguanidine mutagenesis of the effective strain 1062. The pop-1 mutation in strain 116 imparted to it a complex phenotype, characteristic of iron deficiency: the accumulation of porphyrins (precursors of hemes) so that colonies emitted a characteristic pinkish-red fluorescence when excited by UV light, reduced levels of cytochromes b and c, and wild-type growth on high-iron media but low or no growth in low-iron broth and on solid media supplemented with the iron scavenger dipyridyl. Several iron(III)-solubilizing agents, such as citrate, hydroxyquinoline, and dihydroxybenzoate, stimulated growth of 116 on low-iron solid medium; anthranilic acid, the R. leguminosarum siderophore, inhibited low-iron growth of 116. The initial rate of 55Fe uptake by suspensions of iron-starved 116 cells was 10-fold less than that of iron-starved wild-type cells. Electron microscopic observations revealed no morphological abnormalities in the small, white nodules induced by 116. Nodule cortical cells were filled with vesicles containing apparently normal bacteroids. No premature degeneration of bacteroids or of plant cell organelles was evident. We mapped pop-1 by R plasmid-mediated conjugation and recombination to the ade-27-rib-2 region of the R. leguminosarum chromosome. No segregation of pop-1 and the symbiotic defect was observed among the recombinants from these crosses. Cosmid pKN1, a pLAFR1 derivative containing a 24-kilobase-pair fragment of R. leguminosarum DNA, conferred on 116 the ability to grow on dipyridyl medium and to fix nitrogen symbiotically. These results indicate that the insert cloned in pKN1 encodes an element of the iron acquisition system of R. leguminosarum that is essential for symbiotic nitrogen fixation.

Eleven fast-growing strains of Rhizobium japonicum were characterized with respect to indigenous plasmids and abilities to infect (Inf+) and nodulate (Nod+) cowpea, siratro, wild soybean, and three commercial cultivars of soybean. All strains caused infection via infection threads in root hairs and consistently nodulated cowpea, siratro, and wild soybean in growth pouches. Interactions with commercial cultivars of soybean were strikingly strain specific. Some combinations were Nod-, and infection was delayed in others. The ratios of infections to nodules and the distribution of nodules on primary and lateral roots also varied substantially. A modified in-gel lysis procedure was devised for electrophoretic separation of plasmids from the strains. Plasmids (ranging in size from 35 to greater than 300 megadaltons) were reproducibly detected in all strains.

Two Rhizobium phaseoli mutants, isolated previously by Tn5 mutagenesis, elicited infection threads which ceased development prematurely, usually within root hairs. These infection threads were wide, globular, and otherwise altered in morphology, compared with normal infection threads. Anatomy and division of the root cortical cells during initial stages of nodule morphogenesis appeared normal. However, later nodule differentiation deviated considerably from normal development, and release of bacteria from infection threads was not observed. In tryptone-yeast extract medium the mutants sedimented during growth in shaken cultures and formed rough colonies on agar. Electrophoresis of washed cultures solubilized in dodecyl sulfate revealed that the major carbohydrate band was absent from the mutants. The behavior of this carbohydrate in phenol-water extraction and gel chromatography, its apparent ketodeoxyoctonate content, and its susceptibility to mild acid hydrolysis suggested that it was a lipopolysaccharide. From the results of genetic crosses or reversion analysis, the defect in synthesizing this carbohydrate material and the defect in infection could be attributed to a single mutation in each mutant.

Mutant strain ANU2861, a transposon Tn5 mutant of the fast-growing, broad-host-range Rhizobium strain ANU280 (NGR234 Smr Rfr) overproduces polysaccharide, is an ade auxotroph, and induces poorly developed nodules on Leucaena leucocephala and Lablab purpureus (H.C. Chen, M. Batley, J.W. Redmond, and B.G. Rolfe, J. Plant Physiol. 120:331-349, 1985). Strain ANU2861 cannot form nodules on Macroptilium atropurpureum Urb. (siratro) or on Desmodium intortum and D. uncinatum and the nonlegume Parasponia. The parent strain, ANU280, effectively nodulates all these legume species except Parasponia, on which it forms ineffective nodules. Ultrastructural examination of infection sites on the legume siratro showed that mutant strain ANU2861 caused root hair curling (Hac+ phenotype), some cortical cell division (Noi+), but no infection threads (Inf-). Localized cellular responses, known to occur in phytopathological interactions, were observed in electron micrographs of the epidermal tissue at or near the infection zone after inoculation with strain ANU2861 but not the wild-type parental strain. These include (i) the rapid (within 20 h) accumulation of osmiophilic droplets attached to membranes at potential sites of strain ANU2861 penetration and (after 48 h) in the epidermal cells in the immediate region of the curled root hairs, and (ii) localized cell death of the epidermal cells. In addition, strain ANU2861 can initiate a systemic response in split-root siratro plants which prevents the successful nodulation of strain ANU280. A 6.3-kilobase fragment of wild-type genomic DNA, which includes the site of Tn5 insertion in strain ANU2861, was cloned and introduced to strain ANU2861. All the phenotypic defects of the mutant strain were corrected by the introduction of this DNA fragment. This indicates that the original Tn5 insertion is responsible for the phenotype.