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nolX, a nod box-independent nodulation gene from the nitrogen-fixing symbiont, Sinorhizobium fredii, regulates cultivar-specific nodulation of soybean. Expression of this gene is triggered by isoflavonoid cues from host roots but is not mediated by a typical cis-acting nod box promoter. We have found that two isoflavonoids, daidzein and coumestrol, induce expression of a nolX-lacZ gene fusion at pH values between 5.5 and 8.5, with an optimum at 6.5. Expression of nolX is highly dependent on nutritional status but insensitive to salinity. Methionine and casamino acids, amendments that enhance expression of hrpF, a homologous gene from the plant pathogen Xanthomonas campestris biovar vesicatoria, have no and strongly negative effects, respectively, on expression of nolX. Growth of S. fredii on rhamnose, galactose, xylose, or myo-inositol as carbon source greatly elevates inducibility in comparison to a standard yeast extract-mannitol medium. None of the tested variables, however, released nolX from its dependence on isoflavonoids as inducers.

Sinorhizobium fredii strain HH103, a nitrogen-fixing bacterial symbiont of plants, contains an insertion sequence (IS) that cantranspose into plasmid pMUS248 and activate a promoterless TcR gene that is normally not expressed. We have cloned andcharacterized this element, which we designate IS Rf1. The IS is 1002 bp in length, contains a single 513-bp open reading frame(ORF), is flanked by imperfect 36-bp terminal inverted repeats, and creates 5-bp target duplications. Two copies of IS Rf1 arepresent in the genome of HH103, but it is absent from 12 other Sinorhizobium and Rhizobium strains. The element transposes ata frequency of 2.7×10−6per generation per cell. © 1997 Elsevier Science B.V. Keywords: Insertion sequence; Legume– Rhizobium symbiosis; Tetracycline resistance; Tn 5403 1. Introduction 1987), but also as agents of horizontal gene transfer toand from bacteria residing in the soil (Deng et al., 1995;Rhizobia, e.g. bacteria of the genera Rhizobium , Ponsonnet et al., 1995). Although a transposon (Tn)

Rhizobium (now Sinorhizobium) fredii is a symbiotic nitrogen-fixing bacterium that can nodulate soybean in a cultivar-specific manner. This process is governed by a set of negatively acting nodulation genes termed noIXWBTUV. These genes prevent R. fredii strain USDA257 from infecting soybean cultivars such as McCall, but they do not block nodulation of cultivar Peking. R. fredii strain USDA191 contains DNA sequences that hybridize to noIXWBTUV, yet it forms normal nitrogen-fixing nodules on both McCall and Peking soybean. These sequences were isolated and their structure and function examined in comparison to noIXWBTUV of strain USDA257. Restriction maps of the two loci are identical, as is a 2∙4 kb DNA sequence that corresponds to noIX and its promoter region. Expression of noIX by strain USDA191 is flavonoid-dependent in culture and readily detectable in nodules. The gene is not inducible in a mutant of strain USDA191 that lacks the regulatory nodD1 gene, and its expression is greatly attenuated in a nodD2 mutant. noIX is also present and flavonoid-inducible in HH103, a second R. fredii strain that nodulates McCall soybean normally. Inactivation of noIX in strain HH103, USDA191 or USDA257 leads to retardation of initial nodulation rates on soybean cultivars such as Peking and to acquisition of the capacity to form nitrogen-fixing nodules on two species of Erythrina. noIX is thus of symbiotic significance in all three strains, even though it regulates soybean cultivar specificity only in strain USDA257.

The transcripts of nolW and nolB, two divergently oriented cultivar specificity genes of Rhizobium fredii strain USDA257, are known to be initiated 14 bp apart from promoters that face one another. We show here that expression of nolB is dependent both on induction with flavonoid signals and on the regulatory gene, nodD1. Expression of nolW is constitutive and independent of flavonoids and nodD1. Normal expression of nolB is retained with a promoter that extends only 61 bp upstream of the transcript start site, but it is lost if an additional 24 bp are removed. Substantial expression of nolW is retained with a promoter that contains only 34 bp of DNA upstream from the transcript initiation site. The dual control region for the two genes is thus only about 109 bp in length.

Summary We have examined six fast-growing Rhizobium strains isolated from nodulated soybean [Glycine max (L.) Merr.] plants growing in Vietnam, with special emphasis on two of them, SMX11 and SMH12. The abilities of strains SMX11 and SMH12 to utilize 11 carbon sources were indistinguishable from that of control Sinorhizobium fredii strains USDA257 and HH103, but their growth rates in standard media were considerably more rapid. The Vietnamese strains also were more salt-tolerant and antibiotic-resistant than the S. fredii reference strains, and their LPS banding patterns were less complex. Weak immunological cross-reactivity between the Vietnamese strains and S. fredii was evident. RFLP patterns of a series of nodulation genes and a repetitive sequence from S. fredii matched those of the Vietnamese strains, as did the sequence of a portion of a 16S rRNA gene. Analysis of cellular fatty acids was consistent with the assignment of strain SMH 12 and four other Vietnamese strains to S. fredii, but it aligned strain SMX11 more closely to other related groups of rhizobia. The Vietnamese strains have broad host ranges for nodulation of legumes, and their abilities to fix nitrogen in association with five soybean cultivars are equivalent to or surpass that of S. fredii.

Genistein and other flavonoids from host legumes are known to stimulate cells of the nitrogen-fixing soybean symbiont Rhizobium fredii to synthesize Nod factors, which function as signals during nodule initiation. Flavonoids also trigger R. fredii to secrete a set of signal-responsive (SR) proteins into the environment. By insertion mutagenesis, we showed that secretion of SR proteins by this organism has an absolute dependence on the regulatory gene nodD1. We isolated and sequenced nodD1 and nodD2 of R. fredii USDA257 and constructed strains containing additional, plasmid-borne copies of these genes. Extra copies of nodD1 had no effect on secretion of SR proteins, but extra copies of nodD2 rendered the process constitutive. Extracts from seeds of the soybean cultivars McCall and Peking can substitute for purified flavonoids as inducers of SR proteins. The nolXWBTUV locus is known to control cultivar-specific nodulation of McCall soybean in a negative, flavonoid-dependent manner. Inactivation of any of these genes prevented SR proteins from accumulating in culture fluids. Protein secretion in response to host signals was a characteristic of nine out of ten R. fredii strains tested. Immunological probes failed to detect SR3 or SR5 in mature soybean or cowpea nodules. Although the functions of these proteins remain unknown, their potential role in symbiosis is strengthened by the discovery that their accumulation depends on nodD1, nodD2 and nolXWBTUV.

We have isolated and characterized the extracellular Nod factors of Rhizobium fredii USDA257, a nitrogen-fixing symbiont of soybean [Glycine max (L.) Merr.] and several other legume species. These signals are produced upon exposure to the isoflavone genistein and consist of a series of substituted, beta 1,4-linked tri-, tetra-, and pentamers of N-acetylglucosamine. N-Vaccenic acid replaces acetate on the nonreducing residue, and the reducing residue contains alpha-linked 2-O-methylfucose on carbon 6. Small amounts of a fucose-containing tetramer also were present. The Nod factors elicit root-hair deformations on soybean and two other plants at concentrations ranging from 10(-6) to 10(-12) M.

Symbiotic interactions between rhizobia and legumes are largely controlled by reciprocal signal exchange. Legume roots excrete flavonoids which induce rhizobial nodulation genes to synthesize and excrete lipo-oligosaccharide Nod factors. In turn, Nod factors provoke deformation of the root hairs and nodule primordium formation. Normally, rhizobia enter roots through infection threads in markedly curled root hairs. If Nod factors are responsible for symbiosis-specific root hair deformation, they could also be the signal for entry of rhizobia into legume roots. We tested this hypothesis by adding, at inoculation, NodNGR-factors to signal-production-deficient mutants of the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium japonicum strain USDA110. Between 10(-7) M and 10(-6) M NodNGR factors permitted these NodABC- mutants to penetrate, nodulate and fix nitrogen on Vigna unguiculata and Glycine max, respectively. NodNGR factors also allowed Rhizobium fredii strain USDA257 to enter and fix nitrogen on Calopogonium caeruleum, a nonhost. Detailed cytological investigations of V. unguiculata showed that the NodABC- mutant NGR delta nodABC, in the presence of NodNGR factors, entered roots in the same way as the wild-type bacterium. Since infection threads were also present in the resulting nodules, we conclude that Nod factors are the signals that permit rhizobia to penetrate legume roots via infection threads.

Symbiotic rhizobia enter roots of their hosts via infection threads in root-hairs and invade the cortex, where nodules are initiated. We have transferred a cloned cellulase (Cel) gene from Erwinia carotovora subsp. carotovora into rhizobia and examined both the production of the enzyme and its impact on nodulation. Acquisition of a Cel + plasmid allowed cultured cells of Rhizobium fredii ,R. meliloti , and R. tropici to produce extracellular enzymes that degrade carboxymethylcellulose. The Cel + plasmid was retained by R. fredii during nodulation of cowpea, and analysis of a gene fusion confirmed that the Cel promoter was active in planta. Cellulase levels in nodules nevertheless were low, and infection and nodulation were unaffected by the presence of the Cel + plasmid.

Rhizobiumfredii USDA257 is a Gram-negative soil bacterium from China that fixes nitrogen in symbiosis with primitive soybean (Glycine max [L.] Merr.) cultivars such as Peking, but not with advanced cultivars such as McCall. Mutation of either of two bacterial loci, nolBTUVWX or no1C, removes this cultivar-specificity constraint, allowing the bacterium to nodulate advanced cultivars. We have discovered that USDA257 forms nodules on Erythrina costaricensis M. Micheli, but not on six other species of this genus, which includes trees and shrubs from the tropics. Inactivation of nolBU or nolC broadensA4he symbiosis to include other Erythrina species. Whereas nolBU-mutants acquire the capacity to nodulate E. fusca Loureiro, E. variegata L., and E. vespertilio Benth., nolC-mutants could nodulate only E. variegata. Nodules are determinate and often clustered in root axils near the root crown. E. variegata nodules containing a nolBU-mutant consist of well-developed inner and outer cortical layers, as well as a bacteroid zone. The cortical layers are separated from one another by a meristematic region that is associated with large darkly staining cells, and cells of the inner cortex contain numerous starch grains. Histochemical staining of E. variegata roots inoculated with a strain containing a nolB-lacZ gene fusion confirms that nolB and nolC are expressed during the initial stages of bacterial interaction with the host. R. fredii enters elongated root-hairs via infection threads, which ramify within root-hairs and are associated with meristematic activity

Calcium is essential for the growth of rhizobia and the formation of nitrogen-fixing root-nodules on legumes, but its precise role in these processes remains unknown. We have found that Rhizobium fredii USDA208 accumulates a major 38 kDa protein when grown in media supplemented with 0.3-2 microM Cacl2. We have purified this protein and raised polyclonal antibodies against it. The protein initially is synthesized as a 40 kDa precursor which subsequently undergoes calcium-dependent processing to give rise to the mature polypeptide. Subcellular and immunocytochemical localization studies indicate that the 38 kDa protein accumulates preferentially in the periplasmic space. Its N-terminal sequence, AETIKIGVAGPMTG, shows significant homology to the N-termini of amino acid binding proteins from the periplasm, including leucine-, isoleucine-, and valine-specific binding proteins of Pseudomonas aeruginosa and Escherichia coli and a leucine-specific binding protein of E. coli. The R. fredii protein does not, however, bind [3H]-leucine. The 38 kDa protein is encoded by the bacterial chromosome. It is absent in several rhizobia other than R. fredii, but antigenically related polypeptides are present in Escherichia coli and Erwinia carotovora subsp. carotovora.

The nodulation genes of Rhizobium fredii are located on large symbiosis plasmids and induced by flavonoid signals from the host plant. When cultured in the presence of the isoflavone nodulation gene inducer, genistein, R. fredii USDA193 produced extracellular polysaccharide (EPS) with a lower average molecular mass and a reduced uronic acid content in comparison to controls. Genistein treatment also attenuated the yield of these compounds. These genistein-mediated changes are dependent on the presence of the symbiosis plasmid and were not apparent with the plasmid-cured, non-nodulating derivative, IA728. In addition, EPS yields from IA728 were higher than those from USDA193, indicating that the sym plasmid is involved in the regulation of EPS production, independent of the presence of genistein signals.

A constitutively expressed chromosomal gene, nolC, regulates the ability of Rhizobium fredii strain USDA257 to nodulate certain agronomically improved soybean cultivars. Other advanced cultivars, as well as unimproved varieties such as 'Peking', form nitrogen-fixing nodules with parental strain USDA257 and with nolC mutants. We have used 257DH512, a derivative of USDA257 that contains a nolC-lacZ fusion, to directly measure strain competition for nodule occupancy. Seedlings of the soybean cultivar 'Peking' were inoculated with mixtures containing various ratios of 257DH512 and R. fredii USDA208; nodules were harvested 14 days later and stained histochemically with X-gal, a chromogenic substrate for the β-galactosidase product of lacZ. We were able to stain and evaluate batches of nearly 500 nodules in just 4 h, without the use of expensive equipment. Under the conditions of these experiments, USDA208 was considerably more competitive than 257DH512, even when it had been present in the inoculum at a numerical disadvantage. We suggest that gene fusions can be used to greatly simplify the assessment of nodulation competitiveness in rhizobia. Key words: competition, nolC-lacZ fusion, soybean – Rhizobium fredii, nodule occupancy.

We investigated the role of calcium in growth of Rhizobium fredii USDA208 and its influence on nodulation of cv. Peking soybean. The placement and numbers of nodules on soybean roots in growth pouches is relatively insensitive to levels of Ca2+ in the root zone. In contrast, adequate Ca2+ in the Rhizobium culture media is essential for induction of a normal distribution of nodules on the taproot and lateral roots. Ca2+-deprived rhizobia attach in reduced numbers to soybean roots, do not initiate nodules in the most infectible region of the taproot, and induce nodules almost exclusively on lateral roots. Ca2+ starvation also greatly reduces growth of USDA208 in culture, but it has little effect on exopolysaccharide production. Strain USDA208 produces two detectable 45Ca2+-binding proteins in culture. A major 38-kDa bacterial protein is regulated by the availability of Ca2+, but this protein does not itself bind 45Ca2+. Key words: autoregulation, nodule regulation, nodule initiation.

Rhizobium fredii USDA257 does not nodulate McCall soybean (Glycine max (L.) Merr.), but two transposon-mutants derived from it, 257DH4 and 257DH5, do. All three organisms cause curling of McCall root hairs and induce the formation of underlying cortical cell divisions. The mutants produce infection threads, and many of the meristematic foci develop into nodules. In contrast, root hairs that deform in response to USDA257 lack infection threads, and meristematic activity ceases prior to the appearance of nodule meristems. Root systems nodulated by mutant 257DH4 reduce acetylene at rates similar to those of roots nodulated by reference R. fredii strain USDA191. The presence of living cells of USDA257 in inocula leads to strong inhibition of nodulation by 257DH4 but not by 257DH5. This blocking effect depends on the ratio of USDA257 cells to 257DH4 cells in the inoculum; nodules that form contain cells of 257DH4, but not those of parental strain USDA257.

We characterized field isolates of Bradyrhizobium japonicum from soybeans grown in Missouri soil and measured competitiveness of peat-formulated B. japonicum 123 (serogroup 123) and 138 (serogroup c1) in this soil. Nodule occupancy by serogroup 123, but not serogroup c1, varied with plant age in uninoculated. field-grown soybeans. Serogroups c1 and 38–115 were found in 37% of all nodules of these plants. Serogroups 76, 122, 123. c3 and 94 were also present, and rhizobia of unknown serology occupied 17% of the nodules. Inoculation with either strain 123 or 138 produced similar numbers of nodules. Strain 123 as an inoculant increased nodule occupancy by serogroup 123 13-fold, while a 2-fold increase in serogroup c1 nodule occupancy was obtained with strain 138 inoculation. Increases in nodule occupancy by the inoculant was at the expense of serogroups other than 123 and c1. Inoculation of either strain resulted in increased nodule occupancy in first-formed nodules in unsterile soil. Dual inoculation with both strains in unsterile soil allowed serogroup c1 to occupy twice the number of nodules as did serogroup 123. In sterilized soil, strain 123 outcompetcd strain 138, occupying 74% of all nodules. The effect of inoculant carrier on competitiveness is discussed.

Genetically, Rhizobium sp. strain NGR234 and R. fredii USDA257 are closely related. Small differences in their nodulation genes result in NGR234 secreting larger amounts of more diverse lipo-oligosaccharidic Nod factors than USDA257. What effects these differences have on nodulation were analyzed by inoculating 452 species of legumes, representing all three subfamilies of the Leguminosae, as well as the nonlegume Parasponia andersonii, with both strains. The two bacteria nodulated P. andersonii, induced ineffective outgrowths on Delonix regia, and nodulated Chamaecrista fasciculata, a member of the only nodulating genus of the Caesalpinieae tested. Both strains nodulated a range of mimosoid legumes, especially the Australian species of Acacia, and the tribe Ingeae. Highest compatibilities were found with the papilionoid tribes Phaseoleae and Desmodieae. On Vigna spp. (Phaseoleae), both bacteria formed more effective symbioses than rhizobia of the “cowpea” (V. unguiculata) miscellany. USDA257 nodulated an exact subset (79 genera) of the NGR234 hosts (112 genera). If only one of the bacteria formed effective, nitrogen-fixing nodules it was usually NGR234. The only exceptions were with Apios americana, Glycine max, and G. soja. Few correlations can be drawn between Nod-factor substituents and the ability to nodulate specific legumes. Relationships between the ability to nodulate and the origin of the host were not apparent. As both P. andersonii and NGR234 originate from Indonesia/Malaysia/Papua New Guinea, and NGR234's preferred hosts (Desmodiinae/ Phaseoleae) are largely Asian, we suggest that broad host range originated in Southeast Asia and spread outward.

All known R. fredii strains contain two copies of nodD, which are termed nodD1 and nodD2. Mutations in nodD1 abolish the ability to form nodules. Mutations in nodD2 delay nodulation. We have inserted, by marker exchange experiments, the omega cassette (Ω) into the NruI site of nodD1 of R. fredii USDA257 and HH103. Both mutants are unable to nodulate, to induce a nodAp-lacZ fusion in the presence of flavonoids and to produce Nod-factors. DNA hybridization experiments, using nodD1, nodD2 and the Ω. cassette as probes, showed that the nodD2 gene is not affected in the HH103 NodD1− mutant. Hence, nodD2 is not able to replace the nodD1 gene for the trascriptional activation of nodulation genes and nodulation ability. However, a multicopy vector containing the nodD2 gene from USDA257 (called pHBK330) restored nodulation ability to both NodD1− mutants. USDA257 NodD1− carrying pHBK330 produced low quantities of Nod-factors in the presence of flavonoids, or even in their absence. Apparently, the amounts of Nod-factors produced with and without flavonoids are very similar. This fact indicates that, in the presence of extra copies of nodD2, this production is constitutive and independent of flavonoids. Although nodD2 also restored nodulation ability of HH103 NodD1−, we could not demonstrate the production of Nod-factors in the presence of extra copies of nodD2. Moreover, isolates from soybean nodules produced by HH103 NodD1− (pHBK330) did not maintain pHBK330. Instead, they have plasmids than were larger than expected. Because these plasmids code for Tc-resistance (the marker of pHBK330), it is reasonable to assume that they are recombinant derivatives of pHBK330 and the genome of HH103.

Flavonoid cues from the plant host cause symbiotic, nitrogen-fixing rhizobia to synthesize lipochitooligosaccharides (LCOs), which act as return signals to initiate the nodulation process. Rhizobium fredii strain USDA257 is known to produce four LCOs, all substituted with vaccenic acid (C18:1). We show here that a mutant strain can replace vaccenic acid with a C16:0 side chain, and that strain USDA191 synthesizes additional LCOs that differ from one another in the length and unsaturation of their fatty acyl substituents. USDA191 and 257DH4 both produce a novel LCO with glucose substituted for N-acetyl-d-glucosamine in the backbone of the molecule.

Soil bacteria of the genera Azorhizobium, Bradyrhizobium, and Rhizobium are collectively termed rhizobia. They share the ability to penetrate legume roots and elicit morphological responses that lead to the appearance of nodules. Bacteria within these symbiotic structures fix atmosphere nitrogen and thus are of immense ecological and agricultural significance. Although modern genetic analysis of rhizobia began less than 20 years ago, dozens of nodulation genes have now been identified, some in multiple species of rhizobia. These genetic advances have led to the discovery of a host surveillance system encoded by nodD and to the identification of Nod factor signals. These derivatives of oligochitin are synthesized by the protein products of nodABC, nodFE, NodPQ, and other nodulation genes; they provoke symbiotic responses on the part of the host and have generated immense interest in recent years. The symbiotic functions of other nodulation genes are nonetheless uncertain, and there remain significant gaps in our knowledge of several large groups of rhizobia with interesting biological properties. This review focuses on the nodulation genes of rhizobia, with particular emphasis on the concept of biological specificity of symbiosis with legume host plants.

Rhizobium fredii is a nitrogen-fixing bacterial symbiont of soybean and a number of other legume species. We have studied the transcriptional organization of a Sym plasmid locus that restricts the host range of R. fredii USDA257 at both the host species and cultivar level. The genes of this host-specificity locus, noIXWBTUV, are transcribed from three promoters. Two of these, which are upstream of noIW and noIBTUV, are oriented face to face and initiate transcription at sites that are 14 bp apart. The third lies upstream from noIX. The noIW promoter is constitutive, whereas the noIB and noIX promoters are inducible by flavonoid signals. We have attempted to express genes from this locus in Escherichia coli systems, both in vivo and in vitro. We detected the insert- and orientation-specific expression of two genes, noIX and noIW, but we were unable to obtain expression of noIBTUV. Antiserum raised against NoIT nevertheless detected an abundantly expressed polypeptide of the predicted size in protein extracts of USDA257. This observation, as well as RNA dot blot data from a series of mutants, indicates that noIBTUV is expressed as a single transcriptional unit in R. fredii. Immunological detection of NoIT, and of a second protein, NoIX, was strictly dependent on flavonoid induction. The NoIX protein was larger than the size predicted from the previously published nucleotide sequence, and this led to resequencing and revision of the open reading frame.

We determined the nucleotide sequences of 16S rRNA gene segments from five Rhizobium strains that have been isolated from tropical legume species. All share the capacity to nodulate Phaseolus vulgaris L., the common bean. Phylogenetic analysis confirmed that these strains are of two different chromosomal lineages. We defined the host ranges of two strains of Rhizobium etli and three strains of R. tropici, comparing them with those of the two most divergently related new strains. Twenty-two of the 43 tested legume species were nodulated by three or more of these strains. All seven strains have broad host ranges that include woody species such as Albizia lebbeck, Gliricidia maculata, and Leucaena leucocephala.

Rhizobium fredii is a nitrogen-fixing symbiont from China that combines broad host range for nodulation of legume species with cultivar specificity for nodulation of soybean. We have compared 10 R. fredii strains with Rhizobium sp. NGR234, a well known broad host range strain from Papua New Guinea. NGR234 nodulated 16 of 18 tested lugume species, and nodules on 14 of the 16 fixed nitrogen. The R. fredii strains were not distinguishable from one another. They nodulated 13 of the legumes, and in only nine cases were nodules effective. All legumes nodulated by R. fredii were included within the host range of NGR234. Restriction fragment length polymorphisms (RFLPs) were detected with four DNA hybridization probes: the regulatory and common nod genes, nodDABC; the soybean cultivar specificity gene, nolC; the nitrogenase structural genes, nifKDH; and RFRS1, a repetitive sequence from R. fredii USDA257. A fifth locus, corresponding to a second set of soybean cultivar specificity genes, nolBTUVWX, was monomorphic. Using antisera against whole cells of three R. fredii strains and NGR234, we separated the 11 strains into four serogroups. The anti-NGR234 sera reacted with a single R. fredii strain, USDA191. Only one serogroup, which included USDA192, USDA201, USDA217, and USDA257, lacked cross reactivity with any of the others. Although genetic and phenotypic differences among R. fredii strains were as great as those between NGR234 and R. fredii, our results confirm that NGR234 has a distinctly wider host range than R. fredii.

Summary Rhizobium fredii strain USDA257 produces nitrogen-fixing nodules on primitive soybean cultivars such as Peking but fails to nodulate agronomically improved cultivars such as McCall. Transposonmutant 257DH4 has two new phenotypes: it nodulates McCall, and its ability to do so is sensitive to the presence of parental strain U5DA257, i.e. it is subject to competitive nodulation blocking. We have isolated a cosmid containing DNA that corresponds to the site of transposon insertion in 257DH4 and have localized Tn5 on an 8.0 kb EcoRI fragment. The 5596 bp DNA sequence that surrounds the insertion site contains seven open reading frames. Five of these, designated nolBTU, ORF4, and nolV, are closely spaced and of the same polarity. nolWand nolX are of the opposite polarity. The initiation codon for nolW lies 155bp upstream from that of nolB, and it is separated from nolXby 281 bp. The predicted NolT and NolW proteins have putative membrane-spanning regions. The N-terminus of the hypothetical NolW protein also has limited homology to NodH of Rhizobium meliloti, but none of the deduced protein sequences has significant homology to known nodulation gene products. Site-directed mutagenesis with mudll1734 confirms that inactivation of nolB, nolT, nolU, nolV, nolW, or nolX extends host range for nodulation to McCall soybean. This phenotype could not be genetically dissected from sensitivity to competitive nodulation blocking. Expression of nolBTU anti nolX is induced as much as 30-fold by flavonoid signal molecules, even though these genes lack nod-box promoters. Histochemical staining of McCall roots inoculated with nolB–, nolU–, or nolX–lacZ fusions verifies that these genes are expressed continuously from preinfection to the stage of the functional nodule. Although a nolU–ORF4–nolV clone hybridizes to a single 8.0 kb EcoRI fragment from 10 strains of R. fredii and broad-host-range Rhizobium sp. NGR234, hybridizing sequences are not detectable in other rhizobia.

Herewe characterize asecond family member, RFRS9.TheEcoRIfragment containing RFRS9is1,248 bpinlength and contains asingle 666-bp openreading framethat isflanked byperfect 8-bpinverted repeats. Nucleic andamino acid sequencescorresponding totheC terminus oftheputative RFRS9protein arenearly identical tothose of RFRS3,andtheyretain homology toDNA fromA.rhizogenes. Thecentral portion oftheRFRS9protein also appearstoberelated totheSlocus-specific glycoprotein family ofpolenstigma incompatibility glycoproteins fromBrassica oleracea,whichareinvolved insignal perception. Sequences thatdefine theRFRSfamily are restricted totheopenreading frameofRFRS9andassociated upstream sequences.Theseregions also contain a second groupofrepetitive sequences,whichispresent infourcopies within thegenome ofUSDA257.Both families ofrepetitive sequencesareubiquitous inR.fredii, andthey arepreferentially localized on symbiosis plasmids. Southern hybridization confirms that sequenceshomologous toRFRS9arepresent inbroad-host-range Rhizobium sp.strain NGR234,inA.rhizogenes, andintwobiotype 3strains ofAgrobacterium tumefaciens. Thegenomesofnitrogen-fixing rhizobia arecharacterized bythepresenceofvarious sortsofrepetitive DNA sequences (3,5).Theseelements include bothgene and promoterduplications (2,40), aswell asinsertion sequences andotherreiterated regions. Although thebiological significance ofmostoftheseDNA duplications isobscure (1), some multicopy genesinrhizobia arerecognized asplaying pivotal roles inroot-nodule symbiosis. nodD,forexample, whichispresentintwo or threecopies per Rhizobium genome,isknowntoencodea family ofhostrecognition proteins. Thereisevidence thattheinterplay between these related sensorsservestofine tunethebacterial hostrange withlegumes (7). nodH,whichencodes nitrogenase reductase(24), andnodPQ,whichencodesulfation enzymes (29, 34), also arereiterated. Insertion sequencescompose another subclass ofrepetitivesequencesfromrhizobia. Theseelements havetwo diagnostic features. First, theytendtoberestricted intheir occurrencetojust one or afewspecies ofrhizobia. ISRml, whichwas first discovered inthealfalfa andsweetclover symbiont, Rhizobium meliloti (31), isan example. This insertion sequenceispresentin80%ofthestrains froma NorthAmerican collection ofR.meliloti (42) and97%ofthe strains froma similar European collection (10). Onlyone strain ofRhizobium leguminosarum bv.phaseoli andtwo taxonomically intermediate isolates fromalfalfa alsoharbor this sequence(42). ISRm2(4)andISRm3(41) aresimilarly distributed inR meliloti, butbothalso havebeendetected in biovars ofR leguminosarum. A secondcharacteristic of Rhizobium insertion sequences istheir strongassociation withandoftenpreferential transposition intonodulation

Transfer of a cosmid containing nodSU from Rhizobium sp. NGR234 to Rhizobium fredii USDA257 expands the host range for nodulation to include the perennial tropical legumes, Leucaena leucocephala and Leucaena diversifolia. Complementation experiments with a series of subclones established that nodS and its associated nod-box promoter from NGR234 are sufficient to confer this extended host-range phenotype to L. leucocephala. Strain USDA257 contains its own copy of nodSU, including upstream nod-box sequences. Although both nucleotide and deduced amino acid sequences of the reading frames are homologous between the two strains, there are gaps within the promoter region and the 5'-end of nodS of USDA257. Consequently, the deduced NodS protein of USDA257 is shorter than its counterpart from NGR234, and the distance between the nod-box and the initiation codon is greater. A 36 bp deletion encompasses the extreme right border of the USDA257 nod-box and extends into the upstream leader sequence. Transcriptional fusions with both nod-boxes confirmed that the promoter from NGR234 is flavonoid-inducible, and that the nod-box from USDA257 is not. These observations were corroborated by Northern analysis with a nodS-containing Xhol fragment as hybridization probe. Flavonoid-induced cells of NGR234 gave an intense signal, but those of USDA257 yielded only a weak trace of hybridization. EcoRI fragments with homology to nodSU of USDA257 are present in 17 of 35 tested strains, including several representatives of Bradyrhizobium japonicum, Rhizobium sp., R. loti, and R. fredii. Two wild-type, leucaena-nodulating strains of Rhizobium sp. lack this homology. We conclude that a genetic defect in expression of nodS accounts for the inability of USDA257 to nodulate leucaena and that diverse rhizobia may have evolved alternative mechanisms to nodulate this legume species.

Rhizobium species strain NGR234 is the most promiscuous known rhizobium. In addition to the non-legume Parasponia andersonii, it nodulates at least 70 genera of legumes. Here we show that the nodulation genes of this bacterium determine the production of a large family of Nod-factors which are N-acylated chitin pentamers carrying a variety of substituents. The terminal non-reducing glucosamine is N-acylated with vaccenic or palmitic acids, is N-methylated, and carries varying numbers of carbamoyl groups. The reducing N-acetyl-glucosamine residue is substituted on position 6 with 2-O-methyl-L-fucose which may be acetylated or sulphated or non-substituted. All three internal residues are N-acetylated. At pico- to nanomolar concentrations, these signal molecules exhibit biological activities on the tropical legumes Macroptilium and Vigna (Phaseoleae), as well as on both the temperate genera Medicago (Trifoliae) and Vicia (Viciae). These data strongly suggest that the uniquely broad host range of NGR234 is mediated by the synthesis of a family of varied sulphated and non-sulphated lipo-oligosaccharide signals.

Summary Rhizobium fredii strain USDA257 does not nodulate soybean (Glycine max (L.) Merr.) cultivar McCall. Mutant 257DH5, which contains a Tn5 insert in the bacterial chromosome, forms nodules on this cultivar, but acetylene-reduction activity is absent. We have sequenced the region corresponding to the site of Tn5 insertion in this mutant and find that it lies within a 1176 bp open reading frame that we designate nolC. nolC encodes a protein of deduced molecular weight 43564. Nucleotide sequences homologous to nolC are present in several other Rhizobium strains, as well as Agrobacterium tumefaciens, but not in Pseudomonas syringae pathovar glycinea. nolC lacks significant sequence homology with known genes that function in nodulation, but is 61% homologous to dnaJ, an Escherichia coli gene that encodes a 41 kDa heat-shock protein. Both R. fredii USDA257 and mutant 257DH5 produce heat-shock proteins of 78, 70, 22, and 16kDa. A 4.3kb EcoRI–HindIII subclone containing nolC expresses a single 43 kDa polypeptide in mini-cells. A longer, 9.4kb Eco RI fragment expresses both the 43kDa polypeptide and a 78kDa polypeptide that corresponds in size to that of the largest heat-shock protein. Thus, although nolC has strong sequence homology to dnaJ and appears to be linked to another heat-shock gene, it does not directly function in the heat-shock response.

Rhizobium fredii USDA257 forms nitrogen-fixing nodules on soybean cultivar Peking, but not on cultivar McCall. This pattern of nodulation persists when McCall and Peking seedlings are cultivated together in plastic growth pouches. Reciprocal grafting experiments confirm that the root genotype, and not that of the shoot, regulates such cultivar specificity. When Peking roots are grafted onto McCall seedlings, the nodulation responses of roots similarly remain unaffected. Transposon-mutant 257DH4, which is derived from USDA257, can form nitrogen-fixing nodules on McCall. Such nodulation is blocked by the presence of USDA257 in the inoculum. Grafting experiments indicate that blocking is not due to a translocatable inhibitor produced by McCall roots or triggered by their interaction with USDA257. Thus, neither freely diffusible nor graft-transmissible substances are involved in cultivar-specific interactions of soybean with R. fredii and its derivatives.

Inoculation of 2-day-old cowpea (Vigna unguiculata [L.] Walp.) seedlings with Rhizobium fredii USDA257 results in proficient nodulation of the tap root. The most abundant nodulation occurs in a region roughly corresponding to the position of the root tip at the time of inoculation. We have examined plant gene expression in this region, after inoculation with either USDA257 or a nonnodulating mutant, 257B3. After isolation of mRNA and in vitro translation, the protein products were separated by two-dimensional gel electrophoresis. Seven proteins are induced within 2.5 days after inoculation with USDA257. One additional induced protein is detectable by 3.5 days after inoculation, and three more appear by day 6. Three of the proteins that are differentially expressed at 2.5 and 3.5 days after inoculation are produced at equivalent levels after 6 days, indicating transient induction of these genes during early stages of nodule development. Several proteins were more abundant in translations of mRNA from roots that had been inoculated with the nonnodulating mutant. This was particularly true after 6 days, when nine proteins were in this class. Thus, altered plant gene expression in carefully selected, highly responsive tissue can be detected 2 days before emerging nodules are visible on the roots, and 6 to 7 days before acetylene reduction is detectable. Additionally, comparisons of ionically bound cell wall proteins isolated 6 days after inoculation revealed four that were unique to nodulating roots, suggesting that some of the nodulation-induced genes may code for structural proteins.

Rhizobium fredii strains USDA257 and USDA191 both contain a set of nodulation genes termed nolXWBTUV. In the USDA257 background, nolX prevents infection of soybean cultivars such as McCall, and in both backgrounds, it blocks nodulation of Erythrina spp. We report here that expression of nolX is differentially responsive to a panel of flavonoids, and that the most potent inducers are also the most active inducers of nodC, a conventional, nod box-associated gene. Cell-free protein extracts from uninduced and flavonoid-induced cells of strains USDA191 and USDA257 retard the electrophoretic mobility of DNA sequences that lie upstream of nolX. Binding is dependent both on nodD1 and nodD2, and it is abolished by the presence of a double-stranded, 23-bp oligonucleotide that lies within a 114-bp TaqI/SacII restriction fragment. This oligomer has significant sequence homology to A3, a putative negative regulatory element from R.leguminosarum bv. viciae. Deletion of the A3-homologous sequences elevates the basal and flavonoid-inducible expression of nolX by about 50%.

Roots of soybean (Glycine max [L.] Merr. cv Hardee) and cowpea (Vigna unguiculata [L.] Walp. cv Pink Eye Purple Hull) were immersed in suspensions containing 104Rhizobium cells per milliliter of a nitrogen-free solution. After 30 to 120 minutes the roots were rinsed, and the distal 2-centimeter segments excised and homogenized. Portions of the homogenates then were plated on a yeast-extract mannitol medium for bacterial cell counts. The adsorption capacities of four slow-growing rhizobia and a fast-growing R. meliloti strain varied considerably. Adsorption was independent of plant species and of the abilities of the Rhizobium strains to infect and nodulate. R. lupini 96B9 had the greatest adsorption capacity, and Rhizobium sp. 3G4b16 the least. Rhizobium sp. 229, R. japonicum 138, and R. meliloti 102F51 were intermediate, except on cowpea, where the adsorption of strain 102F51 was similar to that of strain 3G4b16. The initial adsorption rates of bacteria cultured in synthetic media and in the presence of soybean roots were about the same. Addition of soybean lectin to the bacterial inoculum failed to influence initial adsorption rates. Both treatments, however, reduced the numbers of bacteria that bound after incubation with roots for 120 minutes. The relationship between the logarithm of the number of strain 138 cells bound per soybean root segment and the logarithm of the density of bacteria in the inoculum was linear over five orders of magnitude. Binding of strain 138 to soybean roots was greatest at room temperature (27°C) and substantially attenuated at both 4 and 37°C. Although R. lupini 96B9 strongly rejected a model hydrophobic plastic surface, there were no simple correlations between bacterial binding to model hydrophobic and hydrophilic plastic surfaces and bacterial adsorption to roots.

Eight lines of soybean (Glycine max), four of wild soybean (Glycine soja), and one cowpea (Vigna unguiculata) cultivar were inoculated with 18 Rhizobium strains. After 4 days, root hairs were examined for infection threads. Threads were produced by all hosts but exclusively in nodulating combinations. Only Rhizobium sp. strains 3G4b9a and 3G4b19 were inconsistent; they nodulated soybean and G. soja in some experiments, but rarely formed infection threads. Soybean and G. soja were indistinguishable in their interactions with rhizobia, as were lele soybean lines (genetically lack soybean lectin), Hardee soybean (contains the noduation-influencing genes Rj2 and Rj3), and several other soybean cultivars. Threads formed in cowpea with all of the R. japonicum strains and most Rhizobium sp. but not with R. lupini. Infection of all three host species occurred in portions of the primary root containing immature or no root hairs at the time of inoculation; proximal tissues having elongated root hairs lacked infection threads. Infected root hairs were short and commonly shaped like question marks. Threads usually branched and sometimes intertwined prior to elongation into the basal portion of root hairs.

Double inoculation (15 h apart) of the soybean cultivar Williams with Bradyrhizobium japonicum I-110ARS reveals a rapid regulatory plant response that inhibits nodulation of distal portions of the primary root (M Pierce, WD Bauer 1984 Plant Physiol 73: 286-290). Only living, homologous rhizobia elicit the response. We conducted similar double inoculation experiments to test the hypothesis that this is a universal phenomenon in soybean symbioses. We investigated interactions of the cultivar McCall with the slow-growing strain Bradyrhizobium sp. 3185 (=3G4b16) and strains of the fast-growing soybean symbiont, Rhizobium fredii (USDA191 [Nod+ on McCall] and USDA257 [Nod− on McCall]). Nodulation was not detectably inhibited when USDA257 was included in various combinations with an inoculum of USDA191. Strain USDA257 cohabited nodules with strain USDA191 when plants were inoculated sequentially with both strains, but USDA257 did not nodulate McCall when a sterile culture filtrate of USDA191 was added to USDA257 inoculum. There was only a slight inhibition of nodulation of distal portions of the primary root in double inoculation experiments with McCall and strain 3185. Because these results were unexpected, we repeated the experiments with Williams and strain I-110ARS. The response was similar to that observed in the McCall × 3185 interaction. Regulation of nodulation on the primary root thus appears to be variable and depend on strain X cultivar interactions.

We labelled cells of tumorigenic and nontumorigenic strains of Agrobacterium tumefaciens with [ 35 S]methionine. The bacteria then were used in binding assays with discs from susceptible potato tubers and from resistant monocotyledon tissues. Approximately 5 × 10 5 cells of tumorigenic strain ACH5 bound to each potato tuber disc. Adsorption of nontumorigenic Agrobacterium strains and of control bacteria ( Rhizobium fredii and Xanthomonas campestris pv. vesicatoria ) to potato was statistically indistinguishable from that of strain ACH5. Cells of strain ACH5 bound to tissue discs from all three monocotyledons. The number of bacteria that bound to discs from rhizomes of Smilax bona-nox was more than 10 times the number that bound to potato. Hydrophilic nonbiological surfaces adsorbed more Agrobacterium cells than did hydrophobic surfaces at pH 4·5 and 7·2. Bacterial binding to hydrophilic surfaces was much greater at pH 4·5 than at pH 7·2 or 9·5. Zeta potential measurements were used to measure the effect of pH on the surface charge characteristics of the bacteria. The isoelectric point of cells of strain ACH5 in distilled water was about 2·9.

Roots of siratro, cowpea, and two cultivars of soybean were inoculated with either Rhizobium sp. 3G4b16 or R. fredii 191. The numbers and distribution of nodules were determined after hydroponic growth of the plants for 10 days in plastic growth pouches. The uppermost nodules on the primary roots of both siratro and cowpea were clustered near the position occupied by the root tip at the time of inoculation. This is the region thought to be maximally susceptible to nodulation. Appreciable numbers of nodules also were scattered down the length of the primary root. Nodulation of McCall and Vicoja soybean (in growth pouches) was very different to that in an autoclaved greenhouse soil mixture. Strain × cultivar-specific differences in the numbers of nodules on primary roots were obliterated by soil. Soil reduced the extent of scattering of nodules along primary roots and shifted the ratios of primary root nodules to lateral root nodules. Soil also caused a 95% reduction in the number of McCall root hairs infected by strain 3G4b16. The soil environment appears to obliterate strain × cultivar-specific variation that is expressed when inoculated plants are grown hydroponically in growth pouches.

Several strains of Rhizobium japonicum have been reported to consist of mixtures of stable derivatives having distinct colony morphologies and physiological characteristics. We isolated derivatives from strains of R. japonicum and systematically compared them with previously isolated derivatives with respect to the utilization of biochemicals, antibiotic sensitivity, and soybean lectin binding. With the exception of a pair of derivatives from 3I1b 110, one of which utilized pyruvate and one of which did not, sibling derivatives had essentially identical biochemical utilization patterns. The sibling derivatives of parental strains 3I1b 110 and 3I1b 140 exhibited marked variation in their sensitivities to several antibiotics, including gentamicin, sulfamethoxazole, and tetracycline. Compared with the derivatives with small colony morphology, derivatives with large colony morphology were in general more sensitive to these antibiotics. With one exception, the binding of soybean lectin to the derivatives was quantitatively the same as that to the parental strain. The anomaly was 110-Y which, in contrast to its parental strain and sibling derivatives, failed to bind detectable amounts of the lectin. 110-Y, as well as all the other derivatives and parental strains, nodulated Disoy soybean.

Highly purified soybean lectin (SBL) was labeled with fluorescein isothiocyanate (FITC-SBL) or tritium ( 3 H-SBL) and repurified by affinity chromatography. FITC-SBL was found to bind to living cells of 15 of the 22 Rhizobium japonicum strains tested. The lectin did not bind to cells of the other seven R. japonicum strains, or to cells of any of the nine Rhizobium strains tested which do not nodulate soybean. The binding of the lectin to the SBL-positive strains of R. japonicum was shown to be specific and reversible by hapten inhibition with d-galactose or N-acetyl-d-galactosamine. The lectin-binding properties of the SBL-positive R. japonicum strains were found to change substantially with culture age. The percentage of cells in a population exhibiting fluorescence after exposure to FITC-SBL varied between 0 and 70%. The average number of SBL molecules bound per cell varied between 0 and 2 × 10 6 . While most strains had their highest percentage of SBL-positive cells and maximum number of SBL-binding sites per cell in the early and midlog phases of growth, one strain had a distinctly different pattern. The SBL-negative strains did not bind lectin at any stage of growth. Quantitative binding studies with 3 H-SBL indicated that the affinity constant for binding of SBL to its receptor sites on R. japonicum is approximately 4 × 10 7 m −1 . Many of the binding curves were biphasic. An inhibitor of SBL binding was found to be present in R. japonicum culture filtrates.

Cells of tumorigenic Agrobacterium tumefaciens strain B6 were labeled with [35S]methionine and used to estimate bacterial adsorption to potato tuber discs. After 10 min at pH 7.2, about 5 × 105 bacteria adsorb to each 9.0-mm disc. Lengthening the incubation period to 90 min increases adsorption to about 11 × 105 bacteria per disc. Bacterial adsorption is reduced under both alkaline and acid pH conditions and increases 2.3-fold if the number of bacterial cells in the inoculum is doubled. Adsorption is increased if citrus pectin, polygalacturonic acid, or demethylated pectin is included in the inoculum; methylated polygalacturonic acid is inactive. The activity of citrus pectin is abolished, however, if the compound is applied to the discs as a pretreatment and then removed prior to inoculation. Lipopolysaccharide preparations from five of six A. tumefaciens strains have no effect on bacterial adsorption. The sixth preparation, from nontumorigenic strain NT-1, reduces adsorption by about 20%.

Seeds of six soybean lines (Glycine max (L.) Merr. cv. Columbia, D68-127, Norredo, Sooty, T-102, Wilson 5) have been reported to lack the 120 000 dalton soybean lectin. Immunodiffusion and radioimmunoassay using anti-soybean lectin immunoglobulin failed to detect the lectin in seeds of five lines, but D68-127 seeds contained as much soybean lectin as the control line, Harosoy 63. The D68-127 seed lectin could be purified by affinity chromatography on Sepharose-N-caproylgalactosamine, and was indistinguishable from the conventional soybean lectin by the following criteria: electrophoretic migration in acidic and alkaline buffers, subunit molecular weight and composition, analytical isoelectric focusing, gel filtration chromatography. Phosphate-buffered saline extracts of roots, hypocotyls, stems, and leaves of 3--66-day-old Norredo and Harosoy 63 plants lacked soybean lectin, as determined by hemagglutination and radioimmunoassay (detection limit: 1.4 micrograms soybean lectin/g dry weight tissue). Cotyledons of Harosoy 63 (but not Norredo) contained large quantities of the lectin, which diminished as the plants aged. 5-day-old roots and hypocotyls of 20 soybean lines did not contain soybean lectin. Roots of Columbia, Norredo, Sooty, T-102, Wilson 5, and Harosoy 63 (control) were nodulated by a variety of strains of Rhizobium japonicum and Rhizobium sp.

We mutagenized Sinorhizobium fredii HH103-1 with Tn5- B20 and screened about 2,000 colonies for increased β-galactosidase activity in the presence of the flavonoid naringenin. One mutant, designated SVQ287, produces lipochitooligosaccharide Nod factors (LCOs) that differ from those of the parental strain. The nonreducing N-acetylglucosamine residues of all of the LCOs of mutant SVQ287 lack fucose and 2-O-methylfucose substituents. In addition, SVQ287 synthesizes an LCO with an unusually long, C20:1 fatty acyl side chain. The transposon insertion of mutant SVQ287 lies within a 1.1-kb HindIII fragment. This and an adjacent 2.4-kb HindIII fragment were sequenced. The sequence contains the 3′ end of noeK, nodZ, and noeL (the gene interrupted by Tn5-B20), and the 5′ end of nolK, all in the same orientation. Although each of these genes has a similarly oriented counterpart on the symbiosis plasmid of the broad-host-range Rhizobium sp. strain NGR234, there are significant differences in the noeK/nodZ intergenic region. Based on amino acid sequence homology, noeL encodes GDP-D-mannose dehydratase, an enzyme involved in the synthesis of GDP-Lfucose, and nolK encodes a NAD-dependent nucleotide sugar epimerase/dehydrogenase. We show that expression of the noeL gene is under the control of NodD1 in S. fredii and is most probably mediated by the nod box that precedes nodZ. Transposon insertion into noeL has two impacts on symbiosis with Williams soybean: nodulation rate is reduced slightly and competitiveness for nodulation is decreased significantly. Mutant SVQ287 retains its ability to form nitrogen-fixing nodules on other legumes, but final nodule number is attenuated on Cajanus cajan.