Rhizobacter

J Bacteriol, 1988 Jul, 170(7), 3249 - 54
Physiology of behavioral mutants of Rhizobium meliloti: evidence for a dual chemotaxis pathway; Bergman K et al.; Wild-type and nonchemotactic mutant strains of Rhizobium meliloti were tested for attraction to localized sites on alfalfa roots and for attraction to numerous small molecules, including sugars, amino acids, and two fractions derived from alfalfa root extracts . Four strains (carrying mutations che-6, che-11, che-12, and che-26) lost all responses and were classified as generally nonchemotactic mutants . One strain (carrying mutation che-7) lost responses to a group of structurally unrelated amino acids but retained all other responses and was classified as a putative sensory transducer mutant . Two strains (carrying mutations che-1 and che-3) lost responses to all the amino acids and sugars tested but retained normal responses to localized sites on roots and to the root fractions . These two mutant strains could not be classified according to the generally accepted model for a sensory pathway, derived from studies of enteric bacteria, and provided evidence for a dual chemotaxis pathway in R . meliloti.

J Bacteriol, 1988 Jul, 170(7), 2994 - 3000
Lectin-enhanced accumulation of manganese-limited Rhizobium leguminosarum cells on pea root hair tips; Kijne JW et al.; The ability of Rhizobium leguminosarum 248 to attach to developing Pisum sativum root hairs was investigated during various phases of bacterial growth in yeast extract-mannitol medium . Direct cell counting revealed that growth of the rhizobia transiently stopped three successive times during batch culture in yeast extract-mannitol medium . These interruptions of growth, as well as the simultaneous autoagglutination of the bacteria, appeared to be caused by manganese limitation . Rhizobia harvested during the transient phases of growth inhibition appeared to have a better attachment ability than did exponentially growing rhizobia . The attachment characteristics of these manganese-limited rhizobia were compared with those of carbon-limited rhizobia (G . Smit, J . W . Kijne, and B . J . J . Lugtenberg, J . Bacteriol . 168:821-827, 1986, and J . Bacteriol . 169:4294-4301, 1987) . In contrast to the attachment of carbon-limited cells, accumulation of manganese-limited rhizobia (cap formation) was already in full progress after 10 min of incubation; significantly delayed by 3-O-methyl-D-glucose, a pea lectin haptenic monosaccharide; partially resistant to sodium chloride; and partially resistant to pretreatment of the bacteria with cellulase . Binding of single bacteria to the root hair tips was not inhibited by 3-O-methyl-D-glucose . Whereas attachment of single R . leguminosarum cells to the surface of pea root hair tips seemed to be similar for both carbon- and manganese-limited cells, the subsequent accumulation of manganese-limited rhizobia at the root hair tips is apparently accelerated by pea lectin molecules . Moreover, spot inoculation tests with rhizobia grown under various culture conditions indicated that differences in attachment between manganese- and carbon-limited R . leguminosarum cells are correlated with a significant difference in infectivity in that manganese-limited rhizobia, in contrast to carbon-limited rhizobia, are infective . This growth-medium-dependent behavior offers and explanation for the seemingly conflicting data on the involvement of host plant lectins in attachment of rhizobia to root hairs of leguminous plants . Sym plasmid-borne genes do not play a role in manganese-limitation-induced attachment of R . leguminosarum.

J Bacteriol, 1988 Jul, 170(7), 3142 - 9
Osmotic control of glycine betaine biosynthesis and degradation in Rhizobium meliloti; Smith LT et al.; Intracellular accumulation of glycine betaine has been shown to confer an enhanced level of osmotic stress tolerance in Rhizobium meliloti . In this study, we used a physiological approach to investigate the mechanism by which glycine betaine is accumulated in osmotically stressed R . meliloti . Results from growth experiments, 14C labeling of intermediates, and enzyme activity assays are presented . The results provide evidence for the pathway of biosynthesis and degradation of glycine betaine and the osmotic effects on this pathway . High osmolarity in the medium decreased the activities of the enzymes involved in the degradation of glycine betaine but not those of enzymes that lead to its biosynthesis from choline . Thus, the concentration of the osmoprotectant glycine betaine is increased in stressed cells . This report demonstrates the ability of the osmolarity of the growth medium to regulate the use of glycine betaine as a carbon and nitrogen source or as an osmoprotectant . The mechanisms of osmoregulation in R . meliloti and Escherichia coli are compared.

Mol Gen Genet, 1988 Jul, 213(1), 155 - 62
Analysis of pss genes of Rhizobium leguminosarum required for exopolysaccharide synthesis and nodulation of peas: their primary structure and their interaction with psi and other nodulation genes; Borthakur D et al.; Strains of Rhizobium leguminosarum (R.l.) biovar viciae containing pss mutations fail to make the acidic exopolysaccharides (EPS) and are unable to nodulate peas . It was found that they also failed to nodulate Vicia hirsuta, another host of this biovar . When peas were co-inoculated with pss mutant derivatives of a strain of R.l . by viciae containing a sym plasmid plus a cured strain lacking a sym plasmid (and which is thus Nod-, but for different reasons) but which makes the acidic EPS, normal numbers of nodules were formed, the majority of which failed to fix nitrogen (the occasional Fix+ nodules were presumably induced by strains that arose as a result of genetic exchange between cells of the two inoculants in the rhizosphere) . Bacteria from the Fix- nodules contained, exclusively, the strain lacking its sym plasmid . When pss mutant strains were co-inoculated with a Nod- strain with a mutation in the regulatory gene nodD (which is on the sym plasmid pRL1JI), normal numbers of Fix+ nodules were formed, all of which were occupied solely by the nodD mutant strain . Since a mutation in nodD abolishes activation of other nod genes required for early stages of infection, these nod genes appear to be dispensable for subsequent stages in nodule development . Recombinant plasmids, containing cloned pss genes, overcame the inhibitory effects of psi, a gene which when cloned in the plasmid vector pKT230, inhibits both EPS production and nodulation ability . Determination of the sequence of the pss DNA showed that one, or perhaps two, genes are required for correcting strains that either carry pss mutations or contain multi-copy psi.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1988 Jun, 212(3), 531 - 5
Identification of nodX, a gene that allows Rhizobium leguminosarum biovar viciae strain TOM to nodulate Afghanistan peas; Davis EO et al.; Gene(s) conferring the ability of Rhizobium leguminosarum biovar viciae strain TOM to nodulate primitive peas (cultivar Afghanistan) had been located in a 2.0 kb region of its sym plasmid, pRL5JI . In this DNA, a single open reading frame of 1101 bp, corresponding to a gene, nodX was found . nodX is downstream of nodJ which is present in strain TOM and also in the sym plasmid of a typical strain of this biovar . nodX specifies a hydrophobic protein (of Mr 41,036) with no clear similarity to other proteins in data bases . Mutations in nodX abolished nodulation of Afghanistan peas but not nodulation of commercial peas . nodX-lacZ fusions were used to show that transcription of nodX was activated by root exudates from both commercial and Afghanistan peas and by defined flavonoids . Exudate from Afghanistan peas activated nod genes of typical strains of R . leguminosarum biovar viciae which fail to nodulate these peas; thus, their failure to nodulate these primitive peas is not due to a lack of activation of their nod genes by exudate from Afghanistan peas . A homologue of nodX exists in R . leguminosarum biovar trifolii (which nodulates clover) but not in typical strains of R . leguminosarum biovar viciae.

Carbohydr Res, 1988 May 1, 176(1), 127 - 35
A core oligosaccharide component from the lipopolysaccharide of Rhizobium trifolii ANU843; Carlson RW et al.; The major oligosaccharide from the core region of the lipopolysaccharide from R . trifolii ANU843 was isolated and its structure determined . It is a trisaccharide consisting of two galacturonic acid residues and one 3-deoxy-D-manno-2-octulosonic acid (KDO) residue . The two galacturonic acid residues are terminally linked alpha to the C-4 and C-7 atoms of KDO . This structure was determined through use of 1H- and 13C-n.m.r . spectroscopy, f.a.b.-m.s., and g.l.c.-m.s . techniques . This oligosaccharide had not previously been reported to be present in the lipopolysaccharides from Gram-negative bacteria.

Mol Microbiol, 1988 May, 2(3), 303 - 14
Identification and characterization of two nitrogen fixation regulatory regions, nifA and nfrX, in Azotobacter vinelandii and Azotobacter chroococcum; Santero E et al.; Five Tn5-induced Nif- mutants of Azotobacter vinelandii were characterized as regulatory mutants because they were restored to Nif+ by the introduction of constitutively expressed nifA from Klebsiella pneumoniae . The mutants fell into two different classes on the basis of hybridization to a Rhizobium leguminosarum nifA gene probe and by complementation with cosmids isolated from pLAFRI gene banks of A . vinelandii and Azotobacter chroococcum . One mutant, MV3, was located in or near a nifA gene . The others, MV12, MV16, MV18 and MV26, defined a new regulatory gene, which has been called nfrX . The lack of expression of different nif-lacZ fusions confirmed the regulatory phenotype of all five mutant strains . The ability of both nifA and nfrX mutants to grow on nitrogen-free medium with vanadium, but not on medium with molybdenum, suggests that neither gene is required for expression of the alternative V-containing nitrogenase of A . vinelandii . A fragment carrying Tn5 and flanking DNA from MV3 was used as a probe to isolate the nifA region of A . chroococcum . Ligation of two adjacent EcoRI fragments of A . chroococcum yielded an intact nifA gene that activated expression of nifH-lac fusions and also restored MV3 to Nif+ . The four nfrX mutants were complemented by pLAFR1 cosmids pLV163 and pLC121 . The nfrX gene was subcloned from pLV163 and located within a 3.2 kb fragment . To determine whether nfrX might be found in other nitrogen-fixing organisms, DNA from 13 different species was hybridized to an nfrX probe . The failure to observe hybridization suggests that nfrX may be specific to nif regulation in Azotobacter.

Mol Microbiol, 1988 May, 2(3), 331 - 7
Transcription of a Rhizobium leguminosarum biovar phaseoli gene needed for melanin synthesis is activated by nifA of Rhizobium and Klebsiella pneumoniae; Hawkins FK et al.; The Rhizobium leguminosarum biovar phaseoli symbiotic plasmid pRP2JI carries a gene, melA, specifying the enzyme tyrosinase, which is responsible for the production of the pigment melanin in these bacteria . Transcription of melA is activated by the nifA gene of Rhizobium and, when the cloned melA gene is transferred to Escherichia coli, melA is expressed if the recipients contain nifA gene of Klebsiella pneumoniae . This nifA-dependent activation was temperature sensitive and required the ntrA gene . The cloned nifA gene of K . pneumoniae, when transferred to a nifA mutant of Rhizobium phaseoli biovar phaseoli, corrected the Mel- but not the Fix- phenotype . nifA of R . leguminosarum biovar phaseoli activated melA at higher levels in cells grown in low concentrations of oxygen . Also, nifA of R . leguminosarum biovar phaseoli activated nifH of K . pneumoniae in Escherichia coli cells grown in low-oxygen concentrations.

Proc Natl Acad Sci U S A, 1988 May, 85(9), 3062 - 5
Common regulatory elements control symbiotic and microaerobic induction of nifA in Rhizobium meliloti; Virts EL et al.; We have previously demonstrated that the nifA promoter (nifAp) of Rhizobium meliloti is inducible under microaerobic conditions in the absence of alfalfa . Here we show that microaerobic activation of nifAp involves both cis- and trans-acting regulatory controls identical to those used symbiotically . The start site for nifA mRNA synthesis was found to be the same during symbiosis and microaerobiosis, and a deletion analysis of nifAp demonstrated that DNA between positions -62 and -45 is essential for induction . Mutants isolated as being unable to induce nifA microaerobically also were found to be defective in symbiotic nitrogen fixation with alfalfa . Such mutants form nodules that are equivalent cytologically to those induced by nifA::Tn5 mutants . Genetic and structural studies have localized the mutations to a cluster of fix genes 200 kilobases distant from the nod-nif region on the pSym megaplasmid {Renalier, M.-H., Batut, J., Ghai, J., Terzaghi, B., Gherardi, M., David, M., Garnerone, A.-M., Vasse, J., Truchet, G., Huguet, T . & Boistard, P . (1987) J . Bacteriol . 169, 2231-2238}.

J Gen Microbiol, 1988 Apr, 134 ( Pt 4), 913 - 9
Symbiotic phenotypes of auxotrophic mutants of Rhizobium meliloti 104A14; Kerppola TK et al.; Auxotrophic mutants of Rhizobium meliloti 104A14 were isolated using nitrous acid mutagenesis followed by penicillin enrichment . Mutants in ornithine transcarbamylase, argininosuccinate synthetase or serine-glycine biosynthesis formed nitrogen-fixing (Fix-nodules on the roost of alfalfa (Medicago sativa) . Mutants with defects in ornithine, pyrimidine, purine, asparagine, leucine, methionine or tyrosine biosynthesis, in one-carbon metabolism or in carbamoylphosphate synthetase formed nodules but these nodules were unable to fix nitrogen . Prototrophic revertants were always Fix inverted question markPlasmids that would complement many of these auxotrophs were isolated by transduction with a P2 cosmid gene bank of R . meliloti 104A14 . These plasmids were then introduced into mutants of the same and different classes and the growth and symbiotic phenotypes of the new strains were determined . In all cases, complementation of the nutritional defect restored symbiotic nitrogen fixation.

J Gen Microbiol, 1988 Apr, 134 ( Pt 4), 931 - 42
Further analysis of nitrogen fixation (nif) genes in Azotobacter chroococcum: identification and expression in Klebsiella pneumoniae of nifS, nifV, nifM, and nifB genes and localization of nifE/N-, nifU-, nifA- and fixABC-like genes; Evans D et al.; The results presented extend previous investigations on the genetics of nitrogen fixation in Azotobacter chroococcum and indicate that nif- and fix-like DNA is located in at least five different regions of the genome . Region I contains functional copies of nifS,V and M, as well as nifH, D and K, all of which complemented mutants of Klebsiella pneumoniae . In addition, nifE- and/or nifN-like and nifU-like DNA is located in this region . The organization of the nif cluster in region I closely resembles that of K . pneumoniae . though spread over 22 kb as compared with 14 kb . Region II contains a functional nifB gene, which complemented a K . pneumoniae nifB mutant, and seems to be adjacent to ap nifA-like gene . Region III harbours nifH*, encoding a second nitrogenase Fe-protein . Region IV contains a reiteration of nifE- on and/or nifN-like sequences, and DNA homologous to Rhizobium meliloti fixABC is present in region V . The apparent complexity of nifDNA in A . chroococcum is probably related to the two systems for N2-fixation pr present in this organism.

Mol Plant Microbe Interact, 1988 Apr, 1(4), 161 - 8
Identification of a nodD-dependent locus in the Rhizobium strain NGR234 activated by phenolic factors secreted by soybeans and other legumes; Bassam BJ et al.; Transfer of the strain NGR234nodD 1 gene into the narrow host range R . trifolii strain ANU843 on either a 6.7-kb HindIII or 17-kb XhoI fragment broadens the host range of this bacterium to include the tropical legumes Vigna unguiculata, Glycine ussuriensis, Leucaena leucocephala, and siratro (Macroptilium atropurpureum) . Contrary to previous data (Bassam et al . 1986), mutagenesis of the 17-kb XhoI fragment with a mini-Mu lac transposon (Mu dII1734) showed that a functional nodD 1 gene was essential for extended host range . Gene expression studies using both Mu dII1734 fusions and a promoter-cloning vector indicated that several loci, including the nodD 1 gene, are constitutively expressed . No evidence was found for regulation of the strain NGR234 nodD 1 gene by its product . Another locus nod-81, was induced only in the presence of exudates from various plant species, including soybean (Glycine max) . Whereas the expression of nod-81 was dependent on the presence of a functional nodD 1 gene product, a regulatory nod-box DNA sequence was not detected 5' to this gene by using available oligonucleotide hybridization probes . The nod-81 locus was induced by genistein, daidzein, naringenin, and coumestrol from both cotyledon and root tissue of freshly germinated soybean seedlings . A broad spectrum of commercially available phenolic compounds stimulated induction of the nod-81 locus, including some that antagonize nod gene induction in other Rhizobium species . The nodD 1 gene product from strain NGR234 was shown to determine the spectrum of compounds that induce nod-81 expression.

J Gen Microbiol, 1988 Apr, 134 ( Pt 4), 921 - 9
Genetic analysis of carbamoylphosphate synthesis in Rhizobium meliloti 104A14; Kerppola TK et al.; We have previously isolated ineffective (Fix-) mutants of Rhizobium meliloti 104A14 requiring both arginine and uracil, and thus probably defective in carbamoylphosphate synthetase . We describe here the molecular and genetic analysis of the R . meliloti genes coding for carbamoylphosphate synthetase . Plasmids that complement the mutations were isolated from a R . meliloti gene bank . Restriction analysis of these plasmids indicated that complementation involved two unlinked regions of the R . meliloti chromosome, carA and carB . Genetic complementation between the plasmids and mutants demonstrated a single complementation group for carA, but two overlapping complementation groups for carB . The cloned R . meliloti genes hybridize to the corresponding E . coli carA and carB genes which encode the two subunits of carbamoylphosphate synthetase . Transposon Tn5 mutagenesis was used to localize the carA and carB genes on the cloned R . meliloti DNA . The cloned R . meliloti carA and carB genes were unable to complement E . coli carA or carB mutants alone or in combination . We speculate on the mechanism of the unusual pattern of genetic complementation at the R . meliloti carB locus.

Mol Gen Genet, 1988 Apr, 212(1), 27 - 37
Genetic characterization and sequence analysis of the duplicated nifA/nifB gene region of Rhodobacter capsulatus; Masepohl B et al.; A DNA region showing homology to Klebsiella pneumoniae nifA and nifB is duplicated in Rhodobacter capsulatus . The two copies of this region are called nifA/nifB copy I and nifA/nifB copy II . Deletion mutagenesis demonstrated that either of the two copies is sufficient for growth in nitrogen-free medium . In contrast, a double deletion mutant turned out to be deficient in nitrogen fixation . The complete nucleotide sequence of a 4838 bp fragment containing nifA/nifB copy I was determined . Two open reading frames coding for a 59,653 (NifA) and a 49,453 (NifB) dalton protein could be detected . Comparison of the amino acid sequences revealed that the R . capsulatus nifA and nifB gene products are more closely related to the NifA and NifB proteins of Rhizobium meliloti and Rhizobium leguminosarum than to those of K . pneumoniae . A rho-independent termination signal and a typical nif promoter region containing a putative NifA binding site and a consensus nif promoter are located within the region between the R . capsulatus nifA and nifB genes . The nifB sequence is followed by an open reading frame (ORF1) coding for a 27721 dalton protein in nifA/nifB copy I . DNA sequence analysis of nifA/nifB copy II showed that both copies differ in the DNA region downstream of nifB and in the noncoding sequence in front of nifA . All other regions compared, i.e . the 5' part of nifA, the intergenic region and the 3' part of nifB, are identical in both copies.

J Bacteriol, 1988 Apr, 170(4), 1848 - 57
Induction of pathogenic-like responses in the legume Macroptilium atropurpureum by a transposon-induced mutant of the fast-growing, broad-host-range Rhizobium strain NGR234; Djordjevic SP et al.; 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.

J Bacteriol, 1988 Apr, 170(4), 1475 - 87
Nucleotide sequence and genetic analysis of the nifB-nifQ region from Azotobacter vinelandii; Joerger RD et al.; A 3.8-kilobase-pair EcoRI fragment which corrects the mutations carried by the NifB- Azotobacter vinelandii strains CA30 and UW45 was cloned, and its nucleotide sequence was determined . Four complete open reading frames (ORFs) and two partial ORFs were found . The translation product of the first partial ORF is the carboxy-terminal end of a protein homologous to the nifA gene product from Klebsiella pneumoniae . A 285-base-pair sequence containing a potential nif promoter and nif regulatory sites separates this nifA gene from the first complete ORF which encodes a protein homologous to nifB gene products from K . pneumoniae and Rhizobium species . The Tn5 insertion in strain CA30 and the nif-45 mutation of strain UW45 are located within this nifB gene . The ORF downstream from nifB predicts an amino acid sequence with a cysteine residue pattern that is characteristic of ferredoxins . No similarities were found between the translation product of the third complete ORF and those of nif genes from other organisms . At the carboxy-terminal end of the predicted translation product of the fourth complete ORF, 30 of 60 amino acid residues were identical with the sequence of the nifQ gene product from K . pneumoniae . The partial ORF located at the end of the fragment encodes the N-terminal part of a potential protein with an unknown function . Northern (RNA) blot analysis indicated that transcripts from the region containing the four complete ORFs were NH4+ repressible and that the transcription products were identical in cells derepressed under conditions of Mo sufficiency or Mo deficiency or in the presence of vanadium . In contrast to the NifB- strain CA30, which is Nif- under all conditions, mutants that carry mutations affecting the C-terminal end of nifB or genes located immediately downstream from nifB, grew under all N2-fixing conditions . However, in the presence of Mo, most of the strains required 1,000 times the amount of molybdate that is sufficient for maximal growth of the wild-type strain CA under N2-fixing conditions . Growth data from strain CA37, which carries a Kanr insertion in nifQ, indicate that nifQ in A . vinelandii is not required for N2 fixation in the presence of V2O5 or under Mo-deficient conditions . Growth studies and acetylene reduction assays performed on two nifEN deletion strains showed that nifE and nifN are required for N2 fixation under Mo sufficiency, as previously observed (K . E . Brigle, M . C . Weiss, W . E . Newton, and D . R . Dean, J . Bacteriol . 169:1547-1553, 1987), but not under conditions of Mo deficiency or in the presence of 50 nM V2O5.

Nucleic Acids Res, 1988 Mar 25, 16(5), 2207 - 24
Essential and non-essential domains in the Bradyrhizobium japonicum NifA protein: identification of indispensable cysteine residues potentially involved in redox reactivity and/or metal binding; Fischer HM et al.; The amino acid sequence of the Bradyrhizobium japonicum nitrogen fixation regulatory protein NifA, as derived from the nucleotide sequence of the nifA gene, was aligned to the corresponding protein sequences from Klebsiella pneumoniae, Rhizobium meliloti and Rhizobium leguminosarum biovar viciae . High conservation was found in the central domain and in the COOH-terminal, putative DNA binding domain, whereas very little homology was present within the first 250 amino acids from the NH2-terminus . Upon deletion of the first 218 amino acids (37% of the protein) and expression of the remainder as a Cat'-'NifA hybrid protein, a fully active, nif-specific transcriptional activator protein was obtained which also retained oxygen sensitivity, a characteristic property of the wild-type B . japonicum NifA protein . In contrast, an unaltered COOH-terminal domain was required for an active NifA protein . Between the central and the DNA binding domains, a so-called interdomain linker region was identified which was conserved in all rhizobial species but missing in the K.pneumoniae NifA protein . Two conserved cysteine residues in this region were changed to serine residues, by oligonucleotide-directed mutagenesis . This resulted in absolutely inactive NifA mutant proteins . Similar null phenotypes were obtained by altering two closely adjacent cysteine residues in the central domain to serine residues . Nif gene activation in vivo by the B.japonicum NifA protein, but not by the K.pneumoniae NifA protein, was sensitive to treatment with chelating agents, and this inhibition could be overcome by the addition of divalent metal ions . On the basis of these observations and previous data on oxygen sensitivity we raise the hypothesis that at least some, if not all, of the four essential cysteine residues may be involved in oxygen reactivity or metal binding or both.

J Bacteriol, 1988 Mar, 170(3), 1191 - 6
Genomic instability in Rhizobium phaseoli; Flores M et al.; Experience from different laboratories indicates that Rhizobium strains can generate variability in regard to some phenotypic characteristics such as colony morphology or symbiotic properties . On the other hand, several reports suggest that under certain stress conditions or genetic manipulations Rhizobium cells can present genomic rearrangements . In search of frequent genomic rearrangements, we analyzed three Rhizobium strains under laboratory conditions that are not considered to cause stress in bacterial populations . DNAs from direct descendants of a single cell were analyzed in regard to the hybridization patterns obtained, using as probes different recombinant plasmids or cosmids; while most of the probes utilized did not show differences in the hybridization patterns, some of them revealed the occurrence of frequent genomic rearrangements . The implications and possible biological significance of these observations are discussed.

Genes Dev, 1988 Mar, 2(3), 282 - 93
Specific binding of proteins from Rhizobium meliloti cell-free extracts containing NodD to DNA sequences upstream of inducible nodulation genes; Fisher RF et al.; Nodulation (nod) genes in Rhizobium meliloti are transcriptionally induced by flavonoid signal molecules, such as luteolin, produced by its symbiotic host plant, alfalfa . This induction depends on expression of nodD . Upstream of three inducible nod gene clusters, nodABC, nodFE, and nodH, is a highly conserved sequence referred to as a 'nod box.' The upstream sequences have no other obvious similarity . We have found that DNA fragments containing the regions upstream of all three inducible transcripts show altered electrophoretic mobility when treated with R . meliloti extracts . The ability of the extracts to interact specifically with these DNAs correlated with the genetic dosage of nodD1 or nodD3 and with the presence and concentration of the nodD1 or nodD3 protein (NodD1 or NodD3) in the extracts . Antiserum specific to NodD was used to construct an immunoaffinity column that permitted a substantial purification of NodD1; this preparation of NodD1 also displayed specific binding to restriction fragments containing DNA sequences found upstream of inducible nod genes . In addition, NodD-specific antiserum removed the specific DNA-binding activity from total Rhizobium cell extracts . The interaction of total extracts and of partially purified NodD protein with nod promoter sequences was competitive with an oligonucleotide representing the 3' 25-bp portion of the nod box . The interaction of R . meliloti extracts and NodD1 protein with nod gene upstream regions occurred independently of exposure of cells or extracts to flavone inducer.

Mol Microbiol, 1988 Mar, 2(2), 173 - 83
Characterization of the Rhizobium leguminosarum genes nodLMN involved in efficient host-specific nodulation; Surin BP et al.; Three nodulation genes, nodL, nodM and nodN, were isolated from Rhizobium leguminosarum and their DNA sequences were determined . The three genes are in the same orientation as the previously described nodFE genes and the predicted molecular weights of their products are 20,105 (nodL), 65,795 (nodM) and 18,031 (nodN) . Analysis of gene regulation using operon fusions showed that nodL, nodM and nodN are induced in response to flavanone molecules and that this induction is nodD-dependent . In addition, it was shown that the nodM and nodN genes are in one operon which is preceded by a conserved 'nod-box' sequence, whereas the nodL gene is in the same operon as the nodFE genes . DNA hybridizations using specific gene probes showed that strongly homologous genes are present in Rhizobium trifolii but not Rhizobium meliloti or Bradyrhizobium japonicum . A mutation within nodL strongly reduced nodulation of peas, Lens and Lathyrus but had little effect on nodulation of Vicia species . A slight reduction in nodulation of Vicia hirsuta was observed with strains carrying mutations in nodM or nodN.

J Bacteriol, 1988 Mar, 170(3), 1153 - 61
A plasmid of Rhizobium meliloti 41 encodes catabolism of two compounds from root exudate of Calystegium sepium; Tepfer D et al.; Our objectives were to identify substances produced by plant roots that might act as nutritional mediators of specific plant-bacterium relationships and to delineate the bacterial genes responsible for catabolizing these substances . We discovered new compounds, which we call calystegins, that have the characteristics of nutritional mediators . They were detected in only 3 of 105 species of higher plants examined: Calystegia sepium, Convolvulus arvensis (both of the Convolvulaceae family), and Atropa belladonna . Calystegins are abundant in organs in contact with the rhizosphere and are not found, or are observed only in small quantities, in aerial plant parts . Just as the synthesis of calystegins is infrequent in the plant kingdom, their catabolism is rare among rhizosphere bacteria that associate with plants and influence their growth . Of 42 such bacteria tested, only one (Rhizobium meliloti 41) was able to catabolize calystegins and use them as a sole source of carbon and nitrogen . The calystegin catabolism gene(s) (cac) in this strain is located on a self-transmissible plasmid (pRme41a), which is not essential to nitrogen-fixing symbiosis with legumes . We suggest that under natural conditions calystegins provide an exclusive carbon and nitrogen source to rhizosphere bacteria which are able to catabolize these compounds . Calystegins (and the corresponding microbial catabolic genes) might be used to analyze and possibly modify rhizosphere ecology.

Mol Plant Microbe Interact, 1988 Mar, 1(3), 145 - 9
Identification and characterization of the nodD gene in Rhizobium leguminosarum strain 1001; Squartini A et al.; A gene library of the symbiotic 240-kb plasmid of Rhizobium leguminosarum strain 1001 was constructed in pUC18 . The clones showing homology with a 6.6-kb fragment containing nodEFDABC from the Sym plasmid pRLlJI were detected by colony hybridization . Additional probes from the symbiotic region of pRLlJI were used to localize the corresponding genes on the map of pRle1001a . The relative positions of nod and nif gene clusters are different than those of pRLlJI . A comparison of the amino acid sequence for NodD from pRle1001a with NodD proteins from other Rhizobium species showed a high degree of sequence conservation at the amino terminus of the protein.

J Cell Biol, 1988 Mar, 106(3), 597 - 607
Rhizobium fix genes mediate at least two communication steps in symbiotic nodule development; Putnoky P et al.; To identify bacterial genes involved in symbiotic nodule development, ineffective nodules of alfalfa (Medicago sativa) induced by 64 different Fix-mutants of Rhizobium meliloti were characterized by assaying for symbiotic gene expression and by morphological studies . The expression of leghemoglobin and nodulin-25 genes from alfalfa and of the nifHD genes from R . meliloti were monitored by hybridizing the appropriate DNA probes to RNA samples prepared from nodules . The mutants were accordingly divided into three groups . In group I none of the genes were expressed, in group II only the plant genes were expressed and in group III all three genes were transcribed . Light and electron microscopical analysis of nodules revealed that nodule development was halted at different stages in nodules induced by different group I mutants . In most cases nodules were empty lacking infection threads and bacteroids or nodules contained infection threads and a few released bacteroids . In nodules induced by a third mutant class bacteria were released into the host cells, however the formation of the peribacteroid membrane was not normal . On this basis we suggest that peribacteroid membrane formation precedes leghemoglobin and nodulin-25 induction, moreover, after induction of nodulation by the nod genes at least two communication steps between the bacteria and the host plants are necessary for the development of the mature nodule . By complementing each mutant of group I with a genomic R . meliloti library made in pLAFRl, four new fix loci were identified, indicating that several bacterial genes are involved in late nodule development.

J Bacteriol, 1988 Feb, 170(2), 1003 - 6
All nod genes of Rhizobium meliloti are involved in alfalfa nodulation by exo mutants; Klein S et al.; 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.

Eur J Biochem, 1988 Feb 1, 171(3), 515 - 22
Identification of cytoplasmic nodule-associated forms of malate dehydrogenase involved in the symbiosis between Rhizobium leguminosarum and Pisum sativum; Appels MA et al.; The malate dehydrogenase activity (EC 1.1.1.37), present in the cytoplasm of Pisum sativum root nodules, can be separated by ion-exchange chromatography into four different fractions . Malate dehydrogenase activity present in the cytoplasm of roots elutes mainly as a single peak . During nodule development an increase in malate dehydrogenase activity per gram of material was observed . This increase occurred concomitantly with the increase in nitrogenase activity . The kinetic properties of the separated malate dehydrogenases of root nodule cytoplasm and root cytoplasm were studied . The Km values for malate (2.6 mM), NAD+ (27 microM), oxaloacetate (18 microM) and NADH (13 microM) of the dominant form of the root nodule cytoplasm are much lower than those of the dominant malate dehydrogenase root form (64 mM, 4.4 mM, 89 microM and 70 microM respectively) . Binding of malate by the enzyme-NADH complex from root nodules results in an abortive complex, thereby blocking the further reduction of oxaloacetate by NADH . The dominant root malate dehydrogenase does not form the abortive complex . From the kinetic data it is concluded, first, that the root nodule forms of the enzyme are capable of catalysing at a high rate the reduction of oxaloacetate, to meet the demands for malate governed by the bacteroid and the infected plant cell . The second conclusion, drawn from the kinetic data, is that under physiological conditions the conversion of oxaloacetate can be controlled just by the malate concentration . Consequently the major root nodule forms of malate dehydrogenase are able to allow a high flux of malate production from oxaloacetate but also to establish a sufficient oxaloacetate concentration necessary for the assimilation and transport of fixed nitrogen.

Mol Plant Microbe Interact, 1988 Feb, 1(2), 94 - 100
Interaction of nod and exo Rhizobium meliloti in alfalfa nodulation; Klein S et al.; Among the genes of Rhizobium meliloti SU47 that affect nitrogen-fixing symbiosis with alfalfa are nod genes, in which mutations block nodule induction, and exo genes, in which mutations allow nodule formation but block rhizobial exopolysaccharide production as well as nodule invasion and nitrogen fixation . To investigate whether an exo+ bacterium can "help" (that is, reverse the symbiotic defect of) an exo mutant in trans, we have coinoculated alfalfa with pairs of rhizobia of different genotypes . Coinoculant genotypes were chosen so that the exo+ helper strain was nif while the exo "indicator" strain was nif+, and thus any fixation observed was carried out by the exo coinoculant . We find that a nod exo+ coinoculant can help an exo mutant both to invade nodules and to fix nitrogen . However, a nod+ exo+ coinoculant cannot help an exo mutant: Few exo bacteria are recovered from nodules, some bacteroids differentiate into bizarre aberrant forms, and the nodules fail to fix nitrogen . In a triple coinoculation, the effect of nod+ helper supersedes that of nod helper . Implications of these results for interaction of nod and exo gene products are discussed.

Mol Plant Microbe Interact, 1988 Feb, 1(2), 66 - 74
Developmental regulation of nodule-specific genes in alfalfa root nodules; Dunn K et al.; We have cloned alfalfa nodule-specific cDNAs that code for leghemoglobin (Lb), glutamine synthetase (GS), and three unidentified nodulins . Hybrid-select translation of nodule RNA followed by 2-D gel electrophoresis showed that the Lb-specific cDNA corresponded to at least four Lb species of 12 kDa . One of the unidentified cDNA clones (N-32/34) corresponded to at least five polypeptides of 32-34 kDa; a second unidentified cDNA clone (N-14) corresponded to an individual polypeptide of 14 kDa . The in vitro translation product(s) of the RNA hybrid selected by the third unidentified cDNA clone (N-22) formed a single band at 22 kDa on a one-dimensional gel . Northern and dot blot analyses of RNA isolated from wild-type nodules and from defective nodules elicited by a variety of Rhizobium meliloti mutants showed that 1) RNAs corresponding to the Lb, nodule-specific GS, and three unidentified nodulins were coordinately expressed during the course of nodule development, and 2) all five nodulins were expressed in Fix- nodules that contained infection threads and bacteroids but were not expressed in nodules that lacked infection threads and intracellular rhizobia.

J Bacteriol, 1988 Feb, 170(2), 985 - 8
Introduction of the Escherichia coli gdhA gene into Rhizobium phaseoli: effect on nitrogen fixation; Bravo A et al.; Rhizobium phaseoli lacks glutamate dehydrogenase (GDH) and assimilates ammonium by the glutamine synthetase-glutamate synthase pathway . A strain of R . phaseoli harboring the Escherichia coli GDH structural gene (gdhA) was constructed . GDH activity was expressed in R . phaseoli in the free-living state and in symbiosis . Nodules with bacteroids that expressed GDH activity had severe impairment of nitrogen fixation . Also, R . phaseoli cells that lost GDH activity and assimilated ammonium by the glutamine synthetase-glutamate synthase pathway preferentially nodulated Phaseolus vulgaris.

J Bacteriol, 1988 Feb, 170(2), 980 - 4
Ammonium assimilation in Rhizobium phaseoli by the glutamine synthetase-glutamate synthase pathway; Bravo A et al.; Evidence from in vitro and in vivo studies showed that in Rhizobium phaseoli ammonium is assimilated by the glutamine synthetase (GS)-glutamate synthase NADPH pathway . No glutamate dehydrogenase activity was detected . R . phaseoli has two GS enzymes, as do other rhizobia . The two GS activities are regulated on the basis of the requirement for low (GSI) or high (GSII) ammonium assimilation . When the 2-oxoglutarate/glutamine ratio decreases, GSI is adenylylated . When GSI is inactivated, GSII is induced . However, induction of GSII activity varied depending on the rate of change of this ratio . GSII was inactivated after the addition of high ammonium concentrations, when the 2-oxoglutarate/glutamine ratio decreased rapidly . Ammonium inactivation resulted in alteration of the catalytic and physical properties of GSII . GSII inactivation was not relieved by shifting of the cultures to glutamate . After GSII inactivation, ammonium was excreted into the medium . Glutamate synthase activity was inhibited by some organic acids and repressed when cells were grown with glutamate as the nitrogen source.

J Bacteriol, 1988 Feb, 170(2), 927 - 34
Rhizobium meliloti genes required for C4-dicarboxylate transport and symbiotic nitrogen fixation are located on a megaplasmid; Watson RJ et al.; A mutant of Rhizobium meliloti unable to transport C4 dicarboxylates (dct) was isolated after Tn5 mutagenesis . The mutant, 4F6, could not grow on aspartate or the tricarboxylic acid cycle intermediates succinate, fumarate, or malate . It produced symbiotically ineffective nodules on Medicago sativa in which bacteroids appeared normal, but the symbiotic zone was reduced and the plant cells contained numerous starch granules at their peripheries . Cosmids containing the dct region were obtained by selecting those which restored the ability of 4F6 to grow on succinate . The Tn5 insertion in 4F6 was found to be within a 5.9-kilobase (kb) EcoRI fragment common to the complementing cosmids . Site-specific Tn5-mutagenesis revealed dct genes in a segment of DNA about 4 kb in size extending from within the 5.9-kb EcoRI fragment into an adjacent 2.9-kb EcoRI fragment . The 4F6 mutation was found to be in a complementation group in which mutations yielded a Fix- phenotype, whereas other dct mutations in the region resulted in mutants which produced effective nodules in most, although not all, plant tests (partially Fix-) . The dct region was found to be located on a megaplasmid known to carry genes required for exopolysaccharide production.

Carbohydr Res, 1988 Jan 15, 172(1), 97 - 112
The complete structure of the trifoliin A lectin-binding capsular polysaccharide of Rhizobium trifolii 843; Hollingsworth RI et al.; The complete structure of the acidic, extracellular, capsular polysaccharide of Rhizobium trifolii 843 has been elucidated by a combination of chemical, enzymic, and spectroscopic methods, confirming an earlier proposed sugar sequence and assigning the locations of the acyl substituents . The polysaccharide was depolymerized by a lyase into octasaccharide units which were uniform in carbohydrate composition and linkage . These units also contained a uniform distribution of acetyl and pyruvic acetal {O-(1-carboxyethylidene)} groups, and half of them were further acylated with D-3-hydroxybutanoyl groups . A much smaller proportion (less than 5%) of the oligomers was further acylated by a second D-3-hydroxybutanoyl group . The locations of the subtituents were determined chemically and by J-correlated, 1H-n.m.r . spectroscopy, proton nuclear Overhauser effect (n.O.e.) measurements, double-rd structure of the carbohydrate chain were determined by methylation analysis using g.l.c.-m.s . fast-atom-bombardment mass spectrometry, and n.m.r . studies on the reduced, deacylated oligomer . Structural studies were supplemented by n.m.r . analyses on the original polymer . The oligosaccharides were found to be branched octasaccharides with four sugar residues in each branch, and the carbohydrate sequence agreed well with that expected from earlier work . In the abbreviated sequence and structure (1a), the sugar residues are labelled "a" through "h" . The main chain (a-d) is composed of a 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid group (a) that is linked to O-4 of a 3-O-acetyl-D-glucosyluronic acid residue (b) which is beta-linked to O-4 of a D-glucosyl residue (c) . Residue c is beta-linked to O-4 of the branching D-glucose residue (d) . The side chain consists of a substituted D-galactosyl group (h) which is beta-linked to O-3 of residue 9 of a beta-(1----4)-linked D-glucose trisaccharide (fragment e-f-g) . The reducing end of the resulting tetrasaccharide (e-f-g-h) is beta-linked to O-6 of the branching D-glucose residue (d) . In the native polymer, this branching residue is alpha-linked to O-4 of the modified D-glucuronic acid residue (a) which is the unsaturated sugar in the oligomer . A small proportion of the O-2 atoms of the acetylated D-glucosyluronic acid residues is acetylated because of ester migration . The two terminal sugars (g and h) of the branch chain bear 4,6-O-(1-carboxyethylidene) groups . The D-galactosyl groups of half of the oligomers are acylated by D-3-hydroxybutanoyl groups at O-3.(ABSTRACT TRUNCATED AT 400 WORDS)

Nature, 1988 Jan 14, 331(6152), 178 - 80
Functioning haemoglobin genes in non-nodulating plants; Bogusz D et al.; Haemoglobin has previously been recorded in plants only in the nitrogen-fixing nodules formed by symbiotic association between Rhizobium or Frankia and legume or non-legume hosts . Structural similarities amongst these and animal haemoglobins at the protein and gene level suggested a common evolutionary origin . This suggests that haemoglobin genes, inherited from an ancestor common to plants and animals, might be present in all plants . We report here the isolation of a haemoglobin gene from Trema tomentosa, a non-nodulating relative of Parasponia (Ulmaceae) . The gene has three introns located at positions identical to those in the haemoglobin genes of nodulating plant species, strengthening the case for a common origin of all plant haemoglobin genes . The data argue strongly against horizontal haemoglobin gene transfer from animals to plants . The Trema gene has a tissue-specific pattern of transcription and translation, producing monomeric haemoglobin in Trema roots . We have also found that the Parasponia haemoglobin gene is transcribed in roots of non-nodulated plants . These results suggest that haemoglobin has a role in the respiratory metabolism of root cells of all plant species . We propose that its special role in nitrogen-fixing nodules has required adaptation of the haemoglobin-gene regulation pathway, to give high expression in the specialized environment of the nodule.

Nucleic Acids Res, 1988 Jan 11, 16(1), 39 - 50
Root nodule specific gene regulation: analysis of the soybean nodulin N23 gene promoter in heterologous symbiotic systems; Jorgensen JE et al.; The nodulin N23 gene promoter was analysed in transgenic plants using the chloramphenicol acetyltransferase (CAT) coding sequence as a reporter . A 5' flanking region of less than 1 kb was sufficient for the organ-specific expression of a chimeric N23-CAT-3'lbc3 gene in root nodules formed on Lotus corniculatus and Trifolium repens after infection by their respective Rhizobium symbionts . Expression was regulated at the level of RNA in both species of transgenic plants . Promoter deletion analysis defined the 5' region required for high level expression and delimited two putative regulatory sequences involved in positive control of the N23 gene in L . corniculatus.

J Bacteriol, 1988 Jan, 170(1), 184 - 9
Dicarboxylic acid transport in Bradyrhizobium japonicum: use of Rhizobium meliloti dct gene(s) to enhance nitrogen fixation; Birkenhead K et al.; A recombinant plasmid encoding Rhizobium meliloti sequences involved in dicarboxylic acid transport (plasmid pRK290:4:46) (E . Bolton, B . Higgisson, A . Harrington, and F . O'Gara, Arch . Microbiol . 144:142-146, 1986) was used to study the relationship between dicarboxylic acid transport and nitrogen fixation in Bradyrhizobium japonicum . The expression of the dct sequences on plasmid pRK290:4:46 in B . japonicum CJ1 resulted in increased growth rates in media containing dicarboxylic acids as the sole source of carbon . In addition, strain CJ1(pRK290:4:46) exhibited enhanced succinate uptake activity when grown on dicarboxylic acids under aerobic conditions . Under free-living nitrogen-fixing conditions, strain CJ1(pRK290:4:46) exhibited higher nitrogenase (acetylene reduction) activity compared with that of the wild-type strain . This increase in nitrogenase activity also correlated with an enhanced dicarboxylic acid uptake rate under these microaerobic conditions . The regulation of dicarboxylic acid transport by factors such as metabolic inhibitors and the presence of additional carbon sources was similar in both the wild-type and the engineered strains . The implications of increasing nitrogenase activity through alterations in the dicarboxylic acid transport system are discussed.

Acta Microbiol Hung, 1988, 35(2), 89 - 92
Selective quantitative determination of tobramycin from fermentation broth; Kiss GB et al.; A derivative of Rhizobium meliloti 41 was constructed (strain GY654) which was resistant to apramycin and kanamycin but remained sensitive to tobramycin . Strain GY654 selectively measures tobramycin and 6"-O-carbamoyltobramycin in the presence of apramycin, kanamycin and 6"-O-carbamoylkanamycin B, therefore it is suitable for the rapid quantitation of 6"-O-carbamoyltobramycin and tobramycin in fermentation broths of Streptomyces tenebrarius and solvents containing antibiotic mixtures.

Acta Biochim Pol, 1988, 35(1), 39 - 50
Isolation and characterization of root nodule proteins from lupin; Strozycki P et al.; A group of root nodule-specific plant proteins (nodulins) has been isolated from yellow lupin (Lupinus luteus) by immunoaffinity chromatography . The cytoplasmic nodule protein extract was initially enriched in nodulins on a column with immobilized IgG fraction . It was then purified by chromatography on Sepharose 4B - bound IgG against uninfected root proteins and finally on Sepharose 4B - bound IgG against Rhizobium lupini proteins . Rocket immunoelectrophoresis showed that the nodulin preparation did not react with antibodies against root or bacterial proteins . SDS gel electrophoresis of lupin nodulins revealed at least 23 polypeptides ranging in Mr, from 7,000 to 70,000, probably representing protein subunits.

Microbiol Sci, 1988 Jan, 5(1), 9 - 12
The genus Frankia: actinomycete symbionts of plants; Benson DR; Biological N2 fixation is performed most effectively by prokaryotic diazotrophs when in mutualistic symbioses with higher plants . The most intensively studied N2-fixing symbioses involve leguminous plants and rhizobia . However, Frankia actinomycetes have attracted attention recently because they form root nodules on a broad range of non-legumes and because such nodules fix N2 as effectively as rhizobial nodules . Since the Frankia symbiosis results from an actinomycetic invasion of plant roots, it has been termed the 'actinorhizal symbiosis'.

Biofactors, 1988 Jan, 1(1), 3 - 10
Flavones and isoflavones as inducing substances of legume nodulation; Rolfe BG; Rhizobia are soil bacteria that can form symbiotic associations with leguminous plants leading to the fixation of atmospheric nitrogen to ammonia which the plant can use . This is an interaction which involves the exchange of many signals between the plant and the bacterium . To start this interaction, rhizobia have adapted to use flavonoid compounds, released by the plant root, as part of a regulatory system to initiate the transcription of their infection (nodulation, nod) genes . The development of an assay system for the detection of plant-derived stimulatory biofactors has now led to the isolation and identification of the compounds which are responsible for the activation of the nod genes . Stimulatory compounds now have been isolated from plants: from clovers, 7,4'-dihydroxyflavone; from alfalfa, luteolin; from peas, apigenin; and from soybeans, the isoflavones daidzein and genistein . These hydroxylated flavonoid compounds are derived from the phenylpropanoid pathways which are responsible for the synthesis of many important plant phenolic compounds, including the phytoalexin molecules which are thought to be involved in plant defence systems . The current hypothesis on the regulation of the nodulation genes in Rhizobium strains is that the gene product of the regulatory nod gene, nodD, requires the presence of the plant signals to convert it to an active form . This altered NodD protein then induces the expression of the other nodulation genes . This bacterium, induced by plant biofactors, now is able to infect legume root hairs.

Biofactors, 1988 Jan, 1(1), 11 - 6
Nickel as a micronutrient element for plants; Dalton DA et al.; The detrimental effects of excessive Ni on plant growth have been well known for many years . More recent evidence indicates that Ni is required in small amounts for normal plant growth and development . Ni is an essential component of urease in plants and microorganisms . A deficiency of Ni in plants is reported to result in necrotic lesions in leaves in response to toxic accumulations of urea . Urease plays an essential role in mobilization of nitrogenous compounds in plants, a process that is especially important during seed germination and fruit formation when protein reserves are degraded into amino acids . Arginine, an abundant amino acid in plants, when degraded produces urea as a product and urease is needed for urea utilization . Theories of urea formation during allantoin degradation in Glycine max have been recently refuted . In G . max ureides apparently are metabolized via an amidohydrolase reaction with subsequent degradation of ureidoglycine, yielding glyoxylate, NH+4 and CO2 . No evidence is available for the formation of urea in this pathway . Nitrogen-fixing symbionts, such as Rhizobium and Bradyrhizobium, contain two known Ni enzymes: urease and hydrogenase . Optimum growth of nodulated legumes and actinorhizal plants may depend on an adequate supply of Ni to meet the requirements of the Ni-requiring enzymes in host plants and endophytes . The seeds of severely Ni-deficient Hordeum are completely inviable, thus providing conclusive evidence for the essentiality of Ni for this species . The evidence indicates that Ni must be added to the list of micronutrient elements generally required by plants.

Arch Microbiol, 1988, 150(4), 320 - 5
Factors affecting the survival and growth of bacteria introduced into lake water; Scheuerman PR et al.; The populations of Pseudomonas sp . B4, Escherichia coli, Klebsiella pneumoniae, Micrococcus flavus, and Rhizobium leguminosarum biovar phaseoli declined rapidly in lake water . The initially rapid decline of the two pseudomonads and R . phaseoli was followed by a period of slow loss of viability, but viable cells of the other species were not found after 10 days . The rapid initial phase of decline was not a result of Bdellovibrio spp., bacteriophages, or toxins in the water since Bdellovibrio spp . were not present and passage of the lake water through filters that should not have removed bacteriophages or soluble toxins led to the elimination of the rapid phase of decline . The addition of 250 micrograms of cycloheximide and 30 micrograms of nystatin per ml eliminated viable protozoa form the lake water, and the population of Pseudomonas sp . B4 did not fall and the decline of E . coli and K . pneumoniae was delayed or slowed under these conditions . Pseudomonas sp . L2 proliferated rapidly in lake water amended with glucose, phosphate, and NH4NO3, but its numbers subsequently fell abruptly; however, in water amended with cycloheximide and nystatin, which killed indigenous protozoa, the population density was higher and the fall in numbers was delayed . Of the nutrients, the chief response was to carbon, but when glucose was added, phosphorus and nitrogen stimulated growth further . Removing other bacteria by filtering the lake water before inoculation with Pseudomonas sp . L2 suggested that competition reduced the extent of response of the pseudomonad to added nutrients.(ABSTRACT TRUNCATED AT 250 WORDS)

Acta Biochim Pol, 1988, 35(2), 119 - 30
Siderophore containing 2,3-dihydroxybenzoic acid and threonine formed by Rhizobium trifolli; Skorupska A et al.; An iron-binding compound was isolated from ethyl acetate extract of culture supernatant fluid of Rhizobium trifolii AR6 and was purified by iron-exchange chromatography . The compound was characterized by UV and IR . It contained 2,3-dihydroxy-benzoic acid and threonine and was accumulated during stationary phase of growth in iron-deficient media . Synthesis of the siderophore was repressed by FeCl3 . In iron limited medium the compound promoted growth of R . trifolii strains.

J Gen Microbiol, 1988 Jan, 134 ( Pt 1), 113 - 21
Distribution of insertion sequence ISRm1 in Rhizobium meliloti and other gram-negative bacteria; Wheatcroft R et al.; An internal 0.9 kb segment of Rhizobium meliloti insertion sequence ISRm1 was used as a probe to determine the distribution of ISRm1 in strains of R . meliloti and other Gram-negative bacteria . The insertion sequence was detected in 80% (12/15) of R . meliloti strains from different parts of the world . Its copy number ranged from one to at least eleven . The ISRm1 copies detected showed variation in their internal restriction sites and their degree of homology to the probe . ISRm1 was found in a variety of genomic restriction fragments, and was detected in plasmids, including the nod and exo megaplasmids of R . meliloti . Other rhizobia found to contain ISRm1 were a strain of R . leguminosarum biovar phaseoli and two Rhizobium isolates capable of nodulating both Medicago sativa and Phaseolus vulgaris . It was also found in a diazotrophic soil bacterium isolated from the roots of wetland rice.

J Bacteriol, 1988 Jan, 170(1), 474 - 7
Genetic and physical analyses of group E exo- mutants of Rhizobium meliloti; Finan TM; Mutants of Rhizobium meliloti which are deficient in exopolysaccharide synthesis have been classified into six different genetic groups (A through F) (J . A . Leigh, E . R . Signer, and G . C . Walker, Proc . Natl . Acad . Sci . USA 82:6231-6235, 1985) . Using physical and genetic techniques, we have demonstrated that the group E Exo- mutants carry deletions in the exoA-exoB region of the megaplasmid pRmeSU47b . We have constructed strains carrying defined deletions which remove up to 200 kilobases of pRmeSU47b, including the exoA-exoB region . These derivatives have the same phenotypes as do the group E mutants.

J Bacteriol, 1988 Jan, 170(1), 12 - 20
Rhizobium japonicum USDA 191 has two nodD genes that differ in primary structure and function; Appelbaum ER et al.; Several Rhizobium genes (designated nod genes) are involved in early steps in nodule formation . Here we present the results of DNA sequence and functional analysis of two nodD genes from the symbiotic plasmid of USDA 191, a fast-growing strain that forms nitrogen-fixing nodules on soybeans . Both genes encoded full-length nodD-related polypeptides, which were 69% homologous to each other . One of these genes, nodD1, complemented a Rhizobium trifolii nodD::Tn5 mutant for clover nodulation; the other gene, nodD2, did not . The nodD1 coding region was preceded by a conserved DNA sequence previously noted in other rhizobia, but no such sequence was found in front of nodD2 . Plants inoculated with a nodD1 insertion mutant appeared to be nitrogen starved and had a greatly reduced nodule number . Plants inoculated with a nodD2 mutant had a partially nitrogen-starved appearance and normal nodule number, were slightly delayed in nodule formation, and formed nodules that contained reduced levels of nodulin-35 and had fewer bacteroids per infected plant cell . Thus, both of these genes are involved in symbiosis . USDA 191 carrying extra copies of nodD2 on a plasmid vector had an altered colony morphology that suggested inhibition of exopolysaccharide synthesis . The predicted gene products of nodD1 and nodD2 both showed homology to LysR, an E . coli regulatory protein . We conclude that nodD1 probably has the same function as nodD in temperate rhizobia, namely, activation of nodABC transcription in the presence of plant signals . nodD2 may be involved in regulation of exopolysaccharide synthetic genes.

J Bacteriol, 1988 Jan, 170(1), 171 - 8
Two host-inducible genes of Rhizobium fredii and characterization of the inducing compound; Sadowsky MJ et al.; Random transcription fusions with Mu d1(Kan lac) generated three mutants in Rhizobium fredii (strain USDA 201) which showed induction of beta-galactosidase when grown in root exudate of the host plants Glycine max, Phaseolus vulgaris, and Vigna ungliculata . Two genes were isolated from a library of total plasmid DNA of one of the mutants, 3F1 . These genes, present in tandem on a 4.2-kilobase HindIII fragment, appear in one copy each on the symbiotic plasmid and do not hybridize to the Rhizobium meliloti common nodulation region . They comprise two separate transcriptional units coding for about 450 and 950 nucleotides, both of which are transcribed in the same direction . The two open reading frames are separated by 586 base pairs, and the 5H regions of the two genes show a common sequence . No similarity was found with the promoter areas of Rhizobium trifolii, R . meliloti, or Bradyrhizobium japonicum nif genes and with any known nodulation genes . Regions homologous to both sequences were detected in EcoRI digests of genomic DNAs from B . japonicum USDA 110, USDA 122, and 61A76, but not in genomic DNA from R . trifolii, Rhizobium leguminosarum, or Rhizobium phaseoli . Mass spectrometry and nuclear magnetic resonance analysis indicated that the inducing compound has properties of 4',7-dihydroxyisoflavone, daidzein . These results suggest that, in addition to common nodulation genes, several other genes appear to be specifically induced by compounds in the root exudate of the host plants.

Nucleic Acids Res, 1987 Dec 10, 15(23), 9677 - 90
Evidence that DNA involved in the expression of nodulation (nod) genes in Rhizobium binds to the product of the regulatory gene nodD; Hong GF et al.; In Rhizobium leguminosarum biovar viciae, the regulatory nodulation nodD gene has at least two functions . It constitutively represses its own transcription and in the presence of inducer flavonoid molecules, it activates the expression of two other nod gene transcriptional units, nodABCIJ and nodFE . Upstream of nodA and nodF is a conserved sequence, the nod box, which has been implicated in nodD-mediated transcriptional activation of these genes . DNA fragments spanning the nod boxes that precede nodA and nodF were end-labelled and were exposed to cell-free extracts obtained from strains of Rhizobium . Using the gel retardation technique, it was shown that a complex between protein and these DNA fragments was formed, but only if the extract contained a functional nodD gene . Evidence that the protein that binds to the regulatory sequences is the nodD gene product came from the observation that a complex was formed between the nod box preceding nodA and protein from a cell-free extract isolated from Escherichia coli containing the cloned nodD gene . Extracts from Rhizobium strains containing mutant forms of nodD which were specifically affected in autoregulation or in flavonoid-dependent activation formed either no protein DNA complex or formed a complex with altered mobility compared to that obtained with extracts from wild-type strains.

Mol Gen Genet, 1987 Dec, 210(2), 323 - 30
The nucleotide sequence of the sigma factor gene ntrA (rpoN) of Azotobacter vinelandii: analysis of conserved sequences in NtrA proteins; Merrick M et al.; The nucleotide sequence of the Azotobacter vinelandii ntrA gene has been determined . It encodes a 56916 Dalton acidic polypeptide (AvNtrA) with substantial homology to NtrA from Klebsiella pneumoniae (KpNtrA) and Rhizobium meliloti (RmNtrA) . NtrA has been shown to act as a novel RNA polymerase sigma factor but the predicted sequence of AvNtrA substantiates our previous analysis of KpNtrA in showing no substantial homology to other known sigma factors . Alignment of the predicted amino acid sequences of AvNtrA, KpNtrA and RmNtrA identified three regions; two showing greater than 50% homology and an intervening sequence of less than 10% homology . The predicted protein contains a short sequence near the centre with homology to a conserved region in other sigma factors . The C-terminal region contains a region of homology to the beta' subunit of RNA polymerase (RpoC) and two highly conserved regions one of which is significantly homologous to known DNA-binding motifs . In A . vinelandii, ntrA is followed by another open reading frame (ORF) which is highly homologous to a comparable ORF downstream of ntrA in K . pneumoniae and R . meliloti.

Proc Natl Acad Sci U S A, 1987 Dec, 84(23), 8558 - 62
Rhizobium meliloti has three functional copies of the nodD symbiotic regulatory gene; Honma MA et al.; We have identified two Rhizobium meliloti genes (nodD2 and nodD3) that are highly homologous and closely linked to the regulatory gene nodD (nodD1) . R . meliloti strains containing mutations in the three nodD genes in all possible combinations were constructed and their nodulation phenotypes were assayed on Medicago sativa (alfalfa) and Melilotus alba (sweet clover) . A triple nodD1-nodD2-nodD3 mutant exhibited a Nod- phenotype on alfalfa and sweet clover, indicating that nodD is an essential nodulation gene in R . meliloti . A nodD2 mutant exhibited no discernable defect in nodulation and nodD3 mutants exhibited a delayed nodulation phenotype of 2-3 days when inoculated onto either host . Alfalfa nodules elicited by a nodD1 mutant appeared 5-6 days after wild-type nodules, and sweet clover nodules elicited by a nodD1 mutant appeared 2-3 days after wild-type nodules . nodD1-nodD2 double mutants formed nodules with the same delay as single nodD1 mutants on both hosts . nodD2-nodD3 double mutants elicited sweet clover nodules at the same rate as single nodD3 mutants, but this same double mutant was slightly more delayed in alfalfa nodule formation than the nodD3 mutant . The nodD1-nodD3 mutant exhibited an extremely delayed nodulation phenotype on alfalfa and elicited no nodules on sweet clover . These experiments indicate that nodD1 and nodD3 have equivalent roles in nodulating sweet clover but that nodD1 plays a more important role than nodD3 in eliciting nodules on alfalfa . The nodD2 gene appears to have some effect on alfalfa nodulation and none on sweet clover . Our results indicate that R . meliloti has three functional nodD genes that modulate the nodulation process in a host-specific manner.

J Bacteriol, 1987 Dec, 169(12), 5393 - 400
Rhizobium meliloti nifN (fixF) gene is part of an operon regulated by a nifA-dependent promoter and codes for a polypeptide homologous to the nifK gene product; Aguilar OM et al.; An essential gene for symbiotic nitrogen fixation (fixF) is located near the common nodulation region of Rhizobium meliloti . A DNA fragment carrying fixF was characterized by hybridization with Klebsiella pneumoniae nif DNA and by nucleotide sequence analysis . The fixF gene was found to be related to K . pneumoniae nifN and was therefore renamed as the R . meliloti nifN gene . Upstream of the nifN coding region a second open reading frame was identified coding for a putative polypeptide of 110 amino acids (ORF110) . By fragment-specific Tn5 mutagenesis it was shown that the nifN gene and ORF110 form an operon . The control region of this operon contains a nif promoter and also the putative nifA-binding sequence . For the deduced amino acid sequence of the nifN gene product a striking homology to the R . meliloti nifK protein was found . One cysteine residue and its adjacent amino acid sequence, which are highly conserved in the R . meliloti nifK, R . meliloti nifN, and K . pneumoniae nifN proteins, may play a role in binding the FeMo cofactor.

Cell, 1987 Nov 20, 51(4), 579 - 87
Rhizobium meliloti mutants that fail to succinylate their calcofluor-binding exopolysaccharide are defective in nodule invasion; Leigh JA et al.; We have identified a set of Tn5-generated mutants of Rhizobium meliloti on the basis of their failure to form a fluorescent halo under UV light when grown on agar medium containing Calcofluor . These mutations define a new genetic locus we have termed exoH . Alfalfa seedlings inoculated with exoH mutants form ineffective nodules that do not contain intracellular bacteria or bacteroids . Root hair curling is significantly delayed and infection threads abort in the nodule cortex . Analyses of exopolysaccharide secreted by exoH mutants have shown that it is identical to the Calcofluor-binding exopolysaccharide secreted by the exoH+ parental strain except for the fact that it completely lacks the succinyl modification . In vitro translation of total RNA isolated from nodules induced by an exoH mutant has shown that only one of the plant-encoded nodulins is induced, as compared with the 17 nodulins induced by wild-type strains . These observations suggest that succinylation of the bacterial polysaccharide is important for its role(s) in nodule invasion and possibly nodule development.

J Bacteriol, 1987 Nov, 169(11), 4923 - 8
Characterization of the lipopolysaccharide from a Rhizobium phaseoli mutant that is defective in infection thread development; Carlson RW et al.; The lipopolysaccharide (LPS) from a Rhizobium phaseoli mutant, CE109, was isolated and compared with that of its wild-type parent, CE3 . A previous report has shown that the mutant is defective in infection thread development, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it has an altered LPS (K . D . Noel, K . A . VandenBosch, and B . Kulpaca, J . Bacteriol . 168:1392-1462, 1986) . Mild acid hydrolysis of the CE3 LPS released a polysaccharide and an oligosaccharide, PS1 and PS2, respectively . Mild acid hydrolysis of CE109 LPS released only an oligosaccharide . Chemical and immunochemical analyses showed that CE3-PS1 is the antigenic O chain of this strain and that CE109 LPS does not contain any of the major sugar components of CE3-PS1 . CE109 oligosaccharide was identical in composition to CE3-PS2 . The lipid A's from both strains were very similar in composition, with only minor quantitative variations . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CE3 and CE109 LPSs showed that CE3 LPS separated into two bands, LPS I and LPS II, while CE109 had two bands which migrated to positions similar to that of LPS II . Immunoblotting with anti-CE3 antiserum showed that LPS I contains the antigenic O chain of CE3, PS1 . Anti-CE109 antiserum interacted strongly with both CE109 LPS bands and CE3 LPS II and interacted weakly with CE3 LPS I . Mild-acid hydrolysis of CE3 LPS I, extracted from the polyacrylamide gel, showed that it contained both PS1 and PS2 . The results in this report showed that CE109 LPS consists of only the lipid A core and is missing the antigenic O chain.

J Bacteriol, 1987 Nov, 169(11), 4929 - 34
Cloning and characterization of hydrogen uptake genes from Rhizobium leguminosarum; Leyva A et al.; A gene library of genomic DNA from the hydrogen uptake (Hup)-positive strain 128C53 of Rhizobium leguminosarum was constructed by using the broad-host-range mobilizable cosmid vector pLAFR1 . The resulting recombinant cosmids contained insert DNA averaging 21 kilobase pairs (kb) in length . Two clones from the above gene library were identified by colony hybridization with DNA sequences from plasmid pHU1 containing hup genes of Bradyhizobium japonicum . The corresponding recombinant cosmids, pAL618 and pAL704, were isolated, and a region of about 28 kb containing the sequences homologous to B . japonicum hup-specific DNA was physically mapped . Further hybridization analysis with three fragments from pHU1 (5.9-kb HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) showed that the overall arrangement of the R . leguminosarum hup-specific region closely parallels that of B . japonicum . The presence of functional hup genes within the isolated cosmid DNA was demonstrated by site-directed Tn5 mutagenesis of the 128C53 genome and analysis of the Hup phenotype of the Tn5 insertion strains in symbiosis with peas . Transposon Tn5 insertions at six different sites spanning 11 kb of pAL618 completely suppressed the hydrogenase activity of the pea bacteroids.

Nucleic Acids Res, 1987 Oct 12, 15(19), 7921 - 34
Deduced products of C4-dicarboxylate transport regulatory genes of Rhizobium leguminosarum are homologous to nitrogen regulatory gene products; Ronson CW et al.; We have sequenced two genes dctB and dctD required for the activation of the C4-dicarboxylate transport structural gene dctA in free-living Rhizobium leguminosarum . The hydropathic profile of the dctB gene product (DctB) suggested that its N-terminal region may be located in the periplasm and its C-terminal region in the cytoplasm . The C-terminal region of DctB was strongly conserved with similar regions of the products of several regulatory genes that may act as environmental sensors, including ntrB, envZ, virA, phoR, cpxA, and phoM . The N-terminal domains of the products of several regulatory genes thought to be transcriptional activators, including ntrC, ompR, virG, phoB and sfrA . In addition, the central and C-terminal regions of DctD were strongly conserved with the products of ntrC and nifA, transcriptional activators that require the alternate sigma factor rpoN (ntrA) as co-activator . The central region of DctD also contained a potential ATP-binding domain . These results are consistent with recent results that show that rpoN product is required for dctA activation, and suggest that DctB plus DctD-mediated transcriptional activation of dctA may be mechanistically similar to NtrB plus NtrC-mediated activation of glnA in E . coli.

Cell Biophys, 1987 Oct, 10(3), 205 - 15
Relationship between Raman spectroscopic lines and growth of Rhizobium japonicum; Chang JJ et al.; The Raman spectroscopic lines of liquid cultures of Rhizobium japonicum have been compared with electron microscopic examinations and growth measurements of these cells . The results showed that the significant Raman lines are related to the reproduction activities of the procaryotic cells.

J Bacteriol, 1987 Sep, 169(9), 4294 - 301
Involvement of both cellulose fibrils and a Ca2+-dependent adhesin in the attachment of Rhizobium leguminosarum to pea root hair tips; Smit G et al.; We have previously described an assay for the attachment of Rhizobium bacteria to pea root hair tips (cap formation) which was used as a model to study the attachment step in the nodulation process . Under all conditions tested, a positive correlation was observed between the percentage of fibrillated cells and the ability of these bacteria to form caps and to adhere to glass, suggesting that fibrils play a role in the attachment of Rhizobium leguminosarum to pea root hair tips and to glass (G . Smit, J . W . Kijne, and B . J . J . Lugtenberg, J . Bacteriol . 168:821-827, 1986) . In the present paper the chemical and functional characterization of the fibrils of R . leguminosarum is described . Characterization of purified fibrils by infrared spectroscopy and cellulase treatment followed by thin-layer chromatography showed that the fibrils are composed of cellulose . Purified cellulose fibrils, as well as commercial cellulose, inhibited cap formation when present during the attachment assay . Incubation of the bacteria with purified cellulase just before the attachment assay strongly inhibited cap formation, indicating that the fibrils are directly involved in the attachment process . Tn5-induced fibril-overproducing mutants showed a greatly increased ability to form caps, whereas Tn5-induced fibril-negative mutants lost this ability . None of these Tn5 insertions appeared to be located on the Sym plasmid . Both types of mutants showed normal nodulation properties, indicating that cellulose fibrils are not a prerequisite for successful nodulation under the conditions used . The ability of the fibril-negative mutants to attach to glass was not affected by the mutations, indicating that attachment to pea root hair tips and attachment to glass are (partly) based on different mechanisms . However, growth of the rhizobia under low Ca2+ conditions strongly reduced attachment to glass and also prevented cap formation, although it had no negative effect on fibril synthesis . This phenomenon was found for several Rhizobium spp . It was concluded that both cellulose fibrils and a Ca2+ -dependent adhesin(s) are involved in the attachment of R . leguminosarum to pea root hair tips . A model cap formation as a two-step process is discussed.

Appl Environ Microbiol, 1987 Aug, 53(8), 1947 - 50
Requirement of succinate dehydrogenase activity for symbiotic bacteroid differentiation of Rhizobium meliloti in alfalfa nodules; Gardiol AE et al.; Transmission electron microscopy was used to study the cellular morphologies of a wild-type Rhizobium meliloti strain (L5-30), a nitrogen fixation-ineffective (Fix-) succinate dehydrogenase mutant (Sdh-) strain, and a Fix+ Sdh+ revertant strain within alfalfa nodules and after free-living growth in a minimal medium containing 27 mM mannitol plus 20 mM succinate . The results showed a requirement of succinate dehydrogenase activity for symbiotic differentiation and maintenance of R . meliloti bacteroids within alfalfa nodules and for succinate-induced cellular pleomorphism in free-living cultures . Also, the Sdh- strain had a 3.5-fold lower rate of oxygen consumption in the defined medium than did the wild type.

J Bacteriol, 1987 Jul, 169(7), 3388 - 91
Flavonoids induce Rhizobium leguminosarum to produce nodDABC gene-related factors that cause thick, short roots and root hair responses on common vetch; Zaat SA et al.; Rhizobium leguminosarum produced a factor(s) that caused thick, short roots (Tsr phenotype) as well as root hair induction (Hai phenotype) and deformation (Had phenotype) in Vicia sativa plants upon incubation with root exudate or with one of the nod gene inducers naringenin or apigenin; this was a nodDABC gene-dependent process . Detection of the Hai and Had phenotypes was much more sensitive than that of the Tsr phenotype.

J Bacteriol, 1987 Jul, 169(7), 3369 - 71
Reexamination of the presence and linkage of 3-hydroxybutyryl substituents in the acidic capsular polysaccharide of Rhizobium trifolii 0403; Hollingsworth RI et al.; We resolved previous conflicting results concerning the presence of 3-hydroxybutyryl substituents on the extracellular acidic polysaccharide from Rhizobium trifolii 0403 . These substituents were indeed present in the polysaccharide and in the oligosaccharide fragments obtained by hydrogen fluoride solvolysis of the extracellular and capsular polysaccharides of the bacteria grown on plates . The 3-hydroxybutyrate substituent could be removed from the polysaccharide by 10 mM sodium deuteroxide without evidence of elimination, indicating that this substituent was ester linked.

J Bacteriol, 1987 Jul, 169(7), 3146 - 50
Rhizobium meliloti swims by unidirectional, intermittent rotation of right-handed flagellar helices; Gotz R et al.; The 5 to 10 peritrichously inserted complex flagella of Rhizobium meliloti MVII-1 were found to form right-handed flagellar bundles . Bacteria swam at speeds up to 60 microns/s, their random three-dimensional walk consisting of straight runs and quick directional changes (turns) without the vigorous angular motion (tumbling) seen in swimming Escherichia coli cells . Observations of R . meliloti cells tethered by a single flagellar filament revealed that flagellar rotation was exclusively clockwise, interrupted by very brief stops (shorter than 0.1 s), typically every 1 to 2 s . Swimming bacteria responded to chemotactic stimuli by extending their runs, and tethered bacteria responded by prolonged intervals of clockwise rotation . Moreover, the motility tracks of a generally nonchemotactic ("smooth") mutant consisted of long runs without sharp turns, and tethered mutant cells showed continuous clockwise rotation without detectable stops . These observations suggested that the runs of swimming cells correspond to clockwise flagellar rotation, and the turns correspond to the brief rotation stops . We propose that single rotating flagella (depending on their insertion point on the rod-shaped bacterial surface) can reorient a swimming cell whenever the majority of flagellar motors stop.

J Bacteriol, 1987 Jul, 169(7), 3217 - 23
The nifA gene of Rhizobium meliloti is oxygen regulated; Ditta G et al.; Experiments using plasmid-borne gene fusions and direct RNA measurements have revealed that expression from the nifA gene is induced in Rhizobium meliloti when the external oxygen concentration is reduced to microaerobic levels . Induction occurs in the absence of alfalfa and in the presence of fixed nitrogen and does not require ntrC . The production of functional nifA gene product (NifA) can be demonstrated by its ability to activate the nitrogenase promoter P1 . Aerobic induction of nifA can also occur during nitrogen starvation at low pH, but in this case induction is dependent on ntrC and does not lead to P1 activation . The data indicate that reduced oxygen tension is potentially a major trigger for symbiotic activation of nitrogen fixation in Rhizobium species.

J Mol Biol, 1987 Jun 20, 195(4), 939 - 43
A set of positively regulated flagellar gene promoters in Caulobacter crescentus with sequence homology to the nif gene promoters of Klebsiella pneumoniae; Mullin D et al.; The study reported here describes nuclease S1 mapping of the in-vivo transcription start sites of transcription units I and III of the hook gene cluster of Caulobacter crescentus . We show that transcription units I and II of this flagellar (fla) gene cluster, which have divergent promoters with transcription start sites separated by 218 nucleotides, are under positive transcriptional control by genes in transcription unit III . The promoters of transcription units I, II, and III were compared with flagellin gene promoters P25, P27 and P29 recently identified in C . crescentus . Promoters PII, P25, and P27, which are under positive regulation by transcription units III to V have strongly conserved sequence elements at -13 and -24 with the consensus sequence (C/T)TGGC(C/G)C-N5-TTGC . The -13, -24 sequence elements are not well conserved in promoter PI, but the promoter does contain a copy of the -13 and -24 consensus sequence 23 base-pairs upstream (PI) . The C . crescentus fla gene promoters are not homologous to the canonical Escherichia coli -10, -35 promoter sequence, but they are very similar to the -12, -24 nif gene promoter sequence reported for Klebsiella pneumoniae and Rhizobium sp . The four positively regulated fla gene promoters examined here also share a third conserved element designated II-1, with the consensus sequence C-C-CGGC--AAA--GC-G, located at approximately -100 . We speculate that the conserved sequence elements mapping at -13, -24 and -100 are cis-acting regulatory elements required for the transcription and periodic regulation of these fla genes in the C . crescentus cell cycle.

J Mol Biol, 1987 Jun 5, 195(3), 603 - 20
Three-dimensional structure of the complex flagellar filament of Rhizobium lupini and its relation to the structure of the plain filament; Trachtenberg S et al.; Electron micrographs of negatively stained preparations were used to obtain a three-dimensional reconstruction of the complex flagellar filament of Rhizobium lupini H13-3 . The complex filament has an organization similar to that of the more common plain filament, but the subunits are perturbed in a pairwise fashion to generate a very distinctive set of three continuous ridges of density along the outer surface of the filament . In the three-dimensional map, the design of the complex filament is similar to that of the plain filament described in the accompanying paper . The structures consist of 11 segmented rods of density lying at a radius of 65 to 70 A . The exterior surfaces of both kinds of filaments consist of features that protrude from the segmented rods . The interiors of both consist of arms that extend inwards from the rods . In the case of the complex filament, but not of the plain filament, the inner arms interact to generate three tubular features, which, together with the three outer ridges, may account for the more brittle and, by implication, stiffer nature of the complex filament.

J Bacteriol, 1987 Jun, 169(6), 2631 - 8
A locus encoding host range is linked to the common nodulation genes of Bradyrhizobium japonicum; Nieuwkoop AJ et al.; By using cloned Rhizobium meliloti, Rhizobium leguminosarum, and Rhizobium sp . strain MPIK3030 nodulation (nod) genes as hybridization probes, homologous regions were detected in the slow-growing soybean symbiont Bradyrhizobium japonicum USDA 110 . These regions were found to cluster within a 25-kilobase (kb) region . Specific nod probes from R . meliloti were used to identify nodA-, nodB-, nodC-, and nodD-like sequences clustered on two adjacent HindIII restriction fragments of 3.9 and 5.6 kb . A 785-base-pair sequence was identified between nodD and nodABC . This sequence contained an open reading frame of 420 base pairs and was oriented in the same direction as nodABC . A specific nod probe from R . leguminosarum was used to identify nodIJ-like sequences which were also contained within the 5.6-kb HindIII fragment . A nod probe from Rhizobium sp . strain MPIK3030 was used to identify hsn (host specificity)-like sequences essential for the nodulation of siratro (Macroptilium atropurpureum) on a 3.3-kb HindIII fragment downstream of nodIJ . A transposon Tn5 insertion within this region prevented the nodulation of siratro, but caused little or no delay in the nodulation of soybean (Glycine max).

J Bacteriol, 1987 Jun, 169(6), 2424 - 31
Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions; Ronson CW et al.; We report the identification and cloning of an ntrA-like (glnF rpoN) gene of Rhizobium meliloti and show that the R . meliloti ntrA product (NtrA) is required for C4-dicarboxylate transport as well as for nitrate assimilation and symbiotic nitrogen fixation . DNA sequence analysis showed that R . meliloti NtrA is 38% homologous with Klebsiella pneumoniae NtrA . Subcloning and complementation analysis suggested that the R . meliloti ntrA promoter lies within 125 base pairs of the initiation codon and may be constitutively expressed.

J Biol Chem, 1987 May 15, 262(14), 6849 - 55
Transcription of Rhizobium meliloti nodulation genes . Identification of a nodD transcription initiation site in vitro and in vivo; Fisher RF et al.; Nodulation genes in Rhizobium are required for invasion of the host plant . The nodABC operon is induced by plant activator molecules; this activation requires the gene product of the constitutively expressed nodD locus, which is transcribed divergently from nodABC . We are employing in vitro transcription to elucidate the molecular mechanism of nod gene activation . We used a micropurification technique to obtain RNA polymerase from Rhizobium meliloti, and here demonstrate that it initiated and terminated accurately at the Escherichia coli trp promoter-leader region . E . coli RNA polymerase, however, apparently fails to recognize R . meliloti promoters . We used the R . meliloti RNA polymerase in a minimal transcription system to attempt to localize the divergent start sites for nodD and nodABC . Transcript sizing and fingerprinting, together with synchronized single-round transcription experiments permit us to designate an in vitro transcription initiation site for nodD . Primer extension analysis of in vivo mRNA demonstrates that the initiation site which is utilized in vitro is the same site used in vivo . While nodABC is not transcribed in our minimal in vitro transcription system, this system should prove useful for the study of factors in induced cells which promote expression of this inducible promoter.

J Bacteriol, 1987 May, 169(5), 2231 - 8
A new symbiotic cluster on the pSym megaplasmid of Rhizobium meliloti 2011 carries a functional fix gene repeat and a nod locus; Renalier MH et al.; A 290-kilobase (kb) region of the Rhizobium meliloti 2011 pSym megaplasmid, which contains nodulation genes (nod) as well as genes involved in nitrogen fixation (nif and fix), was shown to carry at least six sequences repeated elsewhere in the genome . One of these reiterated sequences, about 5 kb in size, had previously been identified as part of a cluster of fix genes located 220 kb downstream of the nifHDK promoter . Deletion of the reiterated part of this fix cluster does not alter the symbiotic phenotype . Deletion of the second copy of this reiterated sequence, which maps on pSym 40 kb upstream of the nifHDK promoter, also has no effect . Deletion of both of these copies however leads to a Fix- phenotype, indicating that both sequences carry functionally reiterated fix gene(s) . The fix copy 40 kb upstream of nifHDK is part of a symbiotic cluster which also carries a nod locus, the deletion of which produces a marked delay in nodulation.

J Bacteriol, 1987 May, 169(5), 2239 - 44
Transcription patterns of Rhizobium meliloti symbiotic plasmid pSym: identification of nifA-independent fix genes; David M et al.; We performed a systematic survey of transcription of a large region of the Rhizobium meliloti symbiotic plasmid pSym . This led to the discovery of two new sequences induced during symbiosis . The first sequence was linked to the known nitrogen fixation (nif-fix) gene cluster, and its expression depended on the nifA gene product . The second sequence was a novel fix locus (M.-H . Renalier, J . Batut, J . Ghai, B . Terzaghi, M . Gherardi, M . David, A.-M . Garnerone, J . Vasse, G . Truchet, T . Huguet, and P . Boistard, J . Bacteriol . 169:2231-2238, 1987) whose expression was independent of the nifA gene product; therefore this fix locus undergoes a novel type of symbiotic regulation.

Mol Gen Genet, 1987 Apr, 207(1), 155 - 60
Identification of two classes of Rhizobium phaseoli genes required for melanin synthesis, one of which is required for nitrogen fixation and activates the transcription of the other; Borthakur D et al.; The symbiotic plasmid pRP2JI of Rhizobium phaseoli strain 8002 was shown to contain two separate regions of DNA which are required and sufficient for the synthesis of the pigment melanin . One of these regions containing the class II mel gene(s) was located to other genes involved in nodulation and in nitrogen fixation . Mutations in this region abolished both the ability to synthesize melanin and to fix nitrogen in Phaseolus bean root nodules . Mutations in the other, unlinked region, containing class I mel gene(s), also abolished melanin synthesis but did not affect symbiotic nitrogen fixation . Transcriptional fusions between the class I mel gene and the Escherichia coli lacZ gene were constructed and it was demonstrated that the class II mel gene(s) activated their transcription in free-living culture . Further, strains containing the cloned regulatory class II gene(s) synthesized melanin when growing in minimal medium, in contrast to wild-type strains which became pigmented only in complete medium containing yeast extract and tryptone . It was shown by hybridization experiments that the regulatory mel gene was closely linked to or may correspond to the regulatory nifA gene; a fragment of R . phaseoli DNA which included the class II gene(s) of R . phaseoli hybridized to a previously identified nifA-like gene of R . leguminosarum, the species that nodulates peas.

Mol Gen Genet, 1987 Apr, 207(1), 149 - 54
Sequence of psi, a gene on the symbiotic plasmid of Rhizobium phaseoli which inhibits exopolysaccharide synthesis and nodulation and demonstration that its transcription is inhibited by psr, another gene on the symbiotic plasmid; Borthakur D et al.; A gene termed psi (polysaccharide inhibition), located close to the nodulation genes of the Rhizobium phaseoli symbiotic plasmid pRP2JI inhibited exopolysaccharide synthesis (EPS) and nodulation ability (Nod) in Rhizobium when it was cloned in a multicopy plasmid . The sequence of psi showed that it specified a polypeptide of mol . wt . 10,000 that may be associated with the membrane of Rhizobium . A second gene, psr (polysaccharide restoration), was located on pRP2JI . When cloned in multicopy plasmids, psr overcame the EPS and Nod defects in strains carrying multicopy psi . Strains with multicopy psr induced non-fixing nodules on Phaseolus beans . Using gene fusions between psi and lacZ, it was found that psr {corrected} inhibited transcription of psi {corrected}.

J Bacteriol, 1987 Apr, 169(4), 1403 - 9
Rhizobium meliloti insertion element ISRm2 and its use for identification of the fixX gene; Dusha I et al.; Two of the three plasmids of the wild-type Rhizobium meliloti 41 (pRme41a and pRme41c) carry a copy of ISRm2, a 2.7-kilobase-long transposable element . ISRm2 is terminated by 22-base-pair (bp) inverted repeat sequences, exhibiting some homology to the inverted repeats of elements generating 9-bp target sequence duplication . Transposition of ISRm2 results in a duplication of 8 bp in length, rather rare among transposable elements . DNA sequences homologous to an internal fragment of ISRm2 were found in several Rhizobium species . Transposition of ISRm2 into fixation and nodulation genes located on the symbiotic plasmid pRme41b was detected at a high frequency . Exact locations of two copies of ISRm2 which transposed into the nod-nif region on the megaplasmid were determined . In one case, integration into the protein-coding region of the hsnD gene that determines a host specificity function of nodulation occurred . In the other mutant, ISRm2 was localized upstream of nifA, where a short open reading frame coding for a new fix gene (fixX) was identified . The product of fixX is a ferredoxin carrying a characteristic cluster of cysteine residues . On the basis of the observation that the arrangement of the ISRm2 copies is identical in the free-living wild-type cells and in nitrogen-fixing nodules, we concluded that the involvement of ISRm2 transposition in the development of nitrogen-fixing symbiosis is unlikely.

J Bacteriol, 1987 Apr, 169(4), 1423 - 32
Identification and characterization of the Rhizobium meliloti ntrC gene: R . meliloti has separate regulatory pathways for activation of nitrogen fixation genes in free-living and symbiotic cells; Szeto WW et al.; We show here that Rhizobium meliloti, the nitrogen-fixing endosymbiont of alfalfa (Medicago sativa), has a regulatory gene that is structurally homologous to previously characterized ntrC genes in enteric bacteria . DNA sequence analysis showed that R . meliloti ntrC is homologous to previously sequenced ntrC genes from Klebsiella pneumoniae and Bradyrhizobium sp . (Parasponia) and that an ntrB-like gene is situated directly upstream from R . meliloti ntrC . Similar to its counterparts in K . pneumoniae and Escherichia coli, R . meliloti ntrC is expressed when the cells are grown in nitrogen-limiting media . In addition, R . meliloti ntrC is required for growth on media containing nitrate as the sole nitrogen source and for the ex planta transcription of several R . meliloti nif genes . On the other hand, root nodules elicited by R . meliloti ntrC mutants fix nitrogen as well as nodules elicited by wild-type R . meliloti . These latter results indicate that R . meliloti has separate regulatory pathways for activating nif gene expression ex planta and during symbiotic nitrogen fixation.

Nucleic Acids Res, 1987 Mar 11, 15(5), 1951 - 64
Tight linkage of glnA and a putative regulatory gene in Rhizobium leguminosarum; Colonna-Romano S et al.; Rhizobium leguminosarum, biovar viceae, strain RCC1001 contains two glutamine synthetase activities, GSI and GSII . We report here the identification of glnA, the structural gene for GSI . A 2 kb fragment of DNA was shown to complement the Gln- phenotype of Klebsiella pneumoniae glnA mutant strains . DNA sequence analysis revealed an open reading frame (ORF) of 469 codons specifying a polypeptide of 52,040 daltons . Its deduced amino acid sequence was found to be highly homologous to other glutamine synthetase sequences . This ORF was expressed in Escherichia coli minicells and the corresponding polypeptide reacted with an antiserum raised against GSI . Upstream of glnA we found an ORF of 111 codons (ORF111) preceded by the consensus sequence for an ntrA-dependent promoter . Minicells experiments showed a protein band, with a molecular weight in good agreement with that (10,469) deduced from the nucleotide sequence . On the basis of homology studies we discuss the possibility that the product of ORF111 is equivalent to the PII protein of E . coli and plays a similar role in regulation of nitrogen metabolism.

J Bacteriol, 1987 Mar, 169(3), 1137 - 46
Effects of Rhizobium meliloti nif and fix mutants on alfalfa root nodule development; Hirsch AM et al.; Ineffective alfalfa nodules were examined at the light and electron microscope level after inoculation with Rhizobium meliloti strains with mutations in nif and fix genes . All the mutant strains induced nodules that contained elongated bacteroids within the host cells, but the bacteroids quickly senesced . The nodules were small and numerous, and the host cells also exhibited symptoms of an ineffective symbiosis . nifB, fixA, and fixB bacteroids appeared to be completely differentiated (by ultrastructural criteria), i.e., as bacteroids developed, they increased in diameter and length and their cytoplasm underwent a change from homogeneous and electron dense to heterogeneous and electron transparent after enlargement . In contrast, nifA bacteroids rarely matured to this state . The bacteroids degenerated at an earlier stage of development and did not become electron transparent.

Mol Gen Mikrobiol Virusol, 1987 Mar, (3), 27 - 32
{Hybridization of DNA from Actinomycetes of the genus Frankia with nitrogenase structural genes (nifHDK) of Klebsiella pneumoniae and nod-genes of Rhizobium melioti}; Tomashevskii AIu et al.; DNA sequence homology with the plasmid pSA30 carrying the cloned nifHDK genes from Klebsiella pneumoniae was revealed in ten Frankia spp . strains, nitrogen-fixing symbionts of non-legumes, irrespective of the strain phenotype (Nod+Fix+, Nod+ Fix- or Nod- Fix?) . None of the Frankia spp . DNAs exhibited homology with the plasmid pRmSL26 harbouring the Rhizobium meliloti nod-genes encoding for early symbiotic functions.

J Bacteriol, 1987 Mar, 169(3), 1345 - 8
Cloning of Rhizobium leguminosarum genes for competitive nodulation blocking on peas; Dowling DN et al.; One type of competitive interaction among rhizobia is that between nonnodulating and nodulating strains of Rhizobium leguminosarum on primitive pea genotypes . Pisum sativum cv . Afghanistan nodulates effectively with R . leguminosarum TOM, and this can be blocked in mixed inoculations by R . leguminosarum PF2, which does not nodulate this cultivar . We termed this PF2 phenotype Cnb+, for competitive nodulation blocking . Strain PF2 contains three large plasmids including a 250-kilobase-pair symbiotic (Sym) plasmid . Transfer of this plasmid, pSymPF2, to nonblocking rhizobia conferred the Cnb+ phenotype on recipients in mixed inoculations on cultivar Afghanistan with TOM . A library of the PF2 genome constructed in the vector pMMB33 was used to isolate two cosmid clones which hybridize to pSymPF2 . These cosmids, pDD50 and pDD58, overlapped to the extent of 23 kilobase pairs and conferred a Cnb+ phenotype on recipient Cnb- rhizobia, as did pSD1, a subclone from the common region.

J Bacteriol, 1987 Mar, 169(3), 1161 - 7
Alteration of surface properties in a Tn5 mutant strain of Rhizobium trifolii 0403; Gardiol AE et al.; A symbiotically defective mutant strain of Rhizobium trifolii, UR251, was obtained by transposon Tn5 mutagenesis of R . trifolii 0403 rif and recognized by its partially ineffective (Fix +/-) phenotype on white clover plants . UR251 had a single Tn5 insertion in plasmid DNA, a wild-type plasmid pattern, and no detectable Mu DNA sequences originally present in the vector used for Tn5 mutagenesis . Agglutination by the clover lectin trifoliin A and attachment to clover root hairs was higher with UR251 than with the wild-type strain . The capsular polysaccharide (CPS) of UR251 was altered, as shown by a slower rate of CPS depolymerization with a CPS beta-lyase, PD-I; more pyruvate and less acetate and 3-hydroxybutanoate noncarbohydrate substitutions as quantitated by 1H nuclear magnetic resonance; and a higher pyruvyl transferase activity (enzymatic pyruvylation of lipid-bound saccharides) . The site of increased pyruvylation in the CPS of UR251 was on the terminal galactose of the branch of the repeating oligosaccharide unit . These results show that the level of noncarbohydrate substitutions of the CPS as well as pyruvyl transferase activity are altered in R . trifolii UR251 and that trifoliin A-binding ability and clover root hair attachment are improved in this mutant strain of R . trifolii 0403 rif.

J Bacteriol, 1987 Mar, 169(3), 1127 - 36
Genetic and structural analysis of the Rhizobium meliloti fixA, fixB, fixC, and fixX genes; Earl CD et al.; The fixA, fixB, fixC, and fixX 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, but fixABC-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 Klebsiella 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 . meliloti fixA, -B, -C, and -X genes was found in the K . pneumoniae nif operon . 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 . The fixABC 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 the fixABCX promoter bears more than 65% homology to the 5' end of the transcribed region, including the first 32 codons of fixA.

J Bacteriol, 1987 Mar, 169(3), 1120 - 6
Conservation of structure and location of Rhizobium meliloti and Klebsiella pneumoniae nifB genes; Buikema WJ et al.; Using transposon Tn5-mediated mutagenesis, an essential Rhizobium meliloti nitrogen fixation (nif) gene was identified and located directly downstream of the regulatory gene nifA . Maxicell and DNA sequence analysis demonstrated that the new gene is transcribed in the same direction as nifA and codes for a 54-kilodalton protein . In Klebsiella pneumoniae, the nifBQ operon is located directly downstream of a gene which is structurally and functionally homologous to the R . meliloti nifA gene . The DNA sequences of the K . pneumoniae nifB and nifQ genes (which code for 51- and 20-kilodalton proteins, respectively) were determined . The DNA sequence of the newly identified R . meliloti gene was approximately 50% homologous to the K . pneumoniae nifB gene . R . meliloti does not contain a gene homologous to nifQ directly downstream of nifB . The R . meliloti nifB product shares approximately 40% amino acid homology with the K . pneumoniae nifB product, and 10 of the 12 cysteine residues of the R . meliloti nifB product are conserved with 10 of the 17 cysteine residues of the K . pneumoniae nifB product.

Nucleic Acids Res, 1987 Jan 12, 15(1), 31 - 49
Organization and partial sequence of a DNA region of the Rhizobium leguminosarum symbiotic plasmid pRL6JI containing the genes fixABC, nifA, nifB and a novel open reading frame; Gronger P et al.; By hybridization and heteroduplex studies the fixABC and nifA genes of the Rhizobium leguminosarum symbiotic plasmid pRL6JI have been identified . DNA sequencing of the region containing nifA showed an open reading frame of 1557 bp encoding a protein of 56, 178 D . Based on sequence homology, this ORF was confirmed to correspond to the nifA gene . Comparison of three nifA proteins (Klebsiella pneumoniae, Rhizobium meliloti, Rhizobium leguminosarum) revealed only a weak relationship in their N-terminal regions, whereas the C-terminal parts exhibited strong homology . Sequence analysis also showed that the R . leguminosarum nifA gene is followed by nifB and preceded by fixC with an open reading frame inserted in between . This novel ORF of 294 bp was found to be highly conserved also in R . meliloti . No known promoter and termination signals could be defined on the sequenced R . leguminosarum fragment.

J Bacteriol, 1987 Jan, 169(1), 198 - 204
Induction of the nodA promoter of Rhizobium leguminosarum Sym plasmid pRL1JI by plant flavanones and flavones; Zaat SA et al.; An expression vector containing the Rhizobium leguminosarum nodA promoter cloned in front of the Escherichia coli lacZ gene was used to characterize the properties of the R . leguminosarum nodA gene-inducing compound(s) present in sterile root exudate of the host plant Vicia sativa L . subsp . nigra (L.) . The major inducing compound was flavonoid in nature, most likely a flavanone . The commercially available flavonoids naringenin (5,7,4'-trihydroxyflavanone), eriodictyol (5,7,3'4'-tetrahydroxyflavanone), apigenin (5,7,4'-trihydroxyflavone), and luteolin (5,7,3',4'-tetrahydroxyflavone) induced the nodA promoter to the same level as the root exudate . On the basis of chromatographic properties, it was concluded that none of these compounds is identical to the inducer that is present in root exudate . The induction of the nodA promoter by root exudate and by the most effective inducer naringenin was very similar, as judged from the genetic requirements and the kinetics of induction.

J Bacteriol, 1987 Jan, 169(1), 53 - 60
Nitrogen fixation ability of exopolysaccharide synthesis mutants of Rhizobium sp . strain NGR234 and Rhizobium trifolii is restored by the addition of homologous exopolysaccharides; Djordjevic SP et al.; Several transposon Tn5-induced mutants of the broad-host-range Rhizobium sp . strain NGR234 produce little or no detectable acidic exopolysaccharide (EPS) and are unable to induce nitrogen-fixing nodules on Leucaena leucocephala var . Peru or siratro plants . The ability of these Exo- mutants to induce functioning nodules on Leucaena plants was restored by coinoculation with a Sym plasmid-cured (Nod- Exo+) derivative of parent strain NGR234, purified EPS from the parent strain, or the oligosaccharide from the EPS . Coinoculation with EPS or related oligosaccharide also resulted in formation of nitrogen-fixing nodules on siratro plants . In addition, an Exo- mutant (ANU437) of Rhizobium trifolii ANU794 was able to form nitrogen-fixing nodules on white clover in the presence of added EPS or related oligosaccharide from R . trifolii ANU843 . These results demonstrate that the absence of Rhizobium EPSs can result in failure of effective symbiosis with both temperate and subtropical legumes.

J Bacteriol, 1987 Jan, 169(1), 410 - 3
Isolation of competition-defective mutants of Rhizobium fredii; McLoughlin TJ et al.; We coupled Tn5 mutagenesis with a competition assay to isolate mutants of Rhizobium fredii USDA 257 that are defective in competition for nodulation of soybeans . Two mutants with single Tn5 inserts in the chromosome showed reduced competitiveness in vermiculite but were identical to the wild-type strain in symbiotic properties when inoculated alone . Recombination of Tn5 and flanking genomic regions cloned from the mutants into the parent strain showed that Tn5 was responsible for the mutant phenotype.

Anal Biochem, 1987 Jan, 160(1), 47 - 56
Universal chemical assay for the detection and determination of siderophores; Schwyn B et al.; A universal method to detect and determine siderophores was developed by using their high affinity for iron(III) . The ternary complex chrome azurol S/iron(III)/hexadecyltrimethylammonium bromide, with an extinction coefficient of approximately 100,000 M-1 cm-1 at 630 nm, serves as an indicator . When a strong chelator removes the iron from the dye, its color turns from blue to orange . Because of the high sensitivity, determination of siderophores in solution and their characterization by paper electrophoresis chromatography can be performed directly on supernatants of culture fluids . The method is also applicable to agar plates . Orange halos around the colonies on blue agar are indicative of siderophore excretion . It was demonstrated with Escherichia coli strains that biosynthetic, transport, and regulatory mutations in the enterobactin system are clearly distinguishable . The method was successfully used to screen mutants in the iron uptake system of two Rhizobium meliloti strains, DM5 and 1021.

EMBO J, 1987 Jan, 6(1), 1 - 9
Glutamine synthetase genes of pea encode distinct polypeptides which are differentially expressed in leaves, roots and nodules; Tingey SV et al.; We have characterized the distinct polypeptides, primary translation products and mRNAs encoding glutamine synthetase (GS) in the various organs of pea . Western blot analysis of soluble protein has identified five distinct GS polypeptides which are expressed at different relative levels in leaves, roots and nodules of pea . Of the two GS polypeptides in leaves (44 and 38 kd), the 44-kd GS polypeptide is predominant and is localized to the chloroplast stroma . In roots, the predominant GS polypeptide is 38 kd . Upon Rhizobium infection of roots, three 37-kd GS polypeptides increase in abundance in the nodules relative to uninfected roots . cDNA clones encoding three different GS mRNAs have been characterized . Hybrid-select translation has identified three different GS primary translation products (49, 38 and 37 kd) . Two cDNA clones (pGS134 and pGS341) are homologous to GS mRNAs most abundant in nodules which encode the 38- and 37-kd GS primary translation products . A third cDNA (pGS197) corresponds to a larger GS mRNA species specific to leaf poly(A) RNA, which encodes a 49-kd putative precursor to the mature chloroplast GS polypeptide . cDNA sequence analysis and Southern blot analysis of pea nuclear DNA identifies at least three genes encoding GS in pea which are related but distinct in structure and in vivo pattern of expression.

Gene, 1987, 55(1), 153 - 6
Nucleotide sequence of a tRNA(leu)CAG gene from Rhizobium meliloti; Boesten B et al.; The nucleotide sequence of a tRNA(leu)CAG gene from Rhizobium meliloti has been determined . Its organization includes a short transcript with a promoter-like structure and two Rho-independent terminators about 10 bp downstream from the cloverleaf structure . The putative promoter shows good homology with the -45 and -35 region of the consensus (sigma 70) promoter sequence of Escherichia coli, but less with the -10 region . A discriminator-like sequence is present between the start of the transcript and the -10 region . The gene shows extensive homology to tRNA(leu)CAG genes from Gram-positive and Gram-negative bacteria . However, significant differences are encountered in the variable loop region . This is the first report of a tRNA sequence from R . meliloti.

J Bacteriol, 1986 Dec, 168(3), 1459 - 62
Biosynthesis of Rhizobium trifolii capsular polysaccharide: enzymatic transfer of pyruvate substitutions into lipid-bound saccharide intermediates; Gardiol AE et al.; The activity of capsular polysaccharide pyruvyltransferase catalyzing the pyruvylation of acidic heteropolysaccharide was measured in Rhizobium trifolii 843 and 0403 rif . This enzyme activity was determined with EDTA-treated cells, uridine diphosphate-sugar precursors, and phosphoenol {1-14C}pyruvate . Activity was measured by the incorporation of radioactivity into organic solvent-soluble glycoconjugates . Enzymatic pyruvylation of capsular polysaccharide occurred from phosphoenolpyruvate at the lipid-bound saccharide stage.

J Bacteriol, 1986 Dec, 168(3), 1392 - 401
Mutations in Rhizobium phaseoli that lead to arrested development of infection threads; Noel KD et al.; 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.

J Bacteriol, 1986 Dec, 168(3), 1075 - 86
Assignment of symbiotic developmental phenotypes to common and specific nodulation (nod) genetic loci of Rhizobium meliloti; Debelle F et al.; Rhizobium meliloti nodulation (nod) genes required for specific infection and nodulation of alfalfa have been cloned . Transposon Tn5 mutagenesis defined three nod regions spanning 16 kilobases of the pSym megaplasmid . Genetic and cytological studies of 62 nodulation-defective mutants allowed the assignment of symbiotic developmental phenotypes to common and specific nod loci . Root hair curling was determined by both common (region I) and specific (region III) nod transcription units; locus IIIb (nodH gene) positively controlled curling on the homologous host alfalfa, whereas loci IIIa (nodFE) and IIIb (nodH) negatively controlled curling on heterologous hosts . Region I (nodABC) was required for bacterial penetration and infection thread initiation in shepherd's crooks, and the nodFE transcription unit controlled infection thread development within the alfalfa root hair . In contrast, induction of nodule organogenesis, which can be triggered from a distance, seemed to be controlled by common nodABC genes and not to require specific nod genes nodFE and nodH . Region II affected the efficiency of hair curling and infection thread formation.

J Bacteriol, 1986 Nov, 168(2), 821 - 7
Correlation between extracellular fibrils and attachment of Rhizobium leguminosarum to pea root hair tips; Smit G et al.; As part of a project meant to characterize molecules involved in nodulation, a semiquantitative microscopic assay was developed for measuring attachment of Rhizobium leguminosarum cells to pea root hair tips, i.e., the site at which R . leguminosarum initiates nodulation . This form of attachment, designated as cap formation, was dependent on the incubation pH and growth phase, with optimal attachment at pH 7.5 and with bacteria in the early stationary phase of growth . Addition of glucose to the growth medium delayed the initiation of the stationary phase and cap formation, suggesting a correlation between cap formation and carbon limitation . Attachment of R . leguminosarum was not inhibited by pea lectin haptens which makes it unlikely that lectins are involved under the tested conditions . Moreover, heterologous fast-growing rhizobia adhered equally well to pea root hair tips . Since the attachment characteristics of a Sym plasmid-cured derivative were indistinguishable from those of the wild-type strain, the Sym plasmidborne nodulation genes are not necessary for attachment . Sodium chloride and various other salts abolished attachment when present during the attachment assay in final concentrations of 100 mM . R . leguminosarum produced extracellular fibrils . A positive correlation between the percentage of fibrillated cells and the ability of the bacteria to form caps and to adhere to glass and erythrocytes was observed under various conditions, suggesting that these fibrils play a role in attachment of the bacteria to pea root hair tips, to glass, and to erythrocytes.

J Bacteriol, 1986 Nov, 168(2), 785 - 90
Mapping and cloning of a fla-che region of the Rhizobium meliloti chromosome; Ziegler RJ et al.; We constructed a genetic map of the fla-che region of the Rhizobium meliloti chromosome using cotransduction with bacteriophage phi M12 . Several other chromosomal markers located in the general area are included in the map . We isolated plasmids carrying wild-type DNA inserts that complement the mapped mutations from a genomic library carried in the broad-host-range vector pLAFR1 . The complementation data obtained from the clones confirmed the contransduction map and clarified the exact order of several of the behavioral genes . A restriction map of this area was developed by using the cloned DNA . One of the five individual EcoRI fragments subcloned from the original clones complemented two of the behavioral mutations.

Carbohydr Res, 1986 Oct 15, 154, 103 - 13
Synthesis of 3,6-dideoxy-3-(methylamino)hexoses for g.l.c.-m.s . identification of Rhizobium lipopolysaccharide components; Hollingsworth RI et al.; A direct synthetic route from methyl alpha-D-glucopyranoside to 3,6-dideoxy-3-(methylamino)hexoses having the D-gluco, D-galacto, and D-manno configurations has been developed . Methyl alpha-D-glucoside was converted into the 4,6-O-benzylidene-2,3-di-O-tosyl derivative, which was then transformed into the 4-O-benzyl-6-deoxy 2,3-ditosylate (5) by successive reductive cleavage of the acetal ring, iodination, and reduction . The intermediate 5 was readily converted into the allo 2,3-epoxide, which yielded the pivotal intermediate methyl 4-O-benzyl-3,6-dideoxy-3-(methylamino)-alpha-D-glucopyranoside (7) by cleavage of the oxirane ring with methylamine . The amino compound 7 can be directly converted into the derivatized galacto and manno derivatives for mass-spectrometric identification by selective inversion at C-4 and C-2, respectively, followed by hydrolysis, reduction, and acetylation.

J Biol Chem, 1986 Oct 15, 261(29), 13624 - 31
The kinetics of the reactions of Parasponia andersonii hemoglobin with oxygen, carbon monoxide, and nitric oxide; Wittenberg JB et al.; Hemoglobin I was isolated from nodules formed on the roots of Parasponia andersonii inoculated with Rhizobium strain CP 283 . The rate of oxygen dissociation from Parasponia hemoglobin increases about 12-fold between pH 4 and 7, with apparent pK 6.4, to reach a limiting value of 14.8s-1 . The optical spectrum of oxyhemoglobin in the visible region is also dependent on pH with pK near 6.4 . The rate constant for oxygen combination with Parasponia hemoglobin increases about 7-8-fold between pH 4 and 7, with apparent pK 5.37, to reach a value of 1.67 X 10(8) M-1 s-1 at pH 7 . The optical spectrum of deoxyhemoglobin in the visible region and the rate constant for carbon monoxide combination are also dependent on pH with apparent pK 5.65 and 5.75, respectively . The rate constant for carbon monoxide dissociation is independent of pH . The oxygen affinity of Parasponia hemoglobin, P50 = 0.049 torr at 20 degrees C, calculated from the kinetic constants at pH 7, is very great . At alkaline pH there is a prominent geminate reaction with oxygen and nitric oxide, with both subnanosecond and tens of nanosecond components . These reactions disappear at acid pH, with pK 6.4, and the effective quantum yield is reduced . In general, the reactions of Parasponia hemoglobin with oxygen and carbon monoxide resemble those of soybean leghemoglobin . In each, great oxygen affinity is achieved by unusually rapid oxygen combination together with a moderate rate of oxygen dissociation . We suggest that protonation of a heme-linked group with pK near 6.4 controls many properties of Parasponia oxyhemoglobin, and protonation of a group with pK near 5.5 controls many properties of Parasponia deoxyhemoglobin.

J Mol Biol, 1986 Oct 5, 191(3), 411 - 20
At least two nodD genes are necessary for efficient nodulation of alfalfa by Rhizobium meliloti; Gottfert M et al.; A Rhizobium meliloti DNA region (nodD1) involved in the regulation of other early nodulation genes has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined . The sequence data indicate a large open reading frame with opposite polarity to nodA, -B and -C, coding for a protein of 308 (or 311) amino acid residues . Tn5 insertion within the gene caused a delay in nodulation of Medicago sativa from four to seven days . Hybridization of nodD1 to total DNA of Rhizobium meliloti revealed two additional nodD sequences (nodD2 and nodD3) and both were localized on the megaplasmid pRme41b in the vicinity of the other nod genes . Genetic and DNA hybridization data, combined with nucleotide sequencing showed that nodD2 is a functional gene, while requirement of nodD3 for efficient nodulation of M . sativa could not be detected under our experimental conditions . The nodD2 gene product consists of 310 amino acid residues and shares 86.4% homology with the nodD1 protein . Single nodD2 mutants had the same nodulation phenotype as the nodD1 mutants, while a double nodD1-nodD2 mutant exhibited a more severe delay in nodulation . These results indicate that at least two functional copies of the regulatory gene nodD are necessary for the optimal expression of nodulation genes in R . meliloti.

Nature, 1986 Oct 2-8, 323(6087), 448 - 50
A family of related ATP-binding subunits coupled to many distinct biological processes in bacteria; Higgins CF et al.; Many biological processes are coupled to ATP hydrolysis . We describe here a class of closely related ATP-binding proteins, from several bacterial species, which are associated with a variety of cellular functions including membrane transport, cell division, nodulation in Rhizobium and haemolysin export . These proteins comprise a family of structurally and functionally related subunits which share a common evolutionary origin, bind ATP and probably serve to couple ATP hydrolysis to each of these biological processes . This finding suggests a specific role for ATP in cell division, nodulation during nitrogen fixation and protein export, and allows us to assign a probable function to one of the protein components from each of these systems.

J Bacteriol, 1986 Oct, 168(1), 270 - 5
Biosynthesis of a galactose-and galacturonic acid-containing polysaccharide in Rhizobium meliloti; Ugalde RA et al.; Previous work showed that two different strains derived from a culture of Rhizobium meliloti 102F51 differed with respect to phage specificity, agglutinability by alfalfa seed lectin, and synthesis of a galactose-containing polysaccharide (R . A . Ugalde, H . Handelsman, and W . J . Brill, J . Bacteriol . 166:148-154, 1986) . Inner membranes from the more competitive strain incorporated galactose from UDP-galactose when a thermostable factor was present . This factor has now been identified as UDP-galacturonic acid . UDP-glucuronic acid was also active as a donor; however, this activity may be due to the presence of a 4-epimerase . Galacturonic acid, together with galactose, is incorporated into the reaction product, which appears to be a polysaccharide formed by several repeating units of these two monosaccharides . Partial acid hydrolysis liberates the disaccharide with galactose at the reducing end.

Mol Gen Mikrobiol Virusol, 1986 Oct, (10), 31 - 3
{Conjugation transfer of the bireplicon plasmid pSPA044 into Rhizobiaceae bacteria}; Stekhin IN et al.; We have demonstrated the possibility of hybrid plasmid pSPA044 conjugative transfer from E . coli cells into different Rhizobium species . The bireplicon plasmid, constructed earlier in our laboratory, consisting of pBR325 and HindIII fragment 13 of the nopaline plasmid pTiC58 was mobilized for transfer by the helper plasmid pRK2013 with the frequency about 10(-4) . We conclude the hybrid plasmid pSPA044 to be able to replicate stably in Rhizobiaceae cells.

Eur J Biochem, 1986 Oct 1, 160(1), 69 - 75
A cell-free system from Rhizobium meliloti to study the specific expression of nodulation genes; Dusha I et al.; An in vitro transcription-translation system was developed using cell-free extracts from the symbiotic nitrogen-fixing bacterium Rhizobium meliloti strain 41 . Conditions for preparation of the 30,000 X g supernatant extract and for measurement of protein-synthesizing activity were determined and compared to the activity of an Escherichia coli cell-free system . Genes expressed in the free-living or in the symbiotic state were studied . The product of a recA-like gene (41-kDa protein) was synthesized both in R . meliloti and E . coli extracts, although less efficiently in the heterologous system . In agreement with earlier results obtained in E . coli minicells, three proteins (44, 28.5 and 23 kDa) were synthesized from a cloned 3.3 X 10(3)-base DNA region carrying genes for nodulation (nod) . However, differences in the transcription-translation of nod and host specificity (hsn) genes were observed when protein expression was compared in R . meliloti and E . coli cell-free extracts, and the possible explanations of these findings are discussed.

Microbiologia, 1986 Oct, 2(2), 89 - 96
{Nitrogenase, hydrogenase and nitrate reductase activities, oxygen consumption, and ATP content in nodules formed by strains of Rhizobium leguminosarum 128C53 and 300 in symbiosis with pea plants}; Bedmar EJ et al.; The nitrogenase activity, nitrate reductase activity and oxygen uptake as well as the hydrogen incorporation and ATP content were examined in the root nodules and bacteroids, respectively, formed by Rhizobium leguminosarum strains 128C53 (hydrogenase positive) and 300 (hydrogenase negative) in symbiosis with Pisum sativum plants grown in the presence of 2 mM KNO3 . The strain 128C53 showed the greatest values for all parameters analyzed, except for the nitrate reductase activity, which was higher for the strain 300 . Similarly, nodule nitrate reductase activity in strain 300 was greater than that in strain 128C53 when plants grew in the absence of combined nitrogen . In general, the highest values were obtained when determinations were made after 7 hours of plant illumination . However, the hydrogenase activity of strain 128C53 and the nitrate reductase activities of both strains increased with the light period, reaching a maximum after 14 hours of illumination . These results suggest that the benefits derived from the superior symbiotic properties and from the presence of hydrogenase activity in strain 128C53 could be counteracted by the higher rates of the nodule nitrate reductase activity in strain 300.

Nucleic Acids Res, 1986 Sep 25, 14(18), 7453 - 72
Nucleotide sequence of Rhizobium meliloti RCR2011 genes involved in host specificity of nodulation; Debelle F et al.; A 6 kb DNA segment of the R . meliloti 2011 pSym megaplasmid, which contains genes controlling host specificity of root hair infection and of nodulation, was cloned and sequenced . The DNA sequence analysis, in conjunction with previous genetic data, allowed identification of four nod genes designated as E, F, G and H . nodH is divergently transcribed with respect to nodFE and nodG . A conserved nucleotide sequence was found around 200 bp upstream of the translation start of nodF, nodH and nodA . This sequence is also present upstream of common nodA and species specific nodF genes of other Rhizobium species . The predicted protein products of nodF and nodG show homology with acyl carrier protein and ribitol dehydrogenase, respectively . The nodH product contains a rare sequence of four contiguous proline residues . Comparison with the nod gene products of R . leguminosarum shows that species specific nodFE products are as well conserved as those of common nodABC and nodD genes.

J Cell Sci, 1986 Sep, 85, 47 - 61
Physical association between the peribacteroid membrane and lipopolysaccharide from the bacteroid outer membrane in Rhizobium-infected pea root nodule cells; Bradley DJ et al.; Monoclonal antibodies were used as cytochemical markers to study surface interactions between endosymbiotic Rhizobium bacteroids from pea root nodules and the encircling peribacteroid membranes, which are of plant origin . Monoclonal antibodies that react with Rhizobium lipopolysaccharide (LPS) or with a plant membrane glycoprotein were used as markers for material from the bacteroid outer membrane or the peribacteroid membrane, respectively . Membrane-enclosed bacteroids were isolated from nodule homogenates by sucrose gradient centrifugation, and the encircling peribacteroid membrane was released by mild osmotic shock treatment . Using an immunochemical technique (sandwich ELISA), it was shown that 1-5% of the LPS antigen released into the peribacteroid fraction by mild osmotic shock treatment was physically associated with peribacteroid membrane through a detergent-sensitive linkage . This association could be visualized when freshly prepared peribacteroid material was immobilized on gold grids and examined by electron microscopy after dual antibody immunogold treatment and subsequent negative staining . The distribution of LPS antigen within infected nodule cells was also investigated by immunogold staining for thin sections of nodule tissue fixed in glutaraldehyde, and a close association between LPS antigen and peribacteroid membrane was often seen.

J Bacteriol, 1986 Sep, 167(3), 1092 - 4
Galactose metabolism in Rhizobium meliloti L5-30; Arias A et al.; Data from previous studies of Rhizobium meliloti mutants have been consistent with the catabolism of hexoses via the Entner-Doudoroff pathway . However, galactose metabolism was not impaired in those mutants . We show here by enzymatic assay and by identification of a galactose mutant lacking 2-keto-3-deoxy-6-phosphogalactonate aldolase that the De Ley-Doudoroff pathway is used for galactose metabolism . Mutants in this pathway have not been previously reported for any organism.

J Bacteriol, 1986 Sep, 167(3), 1083 - 5
Fractionation of Rhizobium leguminosarum cells into outer membrane, cytoplasmic membrane, periplasmic, and cytoplasmic components; de Maagd RA et al.; Rhizobium leguminosarum cells were separated into four distinct fractions by using density gradient centrifugation for the separation of the outer and cytoplasmic membranes and lysozyme-EDTA treatment of whole cells for the isolation of the periplasmic and cytoplasmic fractions . These methods allowed the subcellular localization of R . leguminosarum proteins.

J Cell Biol, 1986 Sep, 103(3), 1043 - 54
Endogenous lectins from cultured soybean cells: isolation of a protein immunologically cross-reactive with seed soybean agglutinin and analysis of its role in binding of Rhizobium japonicum; Ho SC et al.; Incubation of Rhizobium japonicum with the cultured soybean cell line SB-1, originally derived from the roots of Glycine max, resulted in specific adhesion of the bacteria to the plant cells . This binding interaction appears to be mediated via carbohydrate recognition, since galactose can inhibit the heterotypic adhesion but glucose cannot . Affinity chromatography, on a Sepharose column derivatized with N-caproyl-galactosamine, of the supernatant fraction of a SB-1 cell suspension after enzymatic removal of cell wall yielded a single polypeptide (Mr approximately 30,000) on immunoblotting analysis with rabbit antibodies directed against seed soybean agglutinin . Fluorescently labeled rabbit anti-seed soybean agglutinin also yielded specific immunofluorescent staining on the cell wall and plasma membrane of the SB-1 cells . These results suggest that one likely candidate that may mediate the recognition between the Rhizobium and the soybean cells is the endogenously produced SB-1 lectin . This notion is supported by the observation that rabbit anti-seed soybean agglutinin blocked the Rhizobium-soybean cell adhesion, whereas control antibodies did not.

Mol Gen Genet, 1986 Sep, 204(3), 485 - 91
Genetic manipulations in Rhizobium meliloti utilizing two new transposon Tn5 derivatives; De Vos GF et al.; Two derivatives of the prokaryotic transposon Tn5 were constructed in vitro . In Tn5-233, the central area of Tn5, which carries resistance to kanamycin/neomycin, bleomycin and streptomycin, is replaced by a fragment carrying resistance to the aminocyclitol antibiotics gentamycin/kanamycin and streptomycin/spectinomycin . In Tn5-235, the Escherichia coli beta-galactosidase gene is inserted within the streptomycin resistance gene of Tn5, and constitutively expressed from a Tn5 promoter . Both constructs transpose with about the same frequency as Tn5 in Escherichia coli and Rhizobium meliloti . When a Tn5-derivative is introduced into an R . meliloti strain which already contains a different Tn5-derivative, in situ transposon replacement is obtained at high frequency, presumably by a pair of crossovers between the IS50 sequences at the ends of the incoming and resident transposons . In this way we converted a previously isolated recA::Tn5 mutant into the corresponding recA::Tn5-233 strain, which can now be used as a genetic background in the study of complementation of other Tn5-induced mutations . We also replaced the drug markers of several Tn5-induced exo mutants, which we were then able to map relative to each other by transduction with phage phi M12 . In a strain carrying Tn5-235 located near Tn5-233, we were able to isolate deletions of the intervening markers, presumably resulting from general recombination between the two transposons, by screening for loss of the Lac+ phenotype . Unlike Tn5 itself, resident Tn5-233 does not appear to suppress transposition of another incoming Tn5-derivative.

J Gen Microbiol, 1986 Sep, 132 ( Pt 9), 2561 - 9
Characterization and cloning of two Rhizobium leguminosarum genes coding for glutamine synthetase activities; Filser DM et al.; We have demonstrated that Rhizobium leguminosarum strain LPR1105 contains a heat stable and a heat labile glutamine synthetase (EC 6.3.1.2) activity similar to those described for other Rhizobiaceae . Most of the activity is heat stable when this strain is grown on glutamine as sole nitrogen source, but most is heat labile when grown on nitrate . Using a gene bank of R . leguminosarum DNA we have isolated two clones, which code for heat stable (p7D9) and heat labile (p4F7) glutamine synthetase activity, by complementing the glutamine auxotrophy of Klebsiella pneumoniae glnA mutants . Cross-hybridization of p7D9 with a fragment of the glnA gene of K . pneumoniae was observed, but no cross-hybridization between p7D9 and p4F7 was found . Since these two regions hybridize to genomic DNA of R . leguminosarum they are probably the structural genes for GSI and GSII, and the availability of these genes will make it possible to test this hypothesis . Clone p4F7 complements an ntrC+ but not an ntrC K . pneumoniae glnA mutant, suggesting that the ntrC gene is required for the complementation of the glutamine auxotrophy by this plasmid.

Science, 1986 Aug 29, 233(4767), 977 - 80
A plant flavone, luteolin, induces expression of Rhizobium meliloti nodulation genes; Peters NK et al.; The symbiotic interaction of Rhizobium meliloti and alfalfa results in the formation of nitrogen-fixing root nodules . Rhizobium meliloti nodABC genes are required for the early host responses of cortical cell divisions and root hair curling . The induction of nodABC expression by alfalfa exudates demonstrates host-symbiont signaling at an early stage in nodule development . The inducer molecule for nodABC expression was isolated from plant exudate by constructing a nodABC-lacZ fusion to monitor the inducing activity . From ultraviolet-visible absorption spectra, proton nuclear magnetic resonance, and mass spectrometry, the inducer was determined to be 3',4', 5,7-tetrahydroxyflavone (luteolin) . Luteolin is a normal secondary plant metabolite found throughout the plant kingdom that may serve to control nodABC expression during nodule development . This regulatory role for a flavone contrasts with the function of some flavonoids as defense compounds.

J Mol Biol, 1986 Aug 20, 190(4), 569 - 76
Pairwise perturbation of flagellin subunits . The structural basis for the differences between plain and complex bacterial flagellar filaments; Trachtenberg S et al.; Although plain and complex bacterial flagellar filaments differ in their physical properties and helical symmetry, they both appear to derive from a common underlying structure . Analysis of electron micrographs of complex filaments of Rhizobium lupini revealed that the unit cell has twice the length of that of plain filaments, with a corresponding reduction in helical symmetry whereby the six-start helical family present in plain filaments collapses into a three-start family . Mass per unit length measurements were made by scanning transmission electron microscopy . These, together with the unit cell dimensions and the molecular weight of the flagellin monomer, enabled the number of monomers per unit cell to be estimated . Whereas plain filaments have a single monomer per unit cell, complex filaments have two . These results suggest that complex filament structure differs from plain filament structure by a pairwise perturbation, or interaction, of the flagellin monomers . The additional bonding interactions involved in the perturbation in the complex filament may make it more rigid than the plain filament, which has no such perturbation.

J Biol Chem, 1986 Aug 15, 261(23), 10688 - 94
Reversible inactivation of the O2-labile hydrogenases from Azotobacter vinelandii and Rhizobium japonicum; Seefeldt LC et al.; Hydrogenases catalyze the reversible activation of dihydrogen . The hydrogenases from the aerobic, N2-fixing microorganisms Azotobacter vinelandii and Rhizobium japonicum are nickel- and iron-containing dimers that belong to the group of O2-labile enzymes . Exposure of these hydrogenases to O2 results in an irreversible inactivation; therefore, these enzymes are purified anaerobically in a fully active state . We describe in this paper an electron acceptor-requiring and pH-dependent, reversible inactivation of these hydrogenases . These results are the first example of an anaerobic, reversible inactivation of the O2-labile hydrogenases . The reversible inactivation required the presence of an electron acceptor . The rate of inactivation was first-order, with similar rates observed for methylene blue, benzyl viologen, and phenazine-methosulfate . The rate of inactivation was also dependent on the pH . However, increasing the pH of the enzyme in the absence of an electron acceptor did not result in inactivation . Thus, the reversible inactivation was not a result of high pH alone . The inactive enzyme could not be reactivated by H2 or other reductants at high pH . Titration of enzyme inactivated at high pH back to low pH was also ineffective at reactivating the enzyme . However, if reductants were present during this titration, the enzyme could be fully reactivated . The temperature dependence of inactivation yielded an activation energy of 44 kJ X mol-1 . Gel filtration chromatography of active and inactive hydrogenase indicated that neither dissociation nor aggregation of the dimer hydrogenase was responsible for this reversible inactivation . We propose a four-state model to describe this reversible inactivation.

Cell, 1986 Aug 1, 46(3), 335 - 43
Organization, structure and symbiotic function of Rhizobium meliloti nodulation genes determining host specificity for alfalfa; Horvath B et al.; In R . meliloti we have identified four nodulation genes determining plant host-range specificity and have designated them hsnABC and D . The genes code for 9.7, 41.7, 26.7, and 28.6 kd proteins, respectively, and are organized into two transcriptional units . Mutations in these genes affect nodulation of their natural plant hosts Medicago sativa and Melilotus albus to different extents and hsnD mutants have an altered host-range . These Nod- mutations are not complementable by nodulation genes of other Rhizobium species such as R . leguminosarum . The hsn genes determine plant-specific infection through root hairs: hsnD is required for host-specific root hair curling and nodule initiation while the hsnABC genes control infection thread growth from the root hairs.

J Bacteriol, 1986 Aug, 167(2), 487 - 91
Genetic rearrangements of a Rhizobium phaseoli symbiotic plasmid; Soberon-Chavez G et al.; Different structural changes of the Sym plasmid were found in a Rhizobium phaseoli strain that loses its symbiotic phenotype at a high frequency . These rearrangements affected both nif genes and Tn5 mob insertions in the plasmid, and in some cases they modified the expression of the bacterium's nodulation ability . One of the rearrangements was more frequent in heat-treated cells, but was also found under standard culture conditions; other structural changes appeared to be related to the conjugal transfer of the plasmid.

Arch Biochem Biophys, 1986 Jul, 248(1), 183 - 9
Anthranilate-promoted iron uptake in Rhizobium leguminosarum; Rioux CR et al.; Anthranilate promoted the uptake of ferric iron into iron-starved cells of Rhizobium leguminosarum GF160 . The uptake system was a saturable function of the concentration of ferric anthranilate . It was characterized by an apparent Km of 6 microM and a Vmax of 1.6 nmol/min/mg cell protein . Uptake was temperature dependent and inhibited by the metabolic poisons arsenate and iodoacetate . The proton motive force may not be involved since no effect was demonstrated by the respiratory inhibitor sodium azide and by various uncouplers . The anthranilate-promoted iron uptake was lower for cells grown in increasing levels of available iron and the addition of anthranilic acid to low-iron cultures resulted in a stimulation of bacterial growth . During growth under iron deficiency, anthranilic acid was not assimilated by the cells.

Arch Biochem Biophys, 1986 Jul, 248(1), 175 - 82
Iron requirement of Rhizobium leguminosarum and secretion of anthranilic acid during growth on an iron-deficient medium; Rioux CR et al.; Rhizobium leguminosarum GF160 required iron for growth under aerobic conditions in a chemically defined medium . Maximal growth of bacteria previously depleted in iron was obtained with approximately 50 microM unchelated ferric iron and with glucose as the only carbon source . Growth under iron deficiency did not result in the production of detectable levels of siderophores of either the catechol or hydroxamate types . Growing cells released a Fe3+-reducing agent that was identified as anthranilic acid by paper and thin-layer chromatography, ultraviolet and nuclear magnetic resonance spectroscopy, and mass spectrometry . The amount of anthranilic acid secreted per unit of cell growth was inversely related to the iron concentration in the culture medium and reached concentrations up to 1 mM . Ferric but not ferrous ions were solubilized in the growth medium by anthranilic acid.

Plasmid, 1986 Jul, 16(1), 37 - 44
The presence of repeated DNA sequences and a partial restriction map of the pSym of Rhizobium fredii USDA193; Masterson RV et al.; The large, 350-kb Sym (symbiotic) plasmid pRjaUSDA193 of Rhizobium fredii was examined to determine the frequency of repeated sequences present and to produce a physical and genetic map of a large region of the plasmid . A novel hybridization method, the Southern Cross, revealed that the plasmid pRjaUSDA193 contained many repeated sequences and assisted in restriction enzyme mapping of a 100-kb region containing nod genes . A cosmid clone bank was prepared with the broad-host-range cosmid pVK102 . The restriction enzymes HindIII, HpaI, and KpnI were used to construct a physical map of overlapping clones . Labeled nod gene sequences were used to determine their location in the mapped region.

Appl Environ Microbiol, 1986 Jul, 52(1), 206 - 8
Mobilization of Tn5 insertions from Rhizobium fredii by pJB3JI; Thomas PM et al.; Techniques for in vivo cloning were used with the fast-growing nitrogen-fixing soybean microsymbiont R . fredii USDA 191 . Selection for transfer of Tn5 insertions from R . fredii USDA 191 containing the gene-mobilizing plasmid pJB3JI provided recombinants at up to 400 times the background mutation level . These techniques may be useful for future genetic analysis of R . fredii.

J Bacteriol, 1986 Jun, 166(3), 795 - 800
Aerobic purification of hydrogenase from Rhizobium japonicum by affinity chromatography; Stults LW et al.; We purified active hydrogenase from free-living Rhizobium japonicum by affinity chromatography . The uptake hydrogenase of R . japonicum has been treated previously as an oxygen-sensitive protein . In this purification, however, reducing agents were not added nor was there any attempt to exclude oxygen . In fact, the addition of sodium dithionite to aerobically purified protein resulted in the rapid loss of activity . Purified hydrogenase was more stable when stored under O2 than when stored under Ar . Sodium-chloride-washed hydrogen-oxidizing membranes were solubilized in Triton X-100 and deoxycholate and loaded onto a reactive red 120-agarose column . Purified hydrogenase elutes at 0.36 M NaCl, contains a nickel, and has a pH optimum of 6.0 . There was 452-fold purification resulting in a specific activity of 76.9 mumol of H2 oxidized per min per mg of protein and a yield of 17% . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed subunits with estimated molecular weights of 65,000 and 33,000 . Hydrogenase prepared in this manner was used to raise and affinity purify antibodies against both subunits.

J Bacteriol, 1986 May, 166(2), 628 - 34
Genetic locus in Rhizobium japonicum (fredii) affecting soybean root nodule differentiation; Stanley J et al.; A genetic locus in fast-growing Rhizobium japonicum (fredii) USDA 191 (Fix+ on several contemporary soybean cultivars) was identified by random Tn5 mutagenesis as affecting the development and differentiation of root nodules . This mutant (MU042) is prototrophic and shows no apparent alterations in its surface properties . It induces aberrant nodules, arrested at the same early level of differentiation, on all its host plants . An 8.1-kilobase EcoRI fragment containing Tn5 was cloned from MU042 . In USDA 191 as well as another fast-growing strain, USDA 201, the affected locus was found to be unlinked to the large symbiotic plasmid and appears to be chromosomal . An analogous sequence has been shown to be present in Bradyrhizobium japonicum (J . Stanley, G.G . Brown, and D.P.S . Verma, J . Bacteriol . 163:148-154, 1985) as well as in R . trifolii and R . meliloti . MU042 was complemented for effective nodulation of soybean by a cosmid clone from USDA 201, and the complementing locus was delimited to a 6-kilobase EcoRI subfragment . An R . trifolii strain (MU225), whose indigenous symbiotic plasmid was replaced by that of strain USDA 191, induced more highly differentiated nodules on soybean than did MU042 . This suggests that the mutation in MU042 can be functionally substituted by similar loci of other fast-growing rhizobia . Leghemoglobin and nodulin-35 (uricase II) were present in the differentiated Fix- nodules induced by MU225, whereas both were absent in MU042-induced pseudonodule structures.

J Bacteriol, 1986 May, 166(2), 552 - 8
Nodulation inhibition by Rhizobium leguminosarum multicopy nodABC genes and analysis of early stages of plant infection; Knight CD et al.; During analysis of early events in the infection and nodulation of Vicia hirsuta roots inoculated with normal and mutant strains of Rhizobium leguminosarum and strains containing cloned nodulation (nod) genes, a number of novel observations were made . (i) Alternating zones of curled and straight root hairs were seen on roots of V . hirsuta inoculated with the wild-type strain of R . leguminosarum . This phasing of root hair curling was not seen if plants were grown under continuous light or continuous dark conditions . (ii) Reduced nodulation and delayed nodule initiation was observed with a strain carrying a Tn5 mutation in the nodE gene . In addition the phased root hair curling was absent, and root hair curling was observed along the length of the root . (iii) The nodABC genes cloned on a multicopy plasmid in a wild-type strain inhibited nodulation but induced a continuous root hair curling response . Those few nodules that eventually formed were found to contain bacteria which had lost the plasmid carrying the nodABC genes . (iv) With a strain of Rhizobium cured of its indigenous symbiotic plasmid, but containing the cloned nodABCDEF genes, continuous root hair curling on V . hirsuta was observed . However, no infection threads were observed, and surprisingly, it did appear that initial stages of nodule development occurred . Observations of thin sections of these early developing nodules indicated that early nodule meristematic divisions may have occurred but that no bacteria were found within the nodules and no infection threads were observed either within the nodule bumps or within any of the root hairs . It was concluded that for normal infections to occur, precise regulation of the nod genes is required and that overexpression of the root hair curling genes inhibits the normal infection process.

Nucleic Acids Res, 1986 Apr 11, 14(7), 2905 - 19
Conserved nodulation genes from the non-legume symbiont Bradyrhizobium sp . (Parasponia); Scott KF; A nodulation locus from the broad-host-range, non-legume symbiont Bradyrhizobium sp . (Parasponia) strain ANU289, has been identified by hybridisation to cloned Rhizobium trifolii nodulation (nod) genes . Transfer of cloned ANU289 nod genes to R.trifolii nodulation-deficient mutants showed that the locus contains a functional homologue of the R . trifolii nodD gene . DNA sequence analysis revealed the presence of three additional genes nodA, nodB and nodC clustered adjacent to nodD . The four genes from ANU289 share substantial sequence homology with those characterised from narrow-host-range Rhizobium strains . A novel 700-bp sequence inserted between the nodD and nodABC genes encodes an open reading frame designated nodK and is oriented in the same direction as nodABC . nodKABC appear to be organized in a single transcriptional unit and nodD is oriented divergently to nodKABC . A 35-bp sequence containing the ribosome binding site for the nodD gene and an AT-rich core sequence has been identified by comparison with sequences from other Rhizobium strains and is likely to be implicated in the plant-mediated induction of nodulation gene expression.

Nucleic Acids Res, 1986 Apr 11, 14(7), 2891 - 903
DNA sequence of Rhizobium trifolii nodulation genes reveals a reiterated and potentially regulatory sequence preceding nodABC and nodFE; Schofield PR et al.; The Rhizobium trifolii nod genes required for host-specific nodulation of clovers are located on 14 kb of Sym (symbiotic) plasmid DNA . Analysis of the nucleotide sequence of a 3.7 kb portion of this region has revealed open reading frames corresponding to the nodABCDEF genes . A DNA sequencing technique, using primer extension from within Tn5, has been used to determine the precise locations of Tn5 mutations within the nod genes and the phenotypes of the corresponding mutants correlate with their mapped locations . The predicted nodA and nodB genes overlap by four nucleotides and the nod F and nodE genes overlap by a single nucleotide, suggesting that translational coupling may ensure the synthesis of equimolar amounts of these gene products . The nodABC and nodFE genes constitute separate transcriptional units and each is preceded by a conserved 76-bp sequence which may be involved in the regulation of expression of these genes.

J Bacteriol, 1986 Apr, 166(1), 148 - 54
Role of galactosyltransferase activity in phage sensitivity and nodulation competitiveness of Rhizobium meliloti; Ugalde RA et al.; A stock culture of Rhizobium meliloti 102F51 contains colonies of two distinct phenotypes (Handelsman et al., J . Bacteriol . 157:703-707, 1984); one colony type is agglutinated by high dilutions of the alfalfa agglutinin, is sensitive to phage F20, and is resistant to phage 16B, and the other is agglutinated only by low dilutions of the alfalfa agglutinin, is resistant to phage F20, and is sensitive to phage 16B . Cells of the latter phenotype have an inner-membrane-bound galactosyltransferase activity that transfers galactose from UDP-galactose to a water-insoluble anionic polymer . This enzymatic activity is absent in cells of the first phenotype . All of the phage 16B-resistant mutants selected from a sensitive strain were agglutinated by high dilutions of the alfalfa agglutinin, were sensitive to phage F20, and lacked galactosyltransferase activity . The galactose-containing polymer prepared in vitro was immunologically cross-reactive with the cell surface.

Biochemistry, 1986 Mar 11, 25(5), 952 - 8
Isolation and characterization of a soybean lectin having 4-O-methylglucuronic acid specificity; Rutherford WM et al.; A new lectin from soybeans having specificity toward the extracellular 4-O-methyl-D-glucurono-L-rhamnans produced by certain strains of Rhizobium japonicum has been purified and characterized . Isolation was accomplished initially by isoelectric precipitation of contaminating globulins and subsequently by affinity chromatography on partially hydrolyzed glucuronorhamnan covalently coupled to amino-hexylagarose . Residual globulins were removed by adsorption of the lectin on concanavalin A-agarose and elution with methyl alpha-mannoside . The lectin is a glycoprotein (3-5% carbohydrate) with a molecular weight of approximately 175 000 . It is a tetramer with subunit molecular weights of 45 000 when dissociated with sodium dodecyl sulfate . Reverse-phase high-pressure liquid chromatography analysis indicates the presence of two types of subunits, both having equivalent molecular weights . According to amino acid analyses, the lectin is rich in acidic but low in sulfur-containing amino acids . The carbohydrate portion of the lectin contains mannose; no hexosamines could be detected . Chemical modification of the lectin indicated that neither sulfhydryl groups nor amino groups participate in binding . Quantitative binding studies of the lectin with various carbohydrate haptens showed that specificity was directed toward 4-O-methyl-D-glucuronic acid, D-glucuronic acid, and their methyl glycosides with 4-O-methyl-D-glucuronic acid 3-4-fold more effective . In each instance, the methyl glycoside is a more effective hapten.

J Bacteriol, 1986 Feb, 165(2), 517 - 22
Role of plant root exudate and Sym plasmid-localized nodulation genes in the synthesis by Rhizobium leguminosarum of Tsr factor, which causes thick and short roots on common vetch; Van Brussel AA et al.; In a previous paper it was shown that cocultivation of Rhizobium leguminosarum with the plant Vicia sativa subsp . nigra on solid medium causes a changed mode of growth of the plant roots, resulting in thick and short roots (Tsr) . The Sym plasmid present in the bacterium appeared to be essential for causing Tsr (A . A . N . van Brussel, T . Tak, A . Wetselaar, E . Pees, and C . A . Wijffelman, Plant Sci . Lett . 27:317-325, 1982) . In the present paper, we show that a role in causing Tsr is general for Sym plasmids of R . leguminosarum and Rhizobium trifolii . Moreover, mutants with transposon insertions in the Sym plasmid-localized nodulation genes nodA, B, C, and D are unable to cause Tsr, in contrast to nodulation mutants localized in other parts of the Sym plasmid . The observation that Tsr could also be brought about in liquid medium enabled us to show that Tsr is caused by a soluble factor . Experiments in which plants and bacteria were grown separately in the sterile supernatant fluids of each other resulted in establishing the following sequence of events . (i) The plant produces a factor, designated as factor A . (ii) Factor A causes the Sym plasmid-harboring bacteria to produce Tsr factor . (iii) Growth of young plants in the presence of Tsr factor results in the Tsr phenotype . Models explaining this example of molecular signalling between bacteria and plants are discussed.

Arch Microbiol, 1986 Feb, 144(1), 29 - 34
Expression and regulation of the Escherichia coli glutamate dehydrogenase gene (gdh) in Rhizobium japonicum; Lane M et al.; The glutamate dehydrogenase (gdh) gene of Escherichia coli was transferred into an ammonium assimilation deficient mutant (Asm-) of Rhizobium japonicum (CJ9) using plasmid pRP301, a broad host range derivative of RP4 . Exconjugants capable of growth on ammonia as sole N-source occurred at a frequency of 6.8 X 10(-6) . Assimilatory GDH (NADP+) activity was detected in the strain carrying the E . coli gdh gene and the pattern of ammonia assimilation via GDH was similar to that of the Asm+ wild type strain . However, GDH mediated ammonia assimilation was not subject to regulation by L-glutamate . Nitrogenase activity was expressed ex planta in R . japonicum CJ9 harbouring the gdh gene, however, the presence of the gdh gene did not restore symbiotic effectiveness to the CJ9 Asm- strain in nodules . The gdh plasmid was maintained in approximately 90% of the isolates recovered from soybean nodules.

J Bacteriol, 1986 Feb, 165(2), 579 - 84
Immunological homology between the membrane-bound uptake hydrogenases of Rhizobium japonicum and Escherichia coli; Harker AR et al.; Two polypeptides present in aerobic and anaerobic cultures of Escherichia coli HB101 were shown to cross-react with antibodies to the 30- and 60-kilodalton (kDa) subunits of the uptake hydrogenase of Rhizobium japonicum . The cross-reactive polypeptides in a series of different E . coli strains are of Mrs ca . 60,000 and 30,000, and both polypeptides are present in proportion to measurable hydrogen uptake (Hup) activity (r = 0.95) . The 60-kDa polypeptide from E . coli HB101 comigrated on native gels with detectable Hup activity . The exact role of the 30-kDa polypeptide in E . coli is unclear . E . coli MBM7061, a natural Hup- variant, grown anaerobically or aerobically lacked detectable Hup activity and failed to cross-react with the antisera against the hydrogenase from R . japonicum . Anaerobically cultured E . coli MBM7061, however, did express formate hydrogenlyase activity, indicating that the hydrogenases involved in the oxygen-dependent activation of hydrogen and the formate-dependent evolution of hydrogen are biochemically distinct.

EMBO J, 1986 Feb, 5(2), 441 - 7
Sequence and domain relationships of ntrC and nifA from Klebsiella pneumoniae: homologies to other regulatory proteins; Drummond M et al.; We have determined the nucleotide sequences of two genes from Klebsiella pneumoniae, nifA, the nif-specific activator of transcription and ntrC, the bifunctional regulatory protein involved in 'nitrogen control' . These sequences differ significantly from those previously published . In particular, nifA extends 40 codons beyond the stop codon reported earlier . This extension encodes a putative DNA-binding domain strongly homologous to the Rhizobium meliloti nifA protein and to some extent to the ntrC protein . In all three proteins this domain is linked by a segment of variable length to a strongly conserved central domain of 240 residues . A short segment having the properties of an interdomain linker joins the central region to an N-terminal domain, which is weakly related in the case of the two nifA proteins . This homology is not shared by the N-terminal domain of ntrC, which is clearly but unexpectedly related to the N-terminal domains of a diverse set of procaryotic pleiotropic control proteins, including ompR, dye and nusA from Escherichia coli and spoOA and spoOF from Bacillus subtilis.

J Immunol Methods, 1986 Jan 22, 86(1), 45 - 52
Kinetics of monoclonal antibody production in low serum growth medium; Velez D et al.; Factors affecting growth and monoclonal antibody production in vitro by a mouse-mouse hybridoma cell line have been investigated in a series of studies . The goal was to maximize antibody yields and demonstrate that antibodies can be produced efficiently on a large-scale in fermentors . This initial report describes (i) development of a radial immunodiffusion assay for accurate determination of antibody levels in culture, (ii) a culture medium formulation that allowed for reduction in the amount of fetal bovine serum required for good cell growth, and (iii) the kinetics of cell growth and monoclonal antibody production in low-serum media . The radial immunodiffusion assay, employing rabbit anti-mouse IgG antibodies in the immobile phase and the monoclonal antibody (an IgG2a to Rhizobium japonicum cells) as the antigen in the mobile phase, was more reproducible and reliable for determining antibody levels in culture broth than was an indirect enzyme-linked immunosorbent assay . Addition of 0.25% Primatone RL and 0.01% Pluronic F-68 to Dulbecco's modified Eagle medium allowed cells to adapt to growth in medium containing as little as 1% fetal bovine serum; without these additives, 5% serum was the lowest level attained . For the kinetic studies, cells were grown in the low-protein medium in 3 liter spinner flasks . Antibody production occurred during the growth phase, however, significant amounts were also produced during later phases when the cells had stopped growing . Final titers were 100-200 micrograms/ml . It was concluded that maintenance of cell viability is more important than growth rate in production of antibody . This conclusion, confirmed in other studies, has developed into the major underlying strategy employed in subsequent investigations to maximize antibody production in stirred reactors.

Biochem Biophys Res Commun, 1986 Jan 14, 134(1), 205 - 11
In vitro synthesis of a lipid-linked acetylated and pyruvilated oligosaccharide in Rhizobium trifolii; Bossio JC et al.; EDTA-treated Rhizobium trifolii cells (strain NA30) incorporate radioactivity from (14C) labeled UDP-Clc, UDP-ClcA, Acetyl-Coa and/or phosphoenol pyruvate into chloroform: methanol: water (1:2:0.3) extracts . The incorporation products have properties of prenyl-phospho-sugars; mild alkaline hydrolysis of these extracts produce cyclic phosphate esters suggesting the presence of a diphosphate bridge, and mild acid or catalytic reduction-alkaline phosphatase treatments release four main components a, b, c and d, as judged by paper electrophoresis and chromatography and gel filtration studies . The four components can be obtained (14C)acetyl-labeled, but only compound c and to a lesser degree compound b can be (14C)pyruvate-labeled . For the exopolysaccharide produced by this strain the following repeating unit has been proposed (Robertsen et al . (1981), Plant Physiol . 67, 389-400): (Formula: see text) . The results obtained suggest that the octasaccharide repeating unit (compound a) with one (compound b) or two (compound c) ketal pyruvate residues are assembled on a lipid acceptor . All these compounds are assumed to be intermediates in the biosynthesis of R . trifolii exopolysaccharide.

Carbohydr Res, 1986 Jan 1, 145(2), 247 - 65
Location and identity of the acyl substituents on the extracellular polysaccharides of Rhizobium trifolii and Rhizobium leguminosarum; Kuo MS et al.; The basic structures of the extracellular polysaccharides of Rhizobium leguminosarum and Rhizobium trifolii were found to be identical, but their acylation patterns differ . Liquid hydrogen fluoride at -40 degrees degrades the two polysaccharides to a series of oligosaccharides representing the repeating units of the polysaccharides and their higher homologs . At -23 degrees, it degrades the polymers to a mixture of oligosaccharides from which a tetrasaccharide constituting a unit of the backbone of the polysaccharide, and a trisaccharide representing all but the non-reducing terminus of the side chain, could be readily purified . The location and identity of the acyl substituents were determined by 1H-n.m.r . spectroscopy, methylation analysis, and f.a.b . mass spectrometry . The unusual substituent D-3-hydroxybutanoate was found esterified to O-3 of a terminal 4,6-O-pyruvic acetylated D-galactose in both strains of R . leguminosarum, and in one of the three strains of R . trifolii tested . All of the strains tested contained a 3-O-acetyl substituent on the (1----4)-beta-D-glucopyranosyl residues in the backbone of the polysaccharide . Only the R . leguminosarum polysaccharides contained a combination of 2- and 3-O-acetyl groups on the branching sugar of the backbone of the polymer.

Antonie Van Leeuwenhoek, 1986, 52(5), 381 - 6
Congo red absorption and cellulose synthesis by Rhizobiaceae; Zevenhuizen LP et al.; Congo red uptake by Rhizobium colonies from yeast extract-mannitol-mineral salts-Congo red-agar plates was related with the cellulose content in the cell capsule of the bacteria.

J Bacteriol, 1986 Jan, 165(1), 304 - 7
Rhizobium sp . strain ORS571 grows synergistically on N2 and nicotinate as N sources; Ludwig RA; Rhizobium sp . strain ORS571 conducts synergistic, free-living N2 fixation and nicotinate oxidation . Explicitly, ORS571 is able to fix N2 aerobically because 6-OH-nicotinate acts as an intracellular O2 sink . Because 6-OH-nicotinate oxidation is mandatory for aerobic, free-living N2 fixation and because the synergistic processes yield ammonium from substrates (as the nitrogen source for growth), ORS571 is not a diazotroph.

Antonie Van Leeuwenhoek, 1986, 52(1), 85 - 96
The effect of the dissolved oxygen concentration and anabolic limitations on the behaviour of Rhizobium ORS571 in chemostat cultures; de Vries W et al.; Chemostat cultures of Rhizobium ORS571 limited by the supply of oxygen or an anabolic substrate contained poly-beta-hydroxybutyrate (PHB) . Low amounts of PHB (about 10%) were present in ammonia- or nitrate-limited cultures; higher amounts were found in Mg++-limited cultures (about 20%) and in oxygen-limited nitrogen-fixing cultures (37%) . A method is described to calculate YATP values (g PHB-free biomass . mol-1 ATP) from the Ysucc values (g dry wt . mol-1 succinate) measured . Ysucc and YATP values in cultures limited by the supply of an anabolic substrate and in the oxygen-limited ammonia-assimilating culture were much lower than the values found in the PHB-free succinate-limited cultures . This shows that uncoupling of growth and energy production occurred . Therefore, H2/N2 ratio (mol hydrogen formed per mol nitrogen fixed) in nitrogen-fixing cultures could not be calculated from the comparison of the YATP value found in the nitrogen-fixing culture and the value found in the corresponding ammonia-assimilating culture . Although the optimal dissolved oxygen concentration (d.o.c.) for nitrogen-fixing cultures of Rhizobium ORS571 is 5 or 10 microM, nitrogen-fixing cultures could be obtained up to a d.o.c . of 40 microM . Not only nitrogenase but also hydrogenase was active at this d.o.c . However, accumulation of PHB (10%) may indicate that cultures grown at unfavourable oxygen concentrations (15-40 microM O2) were N-limited rather than energy-limited, which may be the result of partial inactivation or repression of nitrogenase at a higher d.o.c.

Gene, 1986, 50(1-3), 141 - 8
Overlapping transcription of the nifA regulatory gene in Rhizobium meliloti; Kim CH et al.; Gene fusions were used to demonstrate complex overlapping transcriptional controls of the regulatory gene nifA in Rhizobium meliloti bacteroids isolated from alfalfa root nodules . Gene nifA has previously been shown to be transcribed from promoter PnifA and to be required for activation of promoters P1 (nifHDK) and P2 (fixABC) during symbiotic nitrogen fixation with alfalfa . P2 is located approximately 4 kb upstream from nifA and is shown in this report to be responsible for at least one-half of the nifA expression observed in bacteroids . Substantial transcription of nifA occurs during symbiosis as evidenced by the fact that PnifA was found to be 53% as active as either P1 or P2 . Together, the data indicate that more than half of the transcripts initiated at P2 fail to terminate before reaching nifA . Additional studies indicated that there may be weak promoter activity in the symbiotically essential region downstream from nifA.

Gene, 1986, 48(2-3), 203 - 9
Construction of a Tn5 derivative determining resistance to gentamicin and spectinomycin using a fragment cloned from R1033; Hirsch PR et al.; A physical and genetic map of the IncP plasmid R1033 was constructed: restriction fragments were subcloned and antibiotic resistance genes were located . The map is consistent with previous reports that R1033 is a derivative of RP4 carrying a 16-kb transposon Tn1696 which contains the antibiotic-resistance determinants present on R1033 but not on RP4 . A BamHI fragment from R1033, determining resistance to gentamicin, spectinomycin and streptomycin, was cloned into Tn5, replacing the central Bg/II fragment that determined kanamycin resistance, producing a recombinant transposon Tn5-GmSpSm . This was shown to transpose in Rhizobium leguminosarum at a frequency similar to that of the parental Tn5.

Gene, 1986, 44(1), 89 - 96
Organization of the adenyl cyclase (cya) locus of Rhizobium meliloti; Lathigra R et al.; A cya-like gene encoding adenyl cyclase from Rhizobium meliloti was localized to a 0.8-kb PstI-EcoRI fragment by subcloning experiments . Experiments in Escherichia coli 'maxicells' identified a R . meliloti cya gene product of 28 kDa, which is significantly smaller than the corresponding protein from enteric bacteria . A control region for the expression of the cya gene in E . coli was found on an adjacent 2.6-kb BglII-BamHI sequence by insertional mutagenesis with Tn5 and phage MudI (ApR lac) . The direction of transcription of the cya gene was also determined using a cya::MudIlac fusion . Promoter activity of this cya::lac fusion was not decreased when glucose was added to the culture . The R . meliloti cya gene is conserved among R . meliloti strains but no homology could be detected to other Rhizobium species or to E . coli in DNA hybridization experiments.

Gene, 1986, 43(1-2), 95 - 101
The nodI gene product of Rhizobium leguminosarum is closely related to ATP-binding bacterial transport proteins; nucleotide sequence analysis of the nodI and nodJ genes; Evans IJ et al.; The nucleotide sequence of a 2-kb fragment immediately downstream of the nodABC genes of the Rhizobium leguminosarum symbiotic plasmid pRL1JI has been determined . Genes corresponding to the two open reading frames identified are named nodI and nodJ . Tn 5 insertions into these genes result in a "nodulation-delayed" phenotype . The predicted amino acid sequence of the nodI gene shows considerable homology to inner-membrane-located gene products involved in active transport systems in Escherichia coli and Salmonella typhimurium . The predicted product of the nodJ gene is very hydrophobic, suggesting that it may be an integral membrane protein.

J Bacteriol, 1986 Jan, 165(1), 72 - 81
Characterization of three genomic loci encoding Rhizobium sp . strain ORS571 N2 fixation genes; Donald RG et al.; Sixty-five independent, N2 fixation-defective (Nif-) vector insertion (Vi) mutants were selected, cloned, and mapped to the ORS571 genome . The recombinant Nif::Vi plasmids obtained in this way were used as DNA hybridization probes to isolate homologous phages from a genomic library of ORS571 constructed in lambda EMBL3 . Genomic maps were drawn for three ORS571 Nif gene loci . Forty-five Nif::Vi mutants in genomic Nif locus 1 defined two gene clusters separated by 8 kilobase pairs (kb) of DNA . In the first cluster, 36 Nif::Vi mutants mapped to a 7-kb DNA segment that showed DNA homology with Klebsiella pneumoniae nifHDKE and encoded at least two Nif operons . In the other cluster, nine Nif::Vi mutants mapped to a 1.5-kb DNA segment that showed homology with K . pneumoniae and Rhizobium meliloti nifA; this DNA segment encoded a separate Nif operon . Fifteen Nif::Vi mutants mapped to a 3.5-kb DNA segment defined as Nif locus 2 and showed DNA homology with the R . meliloti P2 fixABC operon . Nif locus 2 carries a second nifH (nifH2) gene . Four Nif::Vi mutants mapped to a 2-kb DNA segment defined as Nif locus 3 and showed DNA homology with K . pneumoniae nifB . DNA from lambda Nif phages comprising all three genomic Nif loci was subcloned in plasmid vectors able to stably replicate in ORS571 . These plasmid subclones were introduced into ORS571 strains carrying physically mapped Nif::Vi insertions, and genetic complementations were conducted . With the exception of certain mutants mapping to the nifDK genes, all mutants could be complemented to Nif+ when they carried plasmid subclones of defined genomic DNA regions . Conversely, most nifDK mutants behaved as pseudodominant alleles.

J Gen Microbiol, 1985 Dec, 131 ( Pt 12), 3287 - 302
Characterization of the anomalous infection and nodulation of subterranean clover roots by Rhizobium leguminosarum 1020; Hrabak EM et al.; Anomalous nodulation of Trifolium subterraneum (subterranean clover) roots by Rhizobium leguminosarum 1020 was examined as a model of modified host-specificity in a Rhizobium-legume symbiosis . Consistent with previous reports, these nodules (i) appeared most often at sites of secondary root emergence, (ii) were ineffective in nitrogen fixation and (iii) were as numerous as nodules formed by an effective Rhizobium trifolii strain . R . leguminosarum 1020, grown on agar plates or in the clover root environment, did not bind the white clover lectin, trifoliin A . This strain did not attach in high numbers, and did not induce shepherd's crooks or infection threads, in subterranean clover root hairs . However, R . leguminosarum 1020 did cause branching, moderate curling and other deformations of root hairs . The bacteria probably entered the clover root through breaks in the epidermis at sites of lateral root emergence . The anomalous nodulation was inhibited by nitrate . Only trace amounts of leghaemoglobin were detected in the nodules by Western blot analysis . The nodules were of the meristematic type and initially contained well-developed infection, bacteroid and senescent zones . Infection threads were readily found in the infection zone of the nodule . However, the bacteroid-containing tissue senesced more rapidly than in the effective symbiosis between subterranean clover and R . trifolii 0403 . This anomalous nodulation of subterranean clover by R . leguminosarum 1020 suggests a naturally-occurring alternative route of infection that allows Rhizobium to enlarge its host range.

J Bacteriol, 1985 Dec, 164(3), 1383 - 5
Proton motive force in washed cells of Rhizobium japonicum and bacteroids from Glycine max; Bhandari B et al.; The components of the proton motive force (delta p), namely the membrane potential and the transmembrane pH gradient, were measured in washed cells of Rhizobium japonicum CC705 grown in cultures (5% O2-95% N2) in the presence of 10 mM KNO3 and in bacteroids from Glycine max . The delta p and its components remained reasonably constant in cells as well as in bacteroids at various stages of growth . The effects of uncouplers and ATPase inhibitors on the delta p and its components were determined in both cultured cells and bacteroids . The data indicated that a respiration-driven H+ translocation is the source of the delta p in both cultured cells and bacteroids.

J Bacteriol, 1985 Dec, 164(3), 1301 - 8
Isolation and characterization of the DNA region encoding nodulation functions in Bradyrhizobium japonicum; Russell P et al.; The DNA region encoding early nodulation functions of Bradyrhizobium japonicum 3I1b110 (I110) was isolated by its homology to the functionally similar region from Rhizobium meliloti . Isolation of a number of overlapping recombinant clones from this region allowed the construction of a restriction map of the region . The identified nodulation region of B . japonicum shows homology exclusively to those regions of R . meliloti and Rhizobium leguminosarum DNA known to encode early nodulation functions . The region of homology with these two fast-growing Rhizobium species was narrowed to an 11.7-kilobase segment . A nodulation-defective mutant of Rhizobium fredii USDA 201, strain A05B-2, was isolated and found to be defective in the ability to curl soybean root hairs . Some of the isolated recombinant DNA clones of B . japonicum were found to restore wild-type nodulation function to this mutant . Analysis of the complementation results allows the identification of a 1.8-kilobase region as essential for restoration of Hac function.

Appl Environ Microbiol, 1985 Nov, 50(5), 1219 - 24
Isolation and partial characterization of the extracellular polysaccharides and lipopolysaccharides from fast-growing Rhizobium japonicum USDA 205 and its Nod- mutant, HC205, which lacks the symbiotic plasmid; Carlson RW et al.; The extracellular polysaccharides and lipopolysaccharides (LPSs) from two fast-growing Rhizobium japonicum strains, USDA 205 and HC205, were isolated and partially characterized . Strain HC205 is a Nod- mutant of USDA 205 which lacks the symbiotic plasmid . The extracellular polysaccharides from both strains are very similar in composition, having galactose, glucose, glucuronic acid, and acyl groups . The extracellular polysaccharides do not contain detectable levels of pyruvate . Methylation analysis shows that the extracellular polysaccharides from both strains have the same glycosyl linkages . The LPSs were purified by a modified phenol-water extraction procedure and gel filtration chromatography . The LPSs from USDA 205 and HC205 elute as broad peaks from the gel filtration column and contain 2-keto-3-deoxyoctonic acid as one of the major sugar components . Each broad 2-keto-3-deoxyoctonic acid-containing peak has a distinct shoulder on its leading edge . The shoulder and the remainder of the broad peak are separated and labeled LPSI and LPSII, respectively . Glucose (and 2-keto-3-deoxyoctonic acid) is a major sugar in the LPSI fractions . Both the LPSII fractions contain 2-keto-3-deoxyoctonic acid as the major sugar (about 20% of the mass) . There are a number of quantitative differences in these LPS fractions between strain USDA 205 and HC205 . Polyacrylamide gel electrophoresis shows that the LPSs are heterogeneous molecules but less heterogeneous than the LPSs from Salmonella minnesota or Rhizobium leguminosarum . The LPSI fractions from both USDA 205 and HC205 show a single lower-molecular-weight band and a higher-molecular-weight banding region which contains several bands . No bands are observed for the LPSII fractions from either USDA 205 or HC205.

J Bacteriol, 1985 Nov, 164(2), 929 - 31
Measurement of the proton motive force in Rhizobium meliloti with the Escherichia coli lacY gene product; Gober JW et al.; An Escherichia coli lac operon constitutive for lacY was subcloned into the EcoRI site of a wide-host-range plasmid of the Q incompatibility group, and the resulting recombinant plasmid was introduced into Tn5-generated Lac- mutants of Rhizobium meliloti . The R . meliloti transconjugants accumulated lactose about 1,000-fold, equivalent to a proton motive force of -170 to -180 mV, not significantly different from the values calculated from the distributions of weak acids and lipophilic cations.

J Bacteriol, 1985 Nov, 164(2), 757 - 61
Inhibition of growth of Rhizobium japonicum by cyclic GMP; Jones BL et al.; Exogenous cyclic guanosine-3',5'-monophosphate (cGMP) inhibited the growth of Rhizobium japonicum at less than 100 microM . Other nucleotides, including cyclic AMP, cyclic IMP, and cyclic CMP, had no inhibitory effect even at higher concentrations nor was the inhibition by cGMP reversed by cyclic AMP . The inhibitory effect was independent of the carbon and nitrogen source(s) used . cGMP did not inhibit the growth of any other species of bacterium tested, including several fast-growing Rhizobium species . The kinetics of growth inhibition are multiphasic, with no apparent effect for several hours after addition, followed by a period of total inhibition . Subsequently, growth resumed at a slower rate . Resumption of growth was not due to destruction of the nucleotide . Studies of the intracellular cGMP concentration did not reveal significant changes in cells grown under aerobic or microaerobic conditions . No effect of cGMP on the derepression of respiratory nitrate reductase was observed.

J Bacteriol, 1985 Nov, 164(2), 591 - 9
Rhizobium meliloti nodulation genes: identification of nodDABC gene products, purification of nodA protein, and expression of nodA in Rhizobium meliloti; Egelhoff TT et al.; A set of conserved, or common, bacterial nodulation (nod) loci is required for host plant infection by Rhizobium meliloti and other Rhizobium species . Four such genes, nodDABC, have been indicated in R . meliloti 1021 by genetic analysis and DNA sequencing . An essential step toward understanding the function of these genes is to characterize their protein products . We used in vitro and maxicell Escherichia coli expression systems, together with gel electrophoresis and autoradiography, to detect proteins encoded by nodDABC . We facilitated expression of genes on these DNA fragments by inserting them downstream of the Salmonella typhimurium trp promoter, both in colE1 and incP plasmid-based vectors . Use of the incP trp promoter plasmid allowed overexpression of a nodABC gene fragment in R . meliloti . We found that nodA encodes a protein of 21 kilodaltons (kDa), and nodB encodes one of 28 kDa; the nodC product appears as two polypeptide bands at 44 and 45 kDa . Expression of the divergently read nodD yields a single polypeptide of 33 kDa . Whether these represent true Rhizobium gene products must be demonstrated by correlating these proteins with genetically defined Rhizobium loci . We purified the 21-kDa putative nodA protein product by gel electrophoresis, selective precipitation, and ion-exchange chromatography and generated antiserum to the purified gene product . This permitted the immunological demonstration that the 21-kDa protein is present in wild-type cells and in nodB- or nodC-defective strains, but is absent from nodA::Tn5 mutants, which confirms that the product expressed in E . coli is identical to that produced by R . meliloti nodA . Using antisera detection, we found that the level of nodA protein is increased by exposure of R . meliloti cells to plant exudate, indicating regulation of the bacterial nod genes by the plant host.

J Bacteriol, 1985 Oct, 164(1), 78 - 84
Accumulation of alpha,alpha-trehalose by Rhizobium bacteria and bacteroids; Streeter JG; Four strains of Rhizobium japonicum (61A76 and USDA 110, 123, and 138) were grown in eight different defined media . Regardless of the carbon or nitrogen source supplied, alpha, alpha-trehalose was the major carbohydrate (among mono- and disaccharides) accumulated by all four strains . After 7 to 9 days of growth, trehalose generally accounted for 90 to 100% of the mono- and disaccharides detected . None of the four strains would grow with trehalose as a carbon source, but the utilization of endogenous trehalose was demonstrated under carbon starvation conditions in water culture or when the carbon supply in a defined medium was exhausted . Under these conditions, a small amount of trehalose was lost from cells to the medium . In a survey of most of the serogroups of R . japonicum and several strains of other Rhizobium species, all strains tested were found to accumulate some trehalose . Trehalose concentrations varied widely; the highest concentration recorded was 41 micrograms/mg of dry weight . In all but six strains trehalose accounted for greater than 80% of the mono- and disaccharides in cells . Fast-growing strains of R . japonicum also accumulated small amounts trehalose . R . japonicum bacteroids also synthesized trehalose; the quantity in nodules varied in approximate correspondence to accumulation of trehalose by cultured bacteria . In young soybean nodules (29 days after planting), 45 to 80% of the trehalose was recovered in the cytosol . There were differences among R . japonicum strains in the retention of trehalose, and the proportion of trehalose retained by bacteroids increased with increasing plant age for all strains.

Proc Natl Acad Sci U S A, 1985 Oct, 82(19), 6609 - 13
Induction of Rhizobium meliloti nodC expression by plant exudate requires nodD; Mulligan JT et al.; The soil bacterium Rhizobium meliloti invades and establishes a symbiosis with host plants such as alfalfa . Bacterial nodulation (nod) genes are required for this invasion, but their mechanism of action and the timing of their expression are not known . We have used translational lacZ fusions to monitor expression of nodD and nodC, which are located in the cluster of four nod genes on the R . meliloti megaplasmid (pSym) . nodD is expressed at comparable levels by broth-grown bacterial cells and by cells exposed to exudates from aseptically grown plants . Activity of the nodC-lacZ protein fusion in broth-grown bacterial cells is very low . nodC-lacZ activity is increased approximately equal to 30-fold by plant exudate when nodD is expressed at a high level but not when nodD expression is low . Both fusions show differences in expression when borne on inc-P vectors as compared to when located on the pSym megaplasmid . nodD expression from vector-borne copies of the nod segment and response of nodC to plant exudate appear to require additional loci on the megaplasmid . Our results suggest that regulation of bacterial nod gene expression is an important control mechanism early in the symbiosis, and that the biochemical nature of some nod gene products may be cryptic except in cells grown in the presence of plant exudate.

J Bacteriol, 1985 Oct, 164(1), 245 - 54
Characterization of a Rhizobium meliloti fixation gene (fixF) located near the common nodulation region; Aguilar OM et al.; Rhizobium meliloti 2011 DNA from pRmSL26, a plasmid which is known to carry genes involved in the early stages of nodulation, was used to construct Tn5 mutations by site-directed Tn5 mutagenesis . Tn5 mutations located within an 8.7 kilobase EcoRI fragment defined two adjacent loci encoding functions for nodulation (nod) and symbiotic N2 fixation (fix) . We investigated the organization and regulation of the fix locus and the characteristics of alfalfa nodules induced by these Fix- mutants . By monitoring expression in Escherichia coli minicells, we determined that the fix locus encoded a 36-kilodalton polypeptide . The gene corresponding to this locus was designated fixF . Morphological and ultrastructural studies of the ineffective nodules formed by R . meliloti fixF mutants showed infected host cells similar to those of the wild type . The ineffective nodules were able to accumulate leghemoglobin, but at lower levels than those found in the wild-type nodules . Expression of the nifHDK operon was unaffected by Tn5 insertions in the fixF gene . Expression of the fixF gene was monitored in E . coli by using translational lacZ fusions . It was shown that transcription of the fixF gene in E . coli could be activated by Klebsiella pneumoniae nifA and the R . meliloti nifA-like regulatory gene products . Expression of the fixF gene was also studied in free-living and symbiotic R . meliloti cells . It was found that the fixF gene was transcribed in the symbiotic state.

J Bacteriol, 1985 Oct, 164(1), 187 - 91
Further evidence that two unique subunits are essential for expression of hydrogenase activity in Rhizobium japonicum; Harker AR et al.; Eight strains of Rhizobium lacking hydrogenase uptake (Hup) activity and 17 transconjugant strains carrying the hup cosmids pHU1, pHU52, or pHU53 (G . R . Lambert, M . A . Cantrell, F . J . Hanus, S . A . Russell, K . R . Haddad, and H . J . Evans, Proc . Natl . Acad . Sci . USA, 82:3232-3236, 1985) were screened for Hup activity and the presence of immunologically detectable hydrogenase polypeptides . Crude extracts of these strains were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis with affinity-purified antibodies against the two subunits of purified hydrogenase (Mr 60,000 and 30,000) . Derepressed transconjugants carrying the cosmid pHU52 were Hup+ and contained detectable levels of both hydrogenase subunit polypeptides . Non-derepressed strains, Hup- parent strains, and strains carrying cosmids other than pHU52 did not express Hup activity and contained no immunologically detectable protein . These data provide further evidence for the essential involvement of the smaller (Mr 30,000) subunit in the expression of hydrogenase activity in Rhizobium japonicum and suggest that the determinants for hydrogenase subunit synthesis are present on pHU52.

J Bacteriol, 1985 Oct, 164(1), 410 - 3
Cryptic plasmid and rifampin resistance in Rhizobium meliloti influencing nodulation competitiveness; Bromfield ES et al.; An assessment was made of the relative contributions of a spontaneous mutation to rifampin resistance and a cryptic plasmid, pTA2, to competitive nodulation of Medicago sativa by a strain of Rhizobium meliloti . This was facilitated by use of rifampin-resistant derivatives of this strain in which pTA2 was originally present, cured, or reintroduced . Both curing of pTA2 and spontaneous mutation to rifampin resistance significantly influenced nodulating competitiveness, but the effect of rifampin resistance was greater and such that the contribution of pTA2 was evident only in cases in which paired competitors had the common rifampin resistance background . The data suggest that rifampin-resistant derivatives contain an altered RNA polymerase insensitive to the action of rifampin . All R . meliloti derivatives had symbiotic characteristics and phage susceptibility patterns similar to those of the wild type . Plasmid pTA2 transfer or other genetic interchange was not detected in nodules of M . sativa inoculated with paired competitors.

Proc Natl Acad Sci U S A, 1985 Sep, 82(18), 6231 - 5
Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules; Leigh JA et al.; 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.

Clin Lab Med, 1985 Sep, 5(3), 413 - 36
Plasmid fingerprinting . A tool for bacterial strain identification and surveillance of nosocomial and community-acquired infections; Tenover FC; Plasmid fingerprinting provides a rapid and dependable means of identifying bacterial isolates of the same strain . The stability, wide distribution, and diverse nature and size of extrachromosomal elements make it suitable for virtually all bacterial genera . There are many different procedures available for plasmid screening, and the one chosen depends primarily on the types of organisms to be analyzed . Some procedures are better suited to gram-positive organisms; others are better for visualizing the very large plasmids often seen in Pseudomonas and Rhizobium species . The key to the plasmid fingerprinting technique is agarose gel electrophoresis . In this step of the technique, it is important to differentiate open circular from closed circular forms of plasmids and to recognize the "smile effect." Plasmid fingerprinting can be utilized for epidemiologic studies of both nosocomial and community-acquired infections . The use of restriction endonuclease analysis can greatly enhance the ability of the investigator to differentiate strains that harbor only a single plasmid . Plasmid fingerprinting often provides the only differential characteristic for strains involved in epidemics.

J Bacteriol, 1985 Sep, 163(3), 1282 - 4
Catabolite repression and role of cyclic AMP in CO2 fixation and H2 metabolism in Rhizobium spp; McGetrick AM et al.; CO2 fixation in Rhizobium meliloti was repressed by a variety of organic carbon sources . Cellular cyclic AMP levels were similar in repressed and nonrepressed cultures . Exogenous cyclic AMP or additional copies of the adenyl cyclase gene in cells experiencing repression failed to affect the rates of CO2 fixation . However, in R . japonicum catabolite repression of H2 utilization was partially circumvented by the presence of the R . meliloti adenyl cyclase gene.

Nucleic Acids Res, 1985 Aug 26, 13(16), 5965 - 76
Analysis of the 5' regulatory region of the gene for delta-aminolevulinic acid synthetase of Rhizobium meliloti; Leong SA et al.; Transcriptional regulation of the delta-aminolevulinic acid synthetase gene of Rhizobium meliloti was investigated under conditions of normal vegetative growth and during symbiosis with the legume host alfalfa . S1 nuclease mapping and DNA sequence analysis indicated that transcription originates from two sites separated by 238 base pairs . A deletion analysis of the putative promoter regions P1 and P2, corresponding to the proximal and distal RNA start sites, was carried out with Bal-31 nuclease . Promoter function was monitored as beta-galactosidase activity after fusing the deletions to lac Z and introducing them into Rhizobium on a broad host range plasmid . The data obtained suggest that both regions function equivalently as promoters . The DNA sequences of P1 and P2 show considerable homology in the region between -35 and the start of transcription . Both contain a -35 region that is analogous to the consensus E . coli promoter sequence, while the -10 region is dissimilar . No resemblance was found between either P1 or P2 and the promoter regions of genes under general nitrogen control.

J Bacteriol, 1985 Aug, 163(2), 812 - 5
Expression of Rhizobium trifolii early nodulation genes on maize and rice plants; Plazinski J et al.; An IncQ multicopy vector (pKT230) and an IncP1 low-copy-number vector (pRK290), both carrying Rhizobium trifolii root hair curling (Hac) genes, were transferred to a Sym plasmid-cured derivative of R . trifolii ANU843 . The resulting transconjugants were used to inoculate the monocotyledonous plants sorghum, maize, rice, and wheat . Transconjugants carrying the Hac genes on the multicopy vector caused a root hair curling response on maize and rice plants 14 days after inoculation.

J Bacteriol, 1985 Aug, 163(2), 417 - 22
Chemotaxis to aromatic and hydroaromatic acids: comparison of Bradyrhizobium japonicum and Rhizobium trifolii; Parke D et al.; Rhizobia are bacteria well known for their ability to fix nitrogen in symbiosis with leguminous plants . Members of diverse rhizobial species grow at the expense of hydroaromatic and aromatic compounds commonly found in plant cells and plant litter . Using a quantitative capillary assay to measure chemotaxis, we tested the ability of hydroaromatic acids, selected aromatic acids, and their metabolites to serve as chemoattractants for two distantly related rhizobial species, Bradyrhizobium japonicum and Rhizobium trifolii . Slow-growing B . japonicum I-110 demonstrated positive chemotaxis to shikimate, quinate, protocatechuate, and vanillate; threshold concentrations for the compounds were as low as 10(-6) M . The dicarboxylic acids succinate and beta-ketoadipate, metabolites in the catabolism of many aromatic compounds, were positive chemoattractants with low threshold concentrations as well . Taxis to beta-ketoadipate occurred constitutively and, of the tested compounds, beta-ketoadipate gave the strongest peak response . Taxis to shikimate or quinate was induced by growth on either substrate but not by growth on protocatechuate or succinate . In contrast, fast-growing R . trifolii 2066 was only weakly attracted to quinate and other aromatic and dicarboxylic acids that were strong attractants for B . japonicum . The R . trifolii strain exhibited positive chemotaxis to shikimate, but the threshold concentration of shikimate required to elicit a response (10(-4) M) was 2 orders of magnitude higher than that for the B . japonicum strain.

Mol Gen Mikrobiol Virusol, 1985 Aug, (8), 13 - 5
{Analysis of plasmids in Rhizobium meliloti strains of different geographical origin}; Zlotnikov KM et al.; The presence of 1-3 additional plasmids ranging from 16 to 400 Md in mol mass was revealed in bacterial cells besides a megaloplasmid in most of 34 Rhizobium meliloti strains studied . No correlation was found between plasmids pattern and geographical origin of isolation source of the strain analyzed.

J Bacteriol, 1985 Jul, 163(1), 21 - 6
Conservation of symbiotic nitrogen fixation gene sequences in Rhizobium japonicum and Bradyrhizobium japonicum; Masterson RV et al.; Southern hybridization with nif (nitrogen fixation) and nod (nodulation) DNA probes from Rhizobium meliloti against intact plasmid DNA of Rhizobium japonicum and Bradyrhizobium japonicum strains indicated that both nif and nod sequences are on plasmid DNA in most R . japonicum strains . An exception is found with R . japonicum strain USDA194 and all B . japonicum strains where nif and nod sequences are on the chromosome . In R . japonicum strains, with the exception of strain USDA205, both nif and nod sequences are on the same plasmid . In strain USDA205, the nif genes are on a 112-megadalton plasmid, and nod genes are on a 195-megadalton plasmid . Hybridization to EcoRI digests of total DNA to nif and nod probes from R . meliloti show that the nif and nod sequences are conserved in both R . japonicum and B . japonicum strains regardless of the plasmid or chromosomal location of these genes . In addition, nif DNA hybridization patterns were identical among all R . japonicum strains and with most of the B . japonicum strains examined . Similarly, many of the bands that hybridize to the nodulation probe isolated from R . meliloti were found to be common among R . japonicum strains . Under reduced hybridization stringency conditions, strong conservation of nodulation sequences was observed in strains of B . japonicum . We have also found that the plasmid pRjaUSDA193, which possess nif and nod sequences, does not possess sequence homology with any plasmid of USDA194, but is homologous to parts of the chromosome of USDA194 . Strain USDA194 is unique, since nif and nod sequences are present on the chromosome instead of on a plasmid as observed with all other strains examined.

Appl Environ Microbiol, 1985 Jul, 50(1), 41 - 4
Evidence for plasmid- and chromosome-borne multiple nif genes in Rhizobium fredii; Barbour WM et al.; Rhizobium fredii is a fast-growing rhizobium isolated from the primitive Chinese soybean cultivar Peking and from the wild soybean Glycine soja . This rhizobium harbors nif genes on 150- to 200-megadalton plasmids . By passage on acridine orange plates, we obtained a mutant of R . fredii USDA 206 cured of the 197-megadalton plasmid (USDA 206C) which carries both nif and nod genes . This strain, however, has retained its symbiotic effectiveness . Probing EcoRI digests of wild-type and cured plasmid DNA with a 2.2-kilobase nif DH fragment from Rhizobium meliloti has shown four homologous fragments in the wild-type strain (4.2, 4.9, 10, and 11 kilobases) and two fragments in the cured strain (4.2 and 10 kilobases) . EcoRI digests of total DNA show four major bands of homology (4.2, 4.9, 5.8, and 13 kilobases) in both the wild-type and cured strains . The presence of major bands of homology in the total DNA not present in the plasmid DNA indicated chromosomal nif genes . Probing of HindIII digests of total and plasmid DNA led to the same conclusion . Hybridization to the smaller plasmids of USDA 206 and USDA 206C showed the presence of nif genes on at least one of these plasmids, explaining the nif homology in the USDA 206C plasmid digests.

J Bacteriol, 1985 Jul, 163(1), 385 - 8
Expression of symbiotic genes of Rhizobium japonicum USDA 191 in other rhizobia; Appelbaum ER et al.; A 200-megadalton plasmid was mobilized from Rhizobium japonicum USDA 191 to other Rhizobium strains either that cannot nodulate soybeans or that form Fix- nodules on certain cultivars . The symbiotic properties of the transconjugants indicate that both soybean specificity for nodulation and cultivar specificity for nitrogen fixation are plasmid encoded.

J Bacteriol, 1985 Jul, 163(1), 148 - 54
Slow-growing Rhizobium japonicum comprises two highly divergent symbiotic types; Stanley J et al.; We examined the interrelationships of the genomes of 10 slow-growing strains of Rhizobium japonicum to provide a foundation for molecular genetic studies of these agriculturally important endosymbiotic bacteria of commercial soybeans . The degree of base substitution in and around known symbiotic genes (nif and presumptive nod), constitutively expressed genes (glnA and recA), and two other cloned sequences was estimated from restriction site variation by using cloned DNAs as hybridization probes to genomic Southern blots . Two highly divergent patterns of conservation of nifDH genes and nod-homologous sequences were found . On this basis, we classified the strains as the symbiotic genotypes sTI or sTII . Existing maps of the nif genes of R . japonicum apply only to strains of the sTI genotype . This division was further characterized by four other probes which also distinguished two sublines within sTI . Phenograms were constructed depicting interrelationships according to DNA sequence divergence . sTI and sTII are two highly divergent evolutionary lines consistent with the status of individual species . Neither is related to fast-growing Rhizobium strains (PRC strains) nodulating soybeans.

Nucleic Acids Res, 1985 Jun 25, 13(12), 4539 - 55
Nitrogen fixation specific regulatory genes of Klebsiella pneumoniae and Rhizobium meliloti share homology with the general nitrogen regulatory gene ntrC of K . pneumoniae; Buikema WJ et al.; We have determined the complete nucleotide sequences of three functionally related nitrogen assimilation regulatory genes from Klebsiella pneumoniae and Rhizobium meliloti . These genes are: 1) The K . pneumoniae general nitrogen assimilation regulatory gene ntrC (formerly called glnG), 2) the K . pneumoniae nif-specific regulatory gene nifA, and 3) an R . meliloti nif-specific regulatory gene that appears to be functionally analogous to the K . pneumoniae nifA gene . In addition to the DNA sequence data, gel-purified K . pneumoniae nifA protein was used to determine the amino acid composition of the nifA protein . The K . pneumoniae ntrC and nifA genes code for proteins of 52,259 and 53,319 d respectively . The R . meliloti nifA gene codes for a 59,968 d protein . A central region within each polypeptide, consisting of approximately 200 amino acids, is between 52% and 58% conserved among the three proteins . Neither the amino termini nor the carboxy termini show any conserved sequences . Together with data that shows that the three regulatory proteins activate promoters that share a common consensus sequence in the -10 (5'-TTGCA-3') and -23 (5'-CTGG-3') regions, the sequence data presented here suggest a common evolutionary origin for the three regulatory genes.

DNA, 1985 Jun, 4(3), 241 - 8
Nucleotide sequence of Rhizobium meliloti 1021 nodulation genes: nodD is read divergently from nodABC; Egelhoff TT et al.; Nodulation (nod) genes are required for Rhizobium meliloti to invade and stimulate nodule formation in its host, alfalfa . We have established the DNA sequence of nodD, nodA, and nodB, which are part of a gene cluster located 20 kb downstream of nifHDK on the R . meliloti pSym megaplasmid . The nodD open reading frame (308 amino acids) reads from proximal to nifHDK toward distal to nifHDK, divergently from nodA (196 aa) and nodB (217 aa) . These two genes read from distal to nifHDK toward proximal, and are just upstream from the previously defined open reading frame for nodC . Fourteen Tn5 insertion sites have been sequenced in nodD, nodA, and nodB, revealing no major hotspots for insertion, but an overall preference for G/C bases at positions 1 and 9 of the 9-bp repeat.

J Bacteriol, 1985 Jun, 162(3), 950 - 9
Nodule initiation elicited by noninfective mutants of Rhizobium phaseoli; Vandenbosch KA et al.; Rhizobium phaseoli CE106, CE110, and CE115, originally derived by transposon mutagenesis (Noel et al., J . Bacteriol . 158:149-155, 1984), induced the formation of uninfected root nodule-like swellings on bean (Phaseolus vulgaris) . Bacteria densely colonized the root surface, and root hair curling and initiation of root cortical-cell divisions occurred normally in mutant-inoculated seedlings, although no infection threads formed . The nodules were ineffective, lacked leghemoglobin, and were anatomically distinct from normal nodules . Ultrastructural specialization for ureide synthesis, characteristic of legumes that form determinate nodules, was absent . Colony morphology of the mutant strains on agar plates was less mucoid than that of the wild type, and under some cultural conditions, the mutants did not react with Cellufluor, a fluorescent stain for beta-linked polysaccharide . These observations suggest that the genetic lesions in these mutants may be related to extracellular polysaccharide synthesis.

J Bacteriol, 1985 Jun, 162(3), 1261 - 9
Sym plasmid genes of Rhizobium trifolii expressed in Lignobacter and Pseudomonas strains; Plazinski J et al.; A 14-kilobase (kb) fragment of Rhizobium trifolii Sym plasmid containing nodulation (nod) genes or the pSym plasmid of R . trifolii cointegrated with a broad-host-range vector R68.45 (pPN1) were transferred to Lignobacter strain K17 and Pseudomonas aeruginosa strain PAO5 by conjugation . Lignobacter transconjugants carrying Sym plasmid pPN1 formed nodules on white, red, and subterranean clover plants . Lignobacter transconjugants containing a 14-kb fragment of nod genes cloned into a multicopy plasmid nodulated only white and subterranean clover plants, whereas transconjugants carrying the same fragment cloned into a low-copy plasmid vector nodulated only white clover plants . All nodules formed by Lignobacter transconjugants showed bacterial release from the infection threads into the host cytoplasm . Pseudomonas transconjugants with plasmid pPN1 formed nodule-like structures on white clover plants . These structures were not invaded by bacteria; however, a few bacteria were found within the intercellular spaces of the outermost cells of the structures . Pseudomonas transconjugants carrying the 14-kb fragment of R . trifolii nod genes did not form nodules on tested clover plants . All clover plants inoculated with either Pseudomonas or Lignobacter transconjugants containing a 14-kb fragment of nod genes (but not entire Sym plasmid) showed the "thick-and-short-root" response when compared to the control plants inoculated with the R . trifolii wild-type strain.

J Bacteriol, 1985 Jun, 162(3), 1079 - 82
Bacteriocin small of fast-growing rhizobia is chloroform soluble and is not required for effective nodulation; van Brussel AA et al.; Small bacteriocin is a low-molecular-weight bacteriocin which is common in fast-growing rhizobia . As its activity could not be detected in chloroform-sterilized culture supernatants (P.R . Hirsch, J . Gen . Microbiol . 113:219-228, 1979), the bacteriocin could not be purified in order to study its mechanism of action . We report here that small is soluble in chloroform, an observation which led to effective and simple (partial) purification . Other properties of small are its low molecular weight, which is estimated to be between 700 and 1,500, its resistance to proteolytic enzymes, pectinase, and lysozyme, and its heat stability at pH 5.5 but not at pH 7.0 . Its bactericidal action on exponentially growing sensitive cells was not detected until 11 h after its addition . The bactericidal action was preceded by inhibition of cell division . To determine whether small activity is required for nodulation or nitrogen fixation, a transposon Tn5-induced small-negative mutant was isolated . The observation that this strain formed normal, acetylene-reducing root nodules showed that small production is not a prerequisite for the formation of effective nodules.

Microbiol Sci, 1985 Jun, 2(6), 161 - 2, 165-7
Ammonia movements in rhizobia; Glenn AR et al.; When free-living rhizobia are grown under N-excess conditions they appear to take up ammonia by a diffusive mechanism . Under low or limiting N, they derepress an ammonium permease which serves to scavenge NH4+ . Current data suggest that N2-fixing bacteroids lose ammonia by a diffusive movement sustained by the continual removal of ammonia via the plant ammonia assimilatory system(s).

Nucleic Acids Res, 1985 May 24, 13(10), 3407 - 18
Conservation of nif- and species-specific domains within repeated promoter sequences from fast-growing Rhizobium species; Schofield PR et al.; In the fast-growing Rhizobium species, repeated DNA sequences, which include the promoter region of the nif HDK operon have been described . These repeated sequences are promoters which specifically activate transcription in the endosymbiotic state . Hybridization analysis of these sequences from R . trifolii has revealed that they may be involved in the species-specific activation of the various genes whose transcription they promote . Comparative analysis of various copies of these repeated sequences, from R . trifolii (the clover symbiont) and R . meliloti (the alfalfa symbiont), reveals the presence of domains of intra- and interspecific conservation within the promoter regions . We suggest that these promoter elements represent sites which are involved in the species-specific and general, nif-specific activation of Rhizobium symbiotic genes.

FEBS Lett, 1985 May 20, 184(2), 294 - 8
Replacement of the deoxycytidine residues in Rhizobium bacteriophage RL38JI DNA; Swinton D et al.; Rhizobium phage RL38JI DNA is resistant to cleavage by a variety of restriction endonucleases, and is only partially sensitive to digestion by pancreatic DNase I or by micrococcal nuclease . We have found that a mixture of DNase I, P1 nuclease, and bacterial alkaline phosphatase will quantitatively digest RL38JI DNA to deoxyribonucleosides . HPLC analysis revealed that dCyd is nearly totally absent among these digestion products, while dGuo, dAdo, and Thd are readily detected . Three additional peaks are always present; their retention properties correspond to no known modified deoxyribonucleosides . Thus it appears that dCyd is replaced in phage RL38JI DNA by as many as 3 different modified residues.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1985 May, 18(2), 123 - 34
{The influence of acidic conditions on polar exopolysaccharides which bind Rhizobium japonicum to soybean root hairs}; Cheng RH et al.; The influence of acidic conditions on rhizobial exopolysaccharides (EPS) and their symbiotic association in host plants, were investigated . Light micrographs of soybean root hairs were taken with a Fahraeus' slide assembly after the inocubation of rhizobial bacteria; curled and swollen root hairs, and the typical shepherd's crook with an infection thread were observed . The colonizations of soybean root hairs by rhizobia were different under acidic and neutral conditions . For the Rhizobium japonicum strain USDA 136, and the indigenous acidic soil strain KR 23, morphologically distinct types of cells occurred in the acidic and neutral yeast-extract-mannitol broths . The surface structure of R . japonicum, as examined with the carbon replica and negative stain techniques, was rod shaped and polar flagellated only when the cells were incubated under neutral conditions . The materials which reacted with the ruthenium red positive reagent are in all likelihood an acidic EPS, and are believed to effect the adhesive properties of the cells . Bacterial polarity, which could be more readily observed under neutral conditions than under acidic conditions, was related to the distribution of EPS near one end of the bacteria . EPS was found to effect the initial step of polar attachment in the effective infection of host roots . By using DEAE-Sephacel ion exchange chromatography, the capsular polysaccharides from both strains of R . japonicum were also found to have different chemical components when observed in acidic and neutral cultures.

J Bacteriol, 1985 May, 162(2), 784 - 9
Legume agglutinins that bind to Rhizobium meliloti; Seegers R et al.; A protein found in seeds and roots of alfalfa (Medicago sativa) was implicated in the specificity of the infection process, based on its binding to the symbiont Rhizobium meliloti . We found an agglutinin with similar properties in seeds and roots of sweet clover (Melilotis alba) . The sweet clover differed from alfalfa in nodulation by a mutant strain of R . meliloti, but the agglutinins were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Rhizobium agglutination, and cross-reactivity to antibodies . Similar agglutinins binding R . meliloti were found in seeds of legumes from different cross-inoculation groups, including soybean (Glycine max), cowpea (Vigna unguiculata), pea (Pisum sativum L), and mung bean (Vigna mungo) . The agglutinins from these legumes were recognized by antibodies raised against the agglutinins of alfalfa and sweet clover . Seeds of corn (Zea mays) and tomato (Lycopersicon esculentum) contained a protein similar to the legume agglutinin, but it did not react with the antibodies . We conclude that the alfalfa agglutinin is representative of a common legume protein and that there is no evidence for its role in specificity or nodule initiation.

J Bacteriol, 1985 May, 162(2), 645 - 50
Purification and subunit characterization of Rhizobium meliloti RNA polymerase; Nielsen BL et al.; The RNA polymerase of the symbiotic, nitrogen-fixing bacterium Rhizobium meliloti was purified, and its subunit composition was determined . The cells were disrupted in the presence of protease inhibitors, and holoenzyme fractions were purified by fractionation by using DEAE-cellulose and DNA-agarose chromatography . The core polymerase was purified by additional chromatography on phosphocellulose . The subunit structure is beta prime (155,000 molecular weight), beta (151,000), alpha (43,000), and sigma (93,000), with an additional polypeptide of 29,000 molecular weight, which we have designated tau, found associated with both core and holoenzyme fractions . The measured stoichiometry of the holoenzyme complex was found to be 2 alpha:1 beta':1 beta:0.7 sigma:1 tau . The 93,000 molecular-weight protein subunit was identified as the sigma subunit based upon stimulation of specific transcription in assays with reconstituted polymerase.

J Bacteriol, 1985 May, 162(2), 535 - 42
Repeated sequences similar to insertion elements clustered around the nif region of the Rhizobium japonicum genome; Kaluza K et al.; Two different repeated sequences (RSs) were discovered in the Rhizobium japonicum genome: RSRj alpha is 1126 base pairs long and is repeated 12 times; RSRj beta is approximately 950 base pairs long and is repeated at least 6 times . Their arrangement in root nodule bacteroid DNA is the same as in DNA from bacteria grown in culture . Deletion analysis showed that many copies of alpha and beta are clustered around the nitrogenase genes nifDK and nifH, or, in general, they are found within a genomic region harboring genes that are nonessential for growth . One copy each of alpha and beta are located upstream of nifDK and are adjacent to each other . Neither of them, however, is involved in the expression of nifDK . Nucleotide sequence analysis of three copies of RS alpha revealed many characteristics of procaryotic insertion sequence elements: potential inverted repeats at their ends, potential target site duplication, and large open reading frames . Despite this, their genomic positions appear to be stable . One possible function of these RSs is in deletion formation probably via recombination between them.

J Bacteriol, 1985 May, 162(2), 469 - 76
Physical and genetic map of a Rhizobium meliloti nodulation gene region and nucleotide sequence of nodC; Jacobs TW et al.; Infection of alfalfa by the soil bacterium Rhizobium meliloti proceeds by deformation of root hairs and bacterial invasion of host tissue by way of an infection thread . We studied an 8.7-kilobase (kb) segment of the R . meliloti megaplasmid, which contains genes required for infection . Site-directed Tn5 mutagenesis was used to examine this fragment for nodulation genes . A total of 81 R . meliloti strains with mapped Tn5 insertions in the 8.7-kb fragment were evaluated for nodulation phenotype on alfalfa plants; 39 of the insertions defined a 3.5-kb segment containing nodulation functions . Of these 39 mutants, 37 were completely nodulation deficient (Nod-), and 2 at the extreme nif-distal end were leaky Nod- . Complementation analysis was performed by inoculating plants with strains carrying a genomic Tn5 at one location and a plasmid-borne Tn5 at another location in the 3.5-kb nodulation segment . Mutations near the right border of the fragment behaved as two distinct complementation groups . The segment in which these mutations are located was analyzed by DNA sequencing . Several open reading frames were found in this region, but the one most likely to function is 1,206 bases long, reading from left to right (nif distal to proximal) and spanning both mutation groups . The genetic behavior of this segment may be due either to the gene product having two functional domains or to a recombinational hot spot between the apparent complementation groups.

J Bacteriol, 1985 May, 162(2), 698 - 703
Characterization of the gene encoding glutamine synthetase I (glnA) from Bradyrhizobium japonicum; Carlson TA et al.; We have isolated the Bradyrhizobium japonicum gene encoding glutamine synthetase I (glnA) from a phage lambda library by using a fragment of the Escherichia coli glnA gene as a hybridization probe . The rhizobial glnA gene has homology to the E . coli glnA gene throughout the entire length of the gene and can complement an E . coli glnA mutant when borne on an expression plasmid in the proper orientation to be transcribed from the E . coli lac promoter . High levels of glutamine synthetase activity can be detected in cell-free extracts of the complemented E . coli . The enzyme encoded by the rhizobial gene was identified as glutamine synthetase I on the basis of its sedimentation properties and resistance to heat inactivation . DNA sequence analysis predicts a high level of amino acid sequence homology among the amino termini of B . japonicum, E . coli, and Anabaena sp . strain 7120 glutamine synthetases . S1 nuclease protection mapping indicates that the rhizobial gene is transcribed from a single promoter 131 +/- 2 base pairs upstream from the initiation codon . This glnA promoter is active when B . japonicum is grown both symbiotically and in culture with a variety of nitrogen and carbon sources . There is no detectable sequence homology between the constitutively expressed glnA promoter and the differentially regulated nif promoters of the same B . japonicum strain.

J Bacteriol, 1985 Apr, 162(1), 361 - 6
Structure of complex flagellar filaments in Rhizobium meliloti; Krupski G et al.; The complex flagella of Rhizobium meliloti 2011 and MVII-1 were analyzed with regard to serology, fine structure, subunits, and amino acid composition . The serological identities of flagellar filaments of the two strains were demonstrated by double immunodiffusion with antiflagellin antiserum . The filaments had a diameter of 16 nm . Their morphology was dominated by the prominent undulations of an external three-start helix running at a 10-nm axial distance and at an angle of 32 degrees . Faint nearly axial striations indicated the presence of a tubular core of a different helical order . The complex filaments consisted of 40,000-dalton flagellin monomers . Typically, the amino acid composition was 3 to 4% higher in nonpolar residues and 5 to 7% lower in aspartic and glutamic acids (and their amides) than that of plain flagellar proteins . There were no immunochemical relationships among Pseudomonas rhodos, Rhizobium lupini, and R . meliloti complex flagella, suggesting that the latter represent a new class.

Cell, 1985 Apr, 40(4), 869 - 77
Symbiotic mutants of Rhizobium meliloti that uncouple plant from bacterial differentiation; Finan TM et al.; Spontaneous mutants at a new symbiotic locus in Rhizobium meliloti SU47 are resistant to several phages and are conditionally insensitive to a monoclonal antibody to the bacterial surface, apparently because they are deficient in a wild-type exopolysaccharide . On alfalfa, the mutants do not curl root hairs, but penetrate the epidermis directly, forming nodules that contain no visible infection threads or "bacteroids," have a few bacteria in superficial intercellular spaces only and not within the nodule cells, and fail to fix nitrogen (Fix-) . Evidently, infection threads are not essential for cell proliferation and nodule formation, which are here induced by a bacterial signal at a distance and uncoupled from the bacterial differentiation that normally goes on as well.

J Bacteriol, 1985 Apr, 162(1), 335 - 43
Isolation and characterization of transposon Tn5-induced symbiotic mutants of Rhizobium loti; Chua KY et al.; Rhizobium loti NZP2037 and NZP2213, each cured of its single large indigenous plasmid, formed effective nodules on Lotus spp., suggesting that the symbiotic genes are carried on the chromosome of these strains . By using pSUP1011 as a vector for introducing transposon Tn5 into R . loti NZP2037, symbiotic mutants blocked in hair curling (Hac), nodule initiation (Noi), bacterial release (Bar), and nitrogen fixation (Nif/Cof) on Lotus pedunculatus were isolated . Cosmids complementing the Hac, Noi, and Bar mutants were isolated from a pLAFR1 gene library of NZP2037 DNA by in planta complementation and found to contain EcoRI fragments of identical sizes to those into which Tn5 had inserted in the mutants . The cosmids that complemented the mutants of these phenotypic classes did not share common fragments, nor did cosmids that complemented four mutants within the Noi class, suggesting that these symbiotically important regions are not tightly linked on the R . loti chromosome.

J Bacteriol, 1985 Apr, 162(1), 317 - 23
Vector insertion mutagenesis of Rhizobium sp . strain ORS571: direct cloning of mutagenized DNA sequences; Donald RG et al.; When the limited-host-range plasmid pVP2021 carrying Tn5 was mobilized into Rhizobium sp . strain ORS571 and stable acquisition of Tn5 was selected, ORS571 plasmid-genome cointegrates were exclusively obtained; direct Tn5 transposition was never observed . In every case, genomic cointegrates exhibited an additional (third) IS50 element that bordered VP2021 DNA sequences but maintained a single Tn5 element . Genomic cointegrates containing IS50 triplications were stable; neither phenotypic reversion nor resolution was detectable . Auxotrophic mutant strains (vector insertion mutants) were identified at expected frequencies among derivatives carrying ostensibly random genomic pVP2021 insertions; N2 fixation (Nif)-defective vector insertion mutants were observed among these derivatives at a frequency of 10(-3) . The presence of integrated pVP2021 in ORS571 nif::VP2021 mutant genomes enabled VP2021 to constitute an endogenous cloning vector . After EcoRI or KpnI digestions, genomic nif::pVP2021 DNA sequences contiguous with integrated pVP2021 were directly cloned as new replicons without addition of an exogenous vector . Recombinant plasmids derived from two such nif::pVP2021 mutants hybridized to previously analyzed ORS571 Nif DNA sequences . Recombinant plasmid DNA and ORS571 Nif region DNA were found to be colinear; pVP2021 insertions could be accurately mapped . pVP2021 insertion-mutagenesis thus allows the direct cloning of ORS571 gene sequences for which mutant phenotypes can be selected or screened.

Ann Inst Pasteur Microbiol, 1985 Mar-Apr, 136A(2), 261 - 9
Evidence of an active glucose uptake in Rhizobium meliloti; Theodoropoulos PA et al.; Rhizobium meliloti M5N1 showed immediate uptake of glucose . Glucose accumulation was a saturable function of the glucose concentration, and km and Vmax values for uptake were determined . The glucose uptake system was found to be proteic with a turnover of about 6 h . This system was observed to be an active ATP-dependent process, since it was severely inhibited by uncouplers . Glucose analogues and others hexoses had some inhibitory effects on glucose uptake, but not polyols and lactose.

J Bacteriol, 1985 Mar, 161(3), 882 - 7
Isolation of genes (nif/hup cosmids) involved in hydrogenase and nitrogenase activities in Rhizobium japonicum; Hom SS et al.; Recombinant cosmids containing a Rhizobium japonicum gene involved in both hydrogenase (Hup) and nitrogenase (Nif) activities were isolated . An R . japonicum gene bank utilizing broad-host-range cosmid pLAFR1 was conjugated into Hup- Nif- R . japonicum strain SR139 . Transconjugants containing the nif/hup cosmid were identified by their resistance to tetracycline (Tcr) and ability to grow chemoautotrophically (Aut+) with hydrogen . All Tcr Aut+ transconjugants possessed high levels of H2 uptake activity, as determined amperometrically . Moreover, all Hup+ transconjugants tested possessed the ability to reduce acetylene (Nif+) in soybean nodules . Cosmid DNAs from 19 Hup+ transconjugants were transferred to Escherichia coli by transformation . When the cosmids were restricted with EcoRI, 15 of the 19 cosmids had a restriction pattern with 13.2-, 4.0-, 3.0-, and 2.5-kilobase DNA fragments . Six E . coli transformants containing the nif/hup cosmids were conjugated with strain SR139 . All strain SR139 transconjugants were Hup+ Nif+ . Moreover, one nif/hup cosmid was transferred to 15 other R . japonicum Hup- mutants . Hup+ transconjugants of six of the Hup- mutants appeared at a frequency of 1.0, whereas the transconjugants of the other nine mutants remained Hup- . These results indicate that the nif/hup gene cosmids contain a gene involved in both nitrogenase and hydrogenase activities and at least one and perhaps other hup genes which are exclusively involved in H2 uptake activity.

J Bacteriol, 1985 Feb, 161(2), 775 - 7
Role of ubiquinone in hydrogen-dependent electron transport in Rhizobium japonicum; O'Brian MR et al.; Direct evidence for the involvement of ubiquinone in H2 oxidation by Rhizobium japonicum was demonstrated; H2 reduced ca . 80% of the extractable ubiquinone . The inhibitor 2-n-heptyl-4-hydroxyquinoline-N-oxide blocked electron transport at a site between ubiquinone and the cytochromes . The results showed that no cytochrome component mediates electron flow from hydrogen to ubiquinone.

J Bacteriol, 1985 Feb, 161(2), 507 - 14
Expression of cytochrome o in hydrogen uptake constitutive mutants of Rhizobium japonicum; O'Brian MR et al.; Mutant strains of Rhizobium japonicum constitutive for H2 uptake activity (Hupc) contained significantly more membrane-bound b-type cytochrome than did the wild type when grown heterotrophically . The Hupc strains contained approximately three times more dithionite- and NADH-reducible CO-reactive b-type cytochrome than did the wild type; the absorption features of the CO spectra were characteristic of cytochrome o . This component, designated cytochrome b', was not reduced by NADH in the presence of cyanide . Cytochrome o from the wild type (SR) and cytochrome b' from mutants SR476 and SR481 bound to CO with similar dissociation constants of 5.4, 7.4, and 5.6 microM, respectively . NADH-dependent reduction of cytochrome b' from SR476 and SR481 and the cytochrome o from SR followed pseudo-first-order kinetics with similar rate constants . Based on these spectral, ligand-binding, and kinetic measurements, it was concluded that cytochrome b' expressed by the Hupc mutants is equivalent to cytochrome o found in the wild type . H2, NADH, and succinate each reduced the same amount of total b-type cytochrome in membranes from SR481, and the rate of H2-dependent cytochrome o reduction was significantly less than with succinate or NADH as the reductants . It was concluded that neither cytochrome o nor any b-type cytochrome expressed by the Hupc mutants was unique to the H2 oxidation system . At low O2 concentrations, the inhibition of H2 and NADH oxidase activities by CO closely paralleled the binding of CO to cytochrome o rather than cytochromes a3 or c' . This suggested that NADH and H2 oxidation involved primarily cytochrome o as the terminal oxidase at low O2 tensions.

Nucleic Acids Res, 1985 Jan 11, 13(1), 239 - 49
Primary structure of the soybean nodulin-23 gene and potential regulatory elements in the 5'-flanking regions of nodulin and leghemoglobin genes; Mauro VP et al.; The nodulin-23 gene of soybean is one of the most abundantly transcribed genes induced during symbiosis with Rhizobium . Using a plasmid (pNod25) from a nodule cDNA library, we have isolated the nodulin-23 gene from a soybean genomic library . Nucleotide sequence analysis of the cDNA and of the genomic clone indicated that the coding region of this gene is 669 bp long and is interrupted by a single intron of about 530 bp . The deduced protein sequence suggests that nodulin-23 may have a signal sequence . The 5'-flanking sequence of two other nodulin genes, nodulin-24 encoding for a membrane polypeptide and one of the leghemoglobin genes (LbC3), were obtained . Comparison of these sequences revealed three conserved regions, one of which, an octanucleotide (GTTTCCCT), has 100% homology . The conserved sequences are arranged in a unique fashion and have a spatial organization with respect to order and position, which may suggest a potential regulatory role in controlling the expression of nodulin and leghemoglobin genes during symbiosis.

Antonie Van Leeuwenhoek, 1985, 51(3), 249 - 54
Effect of parathion on growth, polysaccharides, lipids and proteins of Rhizobium meliloti; Carranza de Storani MM et al.; Rhizobium meliloti 300hl3 reached the stationary phase of growth very quickly and had an early death when 8.6 muM parathion was added to the growth medium at the start of the culture . Cells also changed their quantitative composition: total carbohydrate content and poly-beta-hydroxybutyrate accumulation diminished, proteins and phospholipids of cellular membranes increased, and some alterations in the proportion of membrane fatty acids were noticed.

Cytobios, 1985, 42(165), 41 - 7
Vacuolation and infection thread in root nodules of soybean; Bal AK; Profuse vacuolation takes place in the soybean root nodule cells where infection threads carry rhizobia . After the rhizobia are released the disappearance of the infection thread is attributed to its degradation within large vacuoles which result from fusion of small vacuoles.

Gene, 1985, 34(2-3), 235 - 41
Cloning of the nodulation (nod) genes of Rhizobium phaseoli and their homology to R . leguminosarum nod DNA; Lamb JW et al.; In Rhizobium phaseoli strain 8002, a large indigenous plasmid, pRP2JI, had previously been shown to carry many of the genes necessary for the induction of nitrogen-fixing nodules on Phaseolus beans . A cosmid clone library was constructed using DNA from strain 8002 . From this library, two overlapping recombinant plasmids (pIJ1097 and pIJ1098) were isolated which spanned about 43 kb of pRP2JI DNA . These plasmids could restore nodulation to some, but not all nodulation-deficient strains of R . phaseoli, indicating that the nodulation genes were not clustered within one small region of pRP2JI . The cloned R . phaseoli nodulation region shared extensive DNA homology with the nodulation genes of R . leguminosarum, and on the basis of DNA hybridization, the nitrogenase genes were found to be within 10 kb of the R . phaseoli nodulation genes . Close to the nodulation genes of R . phaseoli was located a sequence that was repeated on pRP2JI but which was not present elsewhere in the genome of strain 8002.

Acta Biochim Pol, 1985, 32(1), 27 - 34
Identification and synthesis in vitro of plant-specific proteins in yellow lupin root nodules; Strozycki P et al.; Fifteen to twenty specific polypeptides of Mr ranging from 15 000 to 90 000 were detected using immunochemical techniques in the lupin root nodules and cell-free translation products of the nodule polysomal RNA . These polypeptides were characteristic for symbiotic state of the host plant and represented 9-18% of total nodule proteins . They were absent in uninfected roots and in protein extracts of Rhizobium lupini.

Acta Microbiol Hung, 1985, 32(3), 221 - 4
Salt tolerance of Azospirillum brasilense; Rao AV et al.; The effect of various salts on the growth and N2-ase activity of Azospirillum brasilense was tested . Bicarbonate was found to be the most toxic, followed by chlorides and sulphate . Tolerance of A . brasilense to these salts was comparable to that of many species of Rhizobium . SO4-- was stimulatory to growth and N2-ase activity up to 40 meq . The process of N2-fixation (N2-ase activity) was found to be more sensitive to all the salts tested as compared to growth.

Dev Biol Stand, 1985, 60, 185 - 97
Production of monoclonal antibodies in culture; Reuveny S et al.; Factors that affected the production of monoclonal antibodies by a mouse-mouse hybridoma cell line, propagated in vitro in stirred vessels, were investigated . The purpose of the research was to estimate the efficiency of this system for large scale production of monoclonal antibodies . The antibody produced by these hybridoma cells was an IgG2a, specific for a surface antigen on Rhizobium japonicum NR-7 cells . Antibody content in the culture supernatant was determined by a radial-immunodiffusion assay using rabbit anti-mouse IgG antibodies in the immobile phase and mouse IgG (the monoclonal antibody) as the antigen in the mobile phase . This method was found to be more reproducible and reliable compared with an ELISA method . Cells were adapted to grow in an inexpensive, low protein content medium based on Dulbecco's Modified Eagle Medium (DMEM) supplemented with 0.25% Primatone RL, 0.01% Pluronic polyol F-68 and fetal bovine serum as low as 1% . Doubling time for the cells averaged 24 hrs, and final yields reached 2 X 10(6) cells per ml . The hybridoma cells were grown in the newly developed medium in 3 liter fermentors . Monoclonal antibody was produced during the early growth phase (3 days), however, most of the antibody was produced during a later growth phase (3-10 days) when 30 to 90% of the cells were dead . Final antibody yields were estimated to be 100-200 micrograms/ml . A low level of dissolved oxygen (25% air saturation) in the culture was found to increase the amount of antibody produced as compared with cells propagated at 60% air saturation (the optimal level for cell propagation) since the cells were kept alive for longer periods at the lower dissolved oxygen concentration . Using a fed-batch propagation method we were able to keep cells alive for long periods (up to 1 month) at a concentration of about 1 X 10(6) cells per ml, and thus to increase further monoclonal antibody production . Yields of 300-400 micrograms/ml were obtained.

J Cell Sci Suppl, 1985, 2, 333 - 45
Rhizobium attachment to clover roots; Mills KK et al.; The adhesion of rhizobia to surfaces of clover roots was examined by an indirect plate-counting assay and phase-contrast microscopy . The number of Rhizobium trifolii cells attached to clover root segments increased in approximately linear fashion during the first hour of incubation, but did not change appreciably thereafter . The addition of 30 mM-2-deoxy-D-glucose, which effectively inhibits binding of clover root lectin, did not promote the release of previously attached bacteria nor inhibit subsequent attachment to either root segments or root hairs . Rhizobia of several heterologous species attached to clover roots in numbers comparable to those of strains of R . trifolii, the homologous species . These results indicate that rhizobia have effective mechanisms of adhesion to non-host roots and that clover lectin contributed little or nothing to attachment under the conditions examined.

J Cell Sci Suppl, 1985, 2, 317 - 31
The legume-Rhizobium symbiosis: a cell surface interaction; Robertson JG et al.; This review will examine the early stages of infection of legume roots by strains of Rhizobium that induce nitrogen-fixing nodules . The object is to show that, at least in terms of ultrastructure, the interactions between the plant and the rhizobia occur at the cell surface interface between these organisms . This situation exists at all stages, from the time the bacteria attach to the surface of the root hairs to the time they occur as the nitrogen-fixing form in the cytoplasm of the nodule cells, enclosed by peribacteroid membranes.

J Cell Sci Suppl, 1985, 2, 347 - 54
Rhizobium leguminosarum genes involved in early stages of nodulation; Downie JA et al.; Nodulation genes from Rhizobium leguminosarum have been subcloned and transferred to a strain of R . phaseoli with its symbiotic plasmid deleted (and therefore its nodulation and nitrogen fixation genes) . Normal infection and nodule development occurred when these strains were added to the roots of Pisum sativum (peas) and Vicia hirsuta . The pea nodules were examined by electron microscopy; bacteroid forms were seen surrounded by peribacteroid membranes and using immuno-gold labelling it was shown that the nodule cells contained leghaemoglobin within the cytoplasm . By subcloning and analysing the nodulation region it was found that a small (3.2 X 10(3) bases) fragment of DNA contained three genes involved in root hair curling, the first observed step in the interaction between R . leguminosarum and the root hair cells of these legumes.

Mol Gen Genet, 1985, 199(2), 306 - 14
Expression of Rhizobium japonicum nifH and nifDK operons can be activated by the Klebsiella pneumonia nifA protein but not by the product of ntrC; Alvarez-Morales A et al.; Rhizobium japonicum nifH'- and nifD'-'lacZ fusions were constructed using the translational fusion vector pMC1403 . beta-Galactosidase activities from these fusion plasmids were measured in wild-type, ntrA- and delta(ntrBC) Escherichia coli strains carrying plasmids which overproduced the Klebsiella pneumoniae nifA or ntrC gene products . In contrast to results reported in R . meliloti (ref . in the text) neither nifH nor nifD promoters were activated by the ntrC product . In the presence of nifA gene product, however, beta-galactosidase activity from both nifH and nifD fusion plasmids increased substantially . NifA-mediated activation of these Rhizobium promoters was temperature sensitive and was dependent on the host ntrA product . In order to determine the point at which the fusion transcripts were initiated, RNA was extracted from the wild-type E . coli strain carrying each of the R . japonicum fusion plasmids plus the nifA overproducing plasmid . This RNA was used to perform S1 mapping experiments . NifA-mediated transcription from both R . japonicum promoters, began at the same point as previously determined in soybean root-nodule bacteroids (ref . in the text) . The results obtained suggest that there may be differences in the mode of regulation between members of the fast- and slow-growing rhizobia . Also, the results of the S1 mapping experiments indicate that activation of the R . japonicum nitrogenase structural genes may be similar to the activation of nif genes in K . pneumoniae thus raising the possibility that R . japonicum may contain nifA and ntrA-like genes.

Acta Microbiol Pol, 1985, 34(2), 187 - 94
Use of intrinsic antibiotic resistance for characterisation and identification of rhizobia from nodules of Vigna unguiculata (L) Walp . and Phaseolus vulgaris (L); Dakora FD; Intrinsic resistance to low concentrations of antibiotics was used to characterise 83 isolates from nodules of cowpea (Vigna unguiculata) and field bean (Phaseolus vulgaris) . Characterisation and differentiation of isolates from cowpea was made difficult by associated fast-growing bacteria inside the nodule tissue . Thus, reliable pure culture was difficult to secure without repeated isolation and even via nodulation of the appropriate homologous host . Although the technique may be satisfactory for differentiation and identification of fast-growing rhizobia, it is rated inferior to serology on aspects of facility, time and accuracy where rhizobia from cowpea nodules are concerned . Fingerprint patterns of isolates revealed considerable heterogeneity amongst the populations even where there was commonality of location and/or host plant . Pure cultures of slow-growing rhizobia from V . unguiculata nodules were generally more resistant to the concentrations of antibiotics used than fast-growing nodule bacteria from P . vulgaris.

Nucleic Acids Res, 1984 Dec 21, 12(24), 9497 - 508
DNA sequence of the Rhizobium leguminosarum nodulation genes nodAB and C required for root hair curling; Rossen L et al.; A 3.2kb fragment of DNA cloned from Rhizobium leguminosarum has been shown to contain the genes necessary for the induction of root hair curling, the first observed step in the infection of leguminous plants by R . leguminosarum . The DNA sequence of this region has been determined and three open reading frames were identified: genes corresponding to these open reading frames have been called nodA, nodB and nodC and are transcribed in that order . Mutations within the nodC gene completely blocked root hair curling . However, a subcloned fragment containing only the nodC gene did not induce normal root hair curling (although some branching was observed), indicating that the nodA and B genes may also be required for normal root hair curling . From an analysis of the predicted amino acid sequences of the nodAB and C genes it appeared unlikely that their products are secreted; therefore it is concluded that the induction of root hair curling could be due to a secreted metabolite.

Nucleic Acids Res, 1984 Dec 21, 12(24), 9509 - 24
Nucleotide sequence of Rhizobium meliloti nodulation genes; Torok I et al.; A Rhizobium meliloti DNA region, determining nodulation functions common in different Rhizobium species, has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined . The sequence data indicates three large open reading frames with the same polarity coding for three proteins of 196, 217 and 402 (or 426) amino acid residues, respectively . We suggest the existence of three nod genes on this region, which were designated as nodA, B and C, respectively . Comparison of the R . meliloti nodA, B, C nucleotide and amino acid sequences with those from R . leguminosarum, as reported in the accompanying paper, shows 69-72% homology, clearly demonstrating the high degree of conservation of common nod genes in these Rhizobium species.

J Bacteriol, 1984 Dec, 160(3), 1061 - 6
Mode of infection, nodulation specificity, and indigenous plasmids of 11 fast-growing Rhizobium japonicum strains; Heron DS et al.; 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.

J Bacteriol, 1984 Dec, 160(3), 1027 - 30
Glucose-6-phosphate dehydrogenase deficiency in pleiotropic carbohydrate-negative mutant strains of Rhizobium meliloti; Cervenansky C et al.; Several mutant strains of Rhizobium meliloti isolated after nitrosoguanidine mutagenesis were selected as unable to grow on mannose . Some of them also failed to grow on glucose, fructose, ribose, and xylose but grew on L-arabinose, galactose, and many other carbon sources . Biochemical analysis demonstrated that the mutants lacked NAD- and NADP-linked glucose-6-phosphate dehydrogenase activities that reside on a single enzyme species . One such mutant was found to accumulate glucose-6-phosphate, and this could partially explain the inhibition of growth observed on mixtures of permissive and nonpermissive carbon sources . Symbiotic properties remained unaffected in all these mutants.

Appl Environ Microbiol, 1984 Dec, 48(6), 1140 - 50
Specific phases of root hair attachment in the Rhizobium trifolii-clover symbiosis; Dazzo FB et al.; The time course and orientation of attachment of Rhizobium trifolii 0403 to white clover root hairs was examined in slide cultures by light and electron microscopy . Inocula were grown for 5 days on defined BIII agar medium and represented the large subpopulation of fully encapsulated single cells which uniformly bind the clover lectin trifoliin A . When 10(7) cells or more were added per seedling, bacteria attached within minutes, forming randomly oriented clumps at the root hair tips . Several hours later, single cells attached polarly to the sides of the root hair . This sequence of attachment to clover root hairs was selective for R . trifolii at inoculum sizes of 10(7) to 4 X 10(8) per seedling, specifically inhibited if 2-deoxy-D-glucose, a hapten for trifoliin A, was present in the inoculum, and not observed when 4 X 10(8) cells were added to alfalfa seedling roots or to large clover root cell wall fragments which lacked trifoliin A but still had trifoliin A receptors . Once attached, R . trifolii 0403 became progressively less detachable with 2-deoxy-D-glucose . At smaller inoculum sizes (10(5) to 10(6) cells per seedling), there was no immediate clumping of R . trifolii at clover root hair tips, although polar binding of bacteria along the root hair surface was observed after 4 h . The interface between polarly attached bacteria and the root hair cell wall was shown to contain trifoliin A by immunofluorescence microscopy . Also, this interface was shown by transmission electron microscopy to contain electron-dense granules of host origin . Scanning electron microscopy revealed an accumulation of extracellular microfibrils associated with the lateral and polar surfaces of the attached bacteria, detectable after 12 h of incubation with seedling roots . At this same time, there was a significant reduction in the effectiveness of 2-deoxy-D-glucose in dislodging bacteria already attached to root hairs and an increase in firm attachment of bacteria to the root hair surface, which withstood the hydrodynamic shear forces of high-speed vortexing . These results are interpreted as a sequence of phases in attachment, beginning with specific reversible interactions between bacterial and plant surfaces (phase I attachment), followed by production of extracellular microfibrils which firmly anchor the bacterium to the root hair (phase 2 adhesion) . Thus, attachment of R . trifolii to clover root hairs is a specific process requiring more than just the inherent adhesiveness of the bacteria to the plant cell wall.(ABSTRACT TRUNCATED AT 400 WORDS)

J Bacteriol, 1984 Dec, 160(3), 903 - 9
Molecular cloning and genetic organization of C4-dicarboxylate transport genes from Rhizobium leguminosarum; Ronson CW et al.; Cosmids containing C4-dicarboxylate transport (dct) genes were identified from a gene bank of Rhizobium leguminosarum DNA made in the broad-host-range vector pLAFR1 by their ability to complement R . trifolii dct mutants . The dct genes were further characterized by subcloning, restriction site mapping, and transposon Tn5 and Tn7 mutageneses . Three dct loci were identified within a 5.5-kilobase region of DNA, in the order dctA-dctB-dctC . The results suggested that dctA encoded a structural component necessary for C4-dicarboxylate transport, whereas dctB and dctC encoded positive regulatory elements, and that dctA was transcribed divergently from dctB and dctC . Expression of dctA and dctC was obtained from vector promoters in some pLAFR1- and pSUP106-based plasmids.

Nucleic Acids Res, 1984 Nov 26, 12(22), 8329 - 44
Structural analysis of the genes encoding the molybdenum-iron protein of nitrogenase in the Parasponia rhizobium strain ANU289; Weinman JJ et al.; The genes encoding the Molybdenum-Iron protein component of nitrogenase (nifD and nifK) have been identified and fully characterised in the Parasponia Rhizobium strain ANU289 . The two genes are contiguous and are separated from the gene encoding the Fe-protein component of nitrogenase (nifH) by 21 kb of DNA . We present the entire DNA sequence of the nifD and nifK genes, thus completing the characterisation of the primary structure of the nitrogenase genes in this Rhizobium strain . Comparison of the sequence preceding the transcription initiation point of nifDK with that preceding nifH reveals a consensus promoter sequence 5'-PyTGGCAPyG-4 bp-TTGC(T/A)-10 bp-3' . This consensus promoter is found preceding nif genes in both fast-growing and slow-growing Rhizobium strains and shows a structural similarity to that preceding the coordinately-regulated nif operons in the asymbiotic organism Klebsiella pneumoniae.

J Bacteriol, 1984 Nov, 160(2), 517 - 20
Stimulation of clover root hair infection by lectin-binding oligosaccharides from the capsular and extracellular polysaccharides of Rhizobium trifolii; Abe M et al.; A polysaccharide depolymerase isolated from the phage lysate of Rhizobium trifolii 4S was used to fragment capsular polysaccharides (CPS) and extracellular polysaccharides (EPS) of R . trifolii 0403 into oligosaccharides . These products were analyzed for clover lectin (trifoliin A)-binding ability, effect on infection of white clover root hairs, and changes in glycosyl and noncarbohydrate composition with culture age . The oligosaccharides from CPS of cultures grown on agar plates for 3, 5, and 7 days exhibited lectin-binding ability at levels similar to those of the corresponding intact CPS . The intact EPS did not bind to clover lectin, although the oligosaccharide fragments from EPS did . In contrast, oligosaccharides from deacetylated CPS had less than half the lectin-binding ability of the native polysaccharide substrate . The CPS from 5-day-old cultures, its corresponding oligosaccharide fragments, and the oligosaccharide fragments of EPS from 5-day-old cultures, all at a concentration of 2.5 micrograms per seedling, stimulated infection thread formation in root hairs of clover seedlings inoculated with R . trifolii 0403 . Thus, this bacteriophage-induced polysaccharide depolymerase converted the acidic CPS and EPS of R . trifolii 0403 into biologically active oligosaccharides capable of binding trifoliin A and stimulating root hair infection . The amount of the noncarbohydrate substitutions (pyruvate, acetate, and ether-linked 3-hydroxybutyrate) in the CPS oligosaccharides changed with culture age as shown by 1H-nuclear magnetic resonance spectroscopy . The binding of trifoliin A, therefore, appears to be sensitive to changes in the degree of substitution of noncarbohydrate substitutions in the CPS of R . trifolii 0403.

J Bacteriol, 1984 Nov, 160(2), 510 - 6
Bacteriophage-induced acidic heteropolysaccharide lyases that convert the acidic heteropolysaccharides of Rhizobium trifolii into oligosaccharide units; Hollingsworth RI et al.; Acidic heteropolysaccharide lyases from lysates of phages 4S and BY15 grown on Rhizobium trifolii 4S and R . trifolii 0403, respectively, were used to analyze the capsular and excreted extracellular acidic polysaccharides of R . trifolii 0403 . The activities of the enzymes as measured by viscometry were enhanced by the addition of calcium . The oligosaccharide products obtained by depolymerase digestion of the polysaccharides isolated from cells grown on agar plates for 5 days were isolated by gel filtration and had a glycosyl composition of glucose, galactose, glucuronic acid, and alpha-linked 4-deoxy-L-threo-hex-4-enopyranosyluronic acid in an approximate molar ratio of 5:1:1:1 . This latter component was identified by 1H-nuclear magnetic resonance spectroscopy and confirmed by UV spectroscopy, ozonolysis, and its reactivity with thiobarbituric acid . The oligosaccharide had glucose as the reducing terminus, 4-deoxy-L-threo-hex-4-enopyranosyluronic acid as the enzymatically generated nonreducing terminus, and galactose as the terminus of the branched chain . The noncarbohydrate components of the oligosaccharides were acetate, ketal-linked pyruvate, and ether-linked 3-hydroxybutyrate . The mode of action of the enzymes was by beta-elimination from a uronic acid residue with concomitant loss of the glycosyl component substituted at C-4 . The 235-nm absorbing properties of the resulting terminal unsaturated sugar were used to study the kinetics of depolymerization of the capsular and excreted extracellular acidic polysaccharides, using the enzyme from phage BY15 . The two substrates exhibited different kinetics of depolymerization, and the oligosaccharide products differed in the amount of noncarbohydrate substituents, indicating that the acidic capsular and excreted extracellular polysaccharides from 5-day-old cultures of R . trifolii 0403 were different.

J Bacteriol, 1984 Nov, 160(2), 785 - 7
Reiteration of genes involved in symbiotic nitrogen fixation by fast-growing Rhizobium japonicum; Prakash RK et al.; By using cloned Rhizobium meliloti nodulation (nod) genes and nitrogen fixation (nif) genes, we found that the genes for both nodulation and nitrogen fixation were on a plasmid present in fast-growing Rhizobium japonicum strains . Two EcoRI restriction fragments from a plasmid of fast-growing R . japonicum hybridized with nif structural genes of R . meliloti, and three EcoRI restriction fragments hybridized with the nod clone of R . meliloti . Cross-hybridization between the hybridizing fragments revealed a reiteration of nod and nif DNA sequences in fast-growing R . japonicum . Both nif structural genes D and H were present on 4.2- and 4.9-kilobase EcoRI fragments, whereas nifK was present only on the 4.2-kilobase EcoR2 fragment . These results suggest that the nif gene organizations in fast-growing and in slow-growing R . japonicum strains are different.

J Bacteriol, 1984 Oct, 160(1), 454 - 7
Monoclonal antibodies to Rhizobium meliloti and surface mutants insensitive to them; Johansen E et al.; 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.

J Bacteriol, 1984 Oct, 160(1), 483 - 7
Mobilization of a Sym plasmid from a fast-growing cowpea Rhizobium strain; Morrison NA et al.; A large Sym plasmid from a fast-growing cowpea Rhizobium species was made mobilizable by cointegration with plasmid pSUP1011, which carries the oriT region of RP4 . This mobilizable Sym plasmid was transferred to a number of Rhizobium strains, in which nodulation and nitrogen fixation functions for symbiosis with plants of the cowpea group were expressed.

J Bacteriol, 1984 Oct, 160(1), 448 - 50
Coordinate expression of hydrogenase and ribulose bisphosphate carboxylase in Rhizobium japonicum Hupc mutants; Merberg D et al.; In contrast to the wild type, H2 uptake-constitutive mutants of Rhizobium japonicum expressed both hydrogenase and ribulose bisphosphate carboxylase activities when grown heterotrophically . However, as bacteroids from soybean root nodules, the H2 uptake-constitutive mutants, like the wild type, did not express ribulose bisphosphate carboxylase activity.

J Bacteriol, 1984 Oct, 160(1), 216 - 21
H+/ATP stoichiometry of cowpea Rhizobium sp . strain 32H1 cells grown under nitrogen-fixing and nitrogen-nonfixing conditions; Gober JW et al.; The obligate aerobe Cowpea Rhizobium sp . strain 32H1 in axenic culture is able to fix N2 when grown under 0.2% O2 but not when grown under 21% O2 . It was, therefore, of interest to investigate ATP synthesis in these cells grown under the two conditions . When respiring in buffers having pHs ranging from 6 to 8.5, cells grown under either O2 tension maintained an intracellular pH more alkaline than the exterior . The transmembrane chemical gradient of H+ (delta pH) was essentially the same under both conditions of growth, decreasing from ca . 90 mV at medium pH 6 to ca . 30 mV at pH 8.5 . However, the transmembrane electrical gradient (delta psi) was significantly higher in cells grown under 21% O2 (150 to 166 mV) than in cells grown under 0.2% O2, the latter being 16 mV at pH 6 and increasing to 88 mV at pH 8.5 . Therefore, the proton motive force of 21% O2-grown cells ranged from 237 mV at external pH 6 to 185 mV at pH 8.5, compared with a proton motive force of 114 to 121 mV in the 0.2% O2-grown cells . The cells grown in 0.2% O2 had the same proton motive force whether tested at 21 or at 0.2% O2 . The phosphorylation potential, calculated from the intracellular ATP, ADP, and Pi concentrations, was 424 mV in the 21% O2-grown cells and 436 mV in the 0.2% O2-grown cells . Thus, the 21% O2-grown cells translocated 1.8 to 2.3 H+/ATP synthesized by the H+-ATPase, whereas the H+/ATP ratio for 0.2% O2-grown cells was 3.7 to 3.8.

Genetika, 1984 Oct, 20(10), 1594 - 606
{Properties of potential vectors--derivatives of the broad-host--range plasmid RP4}; Riabchenko LE et al.; Nonconjugative deletion and recombinant derivatives of the RP4 plasmid are constructed . The plasmids can be used as vectors because they have relatively small molecular weights, unique cleavage sites for enzymes EcoRI, XhoI, BamHI, PstI, KpnI, BglII, SalGI and HindIII (the plasmids pRP401 and pRP417 having six of these sites), and easily tested phenotypes (Tcr, Apr and Gal+) . In addition, all of them retain the broad host range property . Also, the plasmid pRP420 is a multicopy derivative capable of amplification . The plasmids are mobilized by conjugative plasmids pRK2013 and Flac from Escherichia coli cells into Rhizobium meliloti and Pseudomonas aeruginosa strains . Flac-mediated mobilization of the pRP417 plasmid which has an internal deletion of the transposon Tn1, is decreased, in comparison with the nondeleted plasmid . ColE1 replication machinery is inhibited for RP4--ColE1 recombinant derivatives, if both components are joined via EcoRI restriction site . This inhibition does not depend on the orientation of joined molecules . ColE1 replication machinery is functional, if delta RP4 and ColE1-like plasmids are joined via PstI cleavage site.

J Bacteriol, 1984 Oct, 160(1), 451 - 3
Conjugal transfer of bacterial chromosomes mediated by the RK2 plasmid transfer origin cloned into transposon Tn5; Yakobson EA et al.; We report here a novel system for the conjugal transfer of bacterial chromosomes which utilizes the transfer origin (oriT) of plasmid RK2 cloned into transposon Tn5 . Tn5 with oriT was inserted by transposition into the chromosomes of Escherichia coli and Rhizobium meliloti . The oriT sequence then served as the origin of high-frequency chromosome transfer when a helper RK2 plasmid was present in the same cell . The broad host range features of RK2 make this system of oriented chromosome mobilization applicable to most gram-negative bacteria.

Nucleic Acids Res, 1984 Sep 25, 12(18), 7123 - 34
Identification and DNA sequence of fixZ, a nifB-like gene from Rhizobium leguminosarum; Rossen L et al.; Previously, several mutants which nodulated peas but which failed to fix nitrogen were isolated following Tn5 mutagenesis of pRL 1JI, a symbiotic plasmid of Rhizobium leguminosarum . Two of these alleles, fix52::Tn5 and fix137::Tn5 were in a region of pRL 1JI which hybridized to a probe that contained the nifA gene and the amino-terminal region of the nifB gene of Klebsiella pneumoniae . The nitrogen fixation defect of the fix52::Tn5 mutant strain was corrected by a 2.0kb fragment of the corresponding wild-type DNA cloned in a wide host-range plasmid . The DNA sequence of this region revealed an open reading frame corresponding to the gene within which the fix52::Tn5 allele was located . The polypeptide corresponding to this open reading frame had a deduced molecular weight of 39,936 and the gene was termed fixZ . The deduced amino acid sequence of the fixZ gene product contained two clusters of cysteine residues, suggesting that the protein may contain an iron-sulphur cluster . The sequence of the fixZ polypeptide was very similar to the sequence of the K . pneumoniae nifB gene (provided by W . Arnold and A . Puhler) which is required for the synthesis of the FeMo-cofactor of nitrogenase . It was shown that the previously observed hybridization was due to homology between the amino terminal regions of fixZ and nifB . Upstream from fixZ was found another open reading frame whose 5' terminus was not established, but within which was located the fix137::Tn5 allele . This gene was termed fixY . The deduced amino acid sequence of the sequenced part of fixY showed similarity to that of the regulatory nifA gene of K . pneumoniae (provided by W . J . Buikema and F . M . Ausubel) . Thus in R . leguminoarum the fix genes that correspond to the nifA and nifB genes are in the same relative orientation as in K . pneumoniae.

J Bacteriol, 1984 Sep, 159(3), 850 - 6
Some properties of the nickel-containing hydrogenase of chemolithotrophically grown Rhizobium japonicum; Harker AR et al.; The uptake hydrogenase of chemolithotrophically grown Rhizobium japonicum was purified to apparent homogeneity with a final specific activity of 69 mumol of H2 oxidized per min per mg of protein . The procedure included Triton extraction of broken membranes and DEAE-cellulose and Sephacryl S-200 chromatographies . The purified protein contained two polypeptides separable only by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . They comigrated on native polyacrylamide gels and sucrose density gradients . The molecular weights were ca . 60,000 and 30,000 . Densitometric scans of the sodium dodecyl sulfate gels indicated a molar ratio of 1.03 +/- 0.03 . Antiserum was developed against the 60-kilodalton polypeptide for use in hydrogenase detection by an enzyme-linked immunosorbent assay . The antiserum did not cross-react with the 30-kilodalton polypeptide . Native gel electrophoresis of Triton-extracted cells grown in the presence of 63Ni showed comigration of the hydrogenase and radioactive Ni.

J Bacteriol, 1984 Sep, 159(3), 857 - 62
Physical organization of the Bradyrhizobium japonicum nitrogenase gene region; Adams TH et al.; In Bradyrhizobium japonicum USDA 110 the three genes that encode the nitrogenase enzyme complex are separated into two transcription units, nifH and nifDK . We have physically mapped a 33-kilobase-pair region of the B . japonicum genome that contains both nifH and nifDK . The nifDK operon is located transcriptionally upstream from nifH, and all three genes are transcribed in the same direction . Within the 20-kilobase-pair region that separates the promoters for these two transcription units, we have identified a region homologous to the Klebsiella pneumoniae nifA gene . This nifA homology is situated about 6 kilobase pairs upstream from the nifH transcriptional initiation signal, in an analogous position to the nifA-like locus previously described for Rhizobium meliloti . In addition, we have characterized a second distinct nifA homology in B . japonicum that is not directly linked to the nitrogenase structural gene region.

J Bacteriol, 1984 Sep, 159(3), 1006 - 12
Characterization of Rhizobium japonicum hydrogen uptake genes; Haugland RA et al.; Recombinant cosmids from a gene library of the DNA from Hup+ Rhizobium japonicum 122DES previously have been shown to restore hydrogenase activity when transferred by conjugation into certain Hup- mutants of R . japonicum . We generated a restriction map covering 32.2 kilobases of this cosmid DNA . At least 25.3 kilobases of the cosmid pHU1 were shown to have the same arrangement as those in the genome of strain 122DES . Analysis of Tn5 insertions into the 122DES genome indicates that hup-specific sequences occur in a region spanning about 15 kilobases of insert DNA within pHU1 . Introduction of pHU1 into five out of six R . japonicum Hup- mutants resulted in a Hup+ phenotype in some transconjugants . Three of the mutations appear to be in transcriptional units completely contained within pHU1, whereas the other two must be in genes that are at least partially contained within pHU1 . pBR235 derivatives containing fragments of hup DNA can be transferred into the R . japonicum Hup- mutant PJ18nal if the derivatives contain a region of homology with the R . japonicum genome . The hup mutation in strain PJ18nal appears to be dominant . The hup genes in R . japonicum strain 122DES appear to be organized in at least two, and probably three, transcriptional units.

Eur J Biochem, 1984 Jul 2, 142(1), 37 - 42
Electron allocation to H+ and N2 by nitrogenase in Rhizobium leguminosarum bacteroids; Haaker H et al.; Electron allocation to H+ and N2 by nitrogenase in intact Rhizobium leguminosarum bacteroids has been studied . Nitrogenase activity was measured in intact cells with succinate and oxygen substrates . When whole cell nitrogenase activity was inhibited by oxygen-limitation or by the addition of the H+-conducting ionophore carbonylcyanide m-chlorophenylhydrazone, both inducing a low intracellular ATP/ADP ratio, the electron allocation to H+ was favoured over that to N2 . When whole cell nitrogenase activity was inhibited by excess oxygen or by the addition of the K+-conducting ionophore valinomycin, both inhibiting electron transport to nitrogenase without affecting the intracellular ATP/ADP ratio, no effect upon the electron allocation to H+ and N2 was observed . The whole cell experiments could be confirmed by experiments with bacteroids treated with hexadecyltrimethylammonium bromide . Nitrogenase is highly active in these preparations with Na2S2O4 and MgATP as substrates . No effect was observed upon electron allocation to H+ and N2 when nitrogenase was inhibited by limitation of reductant (Na2S2O4) or MgATP . Only when nitrogenase was inhibited by MgADP, electron allocation to H+ was favoured . The amount of nitrogenase component 1 and 2 in bacteroids was estimated with protein blotting, followed by an immunological detection . It was found that 17% +/- 3% of total bacteroid protein is component 1 and 12% +/- 2% is component 2 . The specific nitrogenase activity of bacteroids treated with hexadecyltrimethylammonium bromide is 178 +/- 62 nmol C2H4 formed X min-1 X mg total protein-1 . Despite the high protein concentrations, nitrogenase is not inhibited . With cell-free extracts or with purified nitrogenase components isolated from R . leguminosarum bacteroids, electron allocation to H+ was favoured over that to N2, independently of the mechanism of inhibition . The discrepancies between the whole cell studies and those with isolated enzyme will be discussed with respect to the present mechanism of action of nitrogenase.

Appl Environ Microbiol, 1984 Jul, 48(1), 68 - 72
Bacteriophage that can distinguish between wild-type Rhizobium japonicum and a non-nodulating mutant; Stacey G et al.; A bacteriophage (phage TN1) that lyses Rhizobium japonicum 3I1b110 was isolated from Tennessee soil . Structurally, this phage resembles the Escherichia coli phage T4, having an icosahedral head (47 by 60 nm) and a contractile tail (17 by 80 nm) . An interesting feature of this phage is that it lyses all of the symbiotic defective mutants derived from R . japonicum 3I1b110 that were tested, except one, mutant strain HS123 . Mutant strain HS123 is a non-nodulating mutant that is defective in attachment to soybean roots . Since Rhizobium attachment to host roots is thought to be mediated by a specific cell surface interaction, it is likely that mutant strain HS123 is defective in some way in its cell surface . Mutant strain HS123 bound soybean lectin to the same extent as the wild type as measured by the binding of tritium-labeled lectin . Phage TN1 did not attach to the surface of strain HS123, nor did cells of strain HS123 inactivate phage TN1 . A hot phenol-water cell extract from the wild-type inactivated phage TN1, whereas a similar cell extract from mutant HS123 did not . Capsular polysaccharide isolated from mutant or wild type did not inactivate the phage . Capsular polysaccharide and exopolysaccharide from the mutant and wild type do not differ in sugar composition . These results indicate that capsular polysaccharide may not play a role in attachment to the plant root surface and that other cell wall components may be important.

Appl Environ Microbiol, 1984 Jul, 48(1), 149 - 52
Rhizobia are attracted to localized sites on legume roots; Gulash M et al.; Clouds of Rhizobium meliloti were attracted to localized sites on the surface of the infectible region of alfalfa roots . This behavior, which required active motility and chemotaxis, was not species specific . Correlation between the behavior of various mutants and their competitiveness for nodulation suggests that cloud formation has a role in the infection of host legume roots by rhizobia.

J Bacteriol, 1984 Jul, 159(1), 153 - 8
Nickel is a component of hydrogenase in Rhizobium japonicum; Stults LW et al.; The derepression of H2-oxidizing activity in free-living Rhizobium japonicum does not require the addition of exogenous metal to the derepression media . However, the addition of EDTA (6 microM) inhibited derepression of H2 uptake activity by 80% . The addition of 5 microM nickel to the derepression medium overcame the EDTA inhibition . The addition of 5 microM Cu or Zn also relieved EDTA inhibition, but to a much lesser extent; 5 microM Fe, Co, Mg, or Mn did not . The kinetics of induction and magnitude of H2 uptake activity in the presence of EDTA plus Ni were similar to those of normally derepressed cells . Nickel also relieved EDTA inhibition of methylene blue-dependent Hup activity, suggesting that nickel is involved directly with the H2-activating hydrogenase enzyme . Adding nickel or EDTA to either whole cells or crude extracts after derepression did not affect the hydrogenase activity . Cells were grown in 63Ni and the hydrogenase was subsequently purified by gel electrophoresis . 63Ni comigrated with the H2-dependent methylene blue reducing activity on native polyacrylamide gels and native isoelectric focusing gels . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the nickel-containing hydrogenase band revealed a single polypeptide with a molecular weight of ca . 67,000 . We conclude that the hydrogenase enzyme in R . japonicum is a nickel-containing metalloprotein.

J Bacteriol, 1984 Jul, 159(1), 145 - 52
Development and trifoliin A-binding ability of the capsule of Rhizobium trifolii; Sherwood JE et al.; The age-dependent lectin-binding ability of Rhizobium trifolii 0403 capsular polysaccharide (CPS) was examined by following the development of the capsule and its ability to interact with the white clover lectin trifoliin A . Bacteria grown on agar plates for 3, 5, 7, 14, and 21 days were examined by electron microscopy and immunofluorescence microscopy with antibodies prepared against either R . trifolii 0403 CPS or trifoliin A after pretreatment with the lectin . The capsule began to develop at one pole by day 3 and completely surrounded the cells in cultures incubated for 5 days or longer . The capsular polysaccharide on cells cultured for 3 and 5 days was completely reactive with trifoliin A, became noticeably less reactive by day 7, and was only reactive with the lectin at one pole of a few cells after that time . The quantity and location of lectin receptors on bacteria of different ages directly correlated with their attachment in short-term clover root hair-binding studies . Cells from 3- or 21-day-old cultures attached almost exclusively in a polar fashion, whereas cells grown for 5 days attached to root hairs randomly and in the highest numbers . CPS isolated from a 5-day-old culture had higher lectin-binding ability than CPS from 3- and 7-day-old cultures, whereas the CPS from a 14-day-old culture had the lowest . Chemical analyses of the isolated CPS showed changes in the levels of uronic acids (as glucuronic acid), pyruvate, and O-acetyl substitutions with culture age, but the neutral sugar composition remained relatively constant . These results provide evidence that the age-dependent distribution of lectin receptors dictates the level and orientation of attachments of R . trifolii 0403 to clover root hairs.

J Bacteriol, 1984 Jul, 159(1), 395 - 9
Host-dependent transposon Tn5-mediated streptomycin resistance; De Vos GF et al.; 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.

J Bacteriol, 1984 Jul, 159(1), 335 - 40
Transposon Tn5-induced mutagenesis of Rhizobium japonicum yielding a wide variety of mutants; Hom SS et al.; When the "suicide" vector pSUP1011, which carries transposon Tn5 (Kmr), was introduced into Rhizobium japonicum USDA 110, kanamycin-resistant (Kmr) colonies were detected at a frequency (4.2 X 10-6) ca . 30 times greater than the spontaneous kanamycin resistance frequency (1.4 X 10-7) . Ten thousand Kmr mutants were isolated and tested for nutritional auxotrophy . Auxotrophs were detected at a frequency of 0.5% . The following classes of auxotrophs were identified: adenine- (three), histidine- (three), glutamate- (five), adenine plus thiamine- (nine), uracil- (three), pantothenic acid- (one), tryptophan- (three), and methionine- (three) . Mutants blocked in symbiotic nitrogen fixation (Fix-) were also identified at a frequency of 3% . The glutamate auxotrophs were studied in more detail, and all five showed an altered expression of nitrogenase activity in free-living cultures.

J Bacteriol, 1984 Jul, 159(1), 125 - 9
Generalized transduction in Rhizobium meliloti; Martin MO et al.; Generalized transduction of Rhizobium meliloti 1021 was carried out by bacteriophage N3 . Genetic markers on the chromosome and the pSym megaplasmid were transduced, along with markers on several IncP plasmids . Cotransduction between transposon Tn5 insertions and integrated recombinant plasmid markers permitted correlation of cotransductional frequencies and known physical distances . Bacteriophage N3 was capable of infecting several commonly used strains of R . meliloti.

J Bacteriol, 1984 Jul, 159(1), 120 - 4
General transduction in Rhizobium meliloti; Finan TM et al.; 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.

J Bacteriol, 1984 Jul, 159(1), 47 - 52
Evaluation of active versus passive uptake of metabolites by Rhizobium japonicum bacteroids; Reibach PH et al.; Rhizobium japonicum bacteroids were isolated anaerobically from soybean {Glycine max (L.) Merr} nodules . The bacteroids, which were capable of acetylene reduction and respiration, were used to study the uptake of metabolites by a method which permits correction for nonspecific adsorption of metabolites and estimation of total cell volume . These determinations permit active uptake to be assessed from metabolite accumulation against a concentration gradient . Succinate, malate, alpha-ketoglutarate, and glutamate were absorbed via an active mechanism . Plots of 1/V versus 1/{S} for succinate and malate indicated the presence of two uptake components: a saturable and presumably active or carrier-mediated component and a nonsaturable and presumably passive component . The uptake of glucose, malonate, D-pinitol, myo-inositol, and glucose 6-phosphate was slow and not active.

Arch Biochem Biophys, 1984 Jul, 232(1), 337 - 47
Mg2+ adenosine triphosphatase from cell envelopes of free-living and bacteroid forms of Rhizobium lupini strain NZP2257; Robertson JG et al.; A procedure for the purification of Mg2+ adenosine triphosphatase (EC 3.6.1.3) from free-living and bacteroid forms of Rhizobium lupini NZP2257 is described . The enzyme was released from cell envelopes using Triton X-100 and purified by gel filtration on Ultrogel AcA 22, followed by preparative gel electrophoresis on agarose . The purified ATPase had a molecular weight of about 355,000, as determined from sedimentation coefficients on sucrose gradients . Kinetic analysis of activity of the enzyme from free-living R . lupini showed it to be typical of F1-type Mg2+ ATPases from bacteria . Mg stimulated activity at pH 7.0, although, when present as the free ion, Mg caused non-competitive inhibition (K1 = 1.5 mM) . Maximum activity with ATP occurred over a broad pH range from 6.0 to 10.5 . ATP, GTP, and UTP, and, to a much lesser degree, CTP and ADP, were hydrolyzed by the enzyme . Hydrolysis of glucose 6-phosphate was not observed . The Km for ATP at pH 7.0 was 0.67 and for GTP 1.4 mM . ATPase activity was inhibited by ADP, and competitive with ATP (KI = 0.18 mM) . Azide also caused inhibition but fluoride and DCCD had no effect . Native and sodium dodecyl sulfate-gel electrophoretic analysis revealed no obvious differences between ATPases from free-living and bacteroid forms of R . lupini.

J Bacteriol, 1984 Jun, 158(3), 1012 - 7
Heterogeneity of Rhizobium lipopolysaccharides; Carlson RW; The lipopolysaccharides ( LPSs ) from strains of Rhizobium leguminosarum, Rhizobium trifolii, and Rhizobium phaseoli were isolated and partially characterized by mild acid hydrolysis and by polyacrylamide gel electrophoresis . Mild acid hydrolysis results in a precipitate which can be removed by centrifugation or extraction with chloroform . The supernatant contains polysaccharides which, in general, are separated into two fractions ( LPS1 and LPS2 ) by Sephadex G-50 gel filtration chromatography . The higher-molecular-weight LPS1 fractions among the various Rhizobium strains are highly variable in composition and reflect the variability reported in the intact LPSs (R . W . Carlson and R . Lee, Plant Physiol . 71:223-228, 1983; Carlson et al., Plant Physiol . 62:912-917, 1978; Zevenhuizen et al., Arch . Microbiol . 125:1-8, 1980) . The LPS1 fraction of R . leguminosarum 128C53 has a higher molecular weight than all other LPS1 fractions examined . All LPS2 fractions examined are oligosaccharides with a molecular weight of ca . 600 . The major sugar component of all LPS2 oligosaccharides is uronic acid . The LPS2 compositions are similar for strains of R . leguminosarum and R . trifolii, but the LPS2 from R . phaseoli was different in that it contained glucose, a sugar not found in the other LPS2 fractions or found only in trace amounts . Polyacrylamide gel electrophoretic analysis shows that each LPS contains two banding regions, a higher-molecular-weight heterogeneous region often containing many bands and a lower-molecular-weight band . The lower-molecular-weight bands of all LPSs have the same electrophoretic mobility, which is greater than that of lysozyme . The banding pattern of the heterogeneous regions varies among the different Rhizobium strains . In the case of R . leguminosarum 128C53 LPS, the heterogeneous region of a higher molecular weight than is this region from all other Rhizobium strains examined and consists of many bands separated from one another by a small and apparently constant molecular weight interval . When the heterogeneous region of R . Leguminosarum 128C53 LPS was cut from the gel and analyzed, its composition was found to be that of the intact LPS, whereas the lower-molecular-weight band contains only sugars found in the LPS2 oligosaccharide . In the case of R . leguminosarum 128C63 and R . trifolii 0403 LPSs, the heterogeneous regions are similar and consist of several band s separated by a large-molecular-weight interval with a the major band of these heterogeneous regions having the lowest molecular weight with an electrophoretic mobility near that of beta-lactoglobulin . The heterogeneous region from R . phaseoli 127K14 consists of several bands with electrophoretic mobilities near that of beta-lactoglobulin, whereas this region from R . trifolii 162S7 shows a continuous staining region, indicating a great deal of heterogeneity . The results described in this paper are discussed with regard to the reported properties of Escherichia coli and Salmonella LPSs.

J Bacteriol, 1984 Jun, 158(3), 1168 - 71
Extracellular polysaccharide composition, ex planta nitrogenase activity, and DNA homology in Rhizobium japonicum; Huber TA et al.; The composition of the major acidic extracellular polysaccharide (EPS) of 25 strains of Rhizobium japonicum was determined . Eight strains synthesized an acidic EPS containing rhamnose and 4-O- methylglucuronic acid and were closely related according to DNA homology . These same strains also expressed high levels of ex planta nitrogenase activity . Sixteen strains produced an acidic EPS containing glucose, mannose, galacturonic acid, and galactose and were also related by DNA homology . These strains developed little or no nitrogenase activity under the experimental conditions employed.

J Bacteriol, 1984 Jun, 158(3), 1204 - 7
Lactose inhibits the growth of Rhizobium meliloti cells that contain an actively expressed Escherichia coli lactose operon; Timblin CR et al.; Expression of the Escherichia coli lactose operon in Rhizobium meliloti 104A14 made the cells sensitive to the addition of the beta-galactosides lactose, phenyl-beta-D-galactoside, and lactobionic acid . Growth stopped when the beta-galactoside was added and viability decreased modestly during the next few hours, but little cell lysis was observed and the cells appeared normal . Protein synthesis was not inhibited . Growth was inhibited only when beta-galactosidase expression was greater than 160 U . Lactose-resistant mutants had defects in the plasmid-carried E . coli beta-galactosidase or beta-galactoside permease and in the R . meliloti genome . We speculate that uncontrolled production of galactose by the action of the lactose operon proteins was responsible for growth inhibition.

J Bacteriol, 1984 Jun, 158(3), 1070 - 7
Gene fusion vehicles for the analysis of gene expression in Rhizobium meliloti; Kahn ML et al.; A set of plasmid cloning vehicles was developed to facilitate the construction of gene or operon fusions in Rhizobium meliloti . The vehicles also contain a broad-host-range replicon and could be introduced into bacteria either by transformation or by transduction, using bacteriophage P2 . Insertion of foreign DNA into a unique restriction endonuclease cleavage site promotes the synthesis of either the Escherichia coli lactose operon or the kanamycin phosphotransferase gene from transposon Tn5 . Expression of the lactose operon could be detected by observing the color of Rhizobium colonies on medium that contained a chromogenic indicator . We also determined the growth conditions that make it possible to select either for or against the expression of the E . coli lactose operon in R . meliloti . Recombinant plasmids were constructed by inserting MboI restriction fragments of R . meliloti DNA into one of the vehicles, pMK353 . Expression of beta-galactosidase by a number of these recombinants was measured in both R . meliloti and E . coli.

J Bacteriol, 1984 Jun, 158(3), 1005 - 11
Rhizobium japonicum nitrogenase Fe protein gene (nifH); Fuhrmann M et al.; A 12.1-kilobase PstI fragment from Rhizobium japonicum, which contains homology to both the Klebsiella pneumoniae and the Rhizobium meliloti nifH genes, was cloned into vector pHE3 . The nifH -homologous region was localized on the restriction enzyme cleavage map by Southern blot hybridization experiments . DNA fragments overlapping the R . japonicum nifH gene were subcloned into plasmid vectors to allow the expression of this region in Escherichia coli minicells . The nifH gene product (the polypeptide of the nitrogenase Fe protein) was thus found to have a molecular weight of 33,000 . The complete nucleotide sequence of the nifH gene was established, and the amino acid sequence of its gene product was deduced . The reading frame is 882 nucleotides long, corresponding to 294 amino acids which add up to a polypeptide with a molecular weight of 31,525 . There was extensive sequence homology with nifH genes or gene products from other nitrogen-fixing bacteria . The transcription initiation site of the R . japonicum nifH gene was found to lie 153 nucleotides upstream from the coding region and was preceded by a characteristic promoter sequence . A potential terminator region was located 13 nucleotides downstream from the coding region.

J Bacteriol, 1984 Jun, 158(3), 1144 - 51
Rhizobium sp . strain ORS571 ammonium assimilation and nitrogen fixation; Donald RG et al.; Among rhizobia studied, Rhizobium sp . strain ORS571 alone grew unambiguously on N2 as sole N source . In ORS571 , only the glutamine synthetase (GS)-glutamate synthase ( GOGAT ) pathway assimilated ammonium . However, ORS571 exhibited two unique physiological aspects of this pathway: ORS571 had only GS I, whereas all other Rhizobiaceae studied had both GS I and GS II, and both NADPH- and NADH-dependent GOGAT activities were present . ORS571 GS-affected and NADPH- GOGAT -affected mutant strains were defective in both ammonium assimilation (Asm-) and N2 fixation (Nif-) in culture and in planta ; NADH- GOGAT mutants were Asm- but Nif+ . "Bacteroid" GS activity was essentially nil, suggesting symbiotic ammonium export . Physiological studies on effects of glutamine, ammonium, methionine sulfoximine, and diazo-oxo-norleucine on nitrogenase induction in culture implied a regulatory role for the intracellular glutamine pool.

J Mol Biol, 1984 May 15, 175(2), 213 - 8
Visualization and exact molecular weight determination of a Rhizobium meliloti megaplasmid; Burkardt B et al.; The entire DNA of Rhizobium meliloti MVII /1 cells was isolated preparatively by gentle lysis and sucrose gradient centrifugation . Fractions of the sucrose gradient were investigated by electron microscopy . We found intact megaplasmids in supercoiled and relaxed form and total chromosomes . The length of the megaplasmid could be measured, which allowed an exact determination of the molecular weight . We found a length of 0.48 +/- 0.019 mm equivalent to a molecular weight of about 1000 X 10(6).

Appl Environ Microbiol, 1984 May, 47(5), 895 - 900
Preservation of Rhizobium viability and symbiotic infectivity by suspension in water; Crist DK et al.; Three Rhizobium japonicum strains and two slow-growing cowpea-type Rhizobium strains were found to remain viable and able to rapidly modulate their respective hosts after being stored in purified water at ambient temperatures for periods of 1 year and longer . Three fast-growing Rhizobium species did not remain viable under the same water storage conditions . After dilution of slow-growing Rhizobium strains with water to 10(3) to 10(5) cells ml-1, the bacteria multiplied until the viable cell count reached levels of between 10(6) and 10(7) cells ml-1 . The viable cell count subsequently remained fairly constant . When the rhizobia were diluted to 10(7) cells ml-1, they did not multiply, but full viability was maintained . If the rhizobia were washed and suspended at 10(9) cells ml-1, viability slowly declined to 10(7) cells ml-1 during 9 months of storage . Scanning electron microscopy showed that no major morphological changes took place during storage . Preservation of slow-growing rhizobia in water suspensions could provide a simple and inexpensive alternative to current methods for the preservation of rhizobia for legume inoculation.

J Bacteriol, 1984 May, 158(2), 621 - 7
Identification of a rhizosphere protein encoded by the symbiotic plasmid of Rhizobium leguminosarum; Dibb NJ et al.; A protein was identified which was made by wild-type strains of Rhizobium leguminosarum but not by nodulation-deficient derivatives which had deletions of their symbiotic plasmids . The protein, which had a subunit molecular weight of ca . 24,000 ( 24K ), was found to be present in large amounts within bacteria that had been reisolated from the surface of inoculated pea roots but was not detected in bacteroids isolated from nodules . The protein could also be induced during growth of R . leguminosarum on nutrient medium and was purified from the cytoplasmic fraction of broken cells . Antiserum raised against the purified protein was used to screen transposon-induced mutants of R . leguminosarum, and four independent mutants were isolated which lacked the protein . The sites of the Tn5 insertions were found to map between the nitrogenase and nodulation genes on symbiotic plasmid pRL1JI , ca . 5 kilobases from the nitrogenase genes and 13 kilobases from the nodulation genes . Genetic determinants for the 24K protein were found to be closely linked to plasmid-borne nodulation genes for all strains of R . leguminosarum tested . However, the mutants which lacked the 24K protein still formed normal nitrogen-fixing nodules on peas, and the function of the protein is unknown.

J Bacteriol, 1984 May, 158(2), 580 - 9
Transposon Tn5 specifies streptomycin resistance in Rhizobium spp; Selvaraj G et al.; Transposon Tn5 conferred streptomycin resistance on different strains of Rhizobium meliloti, Rhizobium leguminosarum, and Rhizobium trifolii but not on Escherichia coli . A gene (str) specifying this phenotype has been identified and localized on the physical and genetic map of Tn5 . It is transcribed from the promoter of neo, the gene that encodes neomycin phosphotransferase . The str gene is downstream from neo in a single transcriptional unit, as revealed by molecular cloning of different segments of Tn5 and by cloning of the neo-str region of Tn5 downstream from a lac promoter . Fusion of the SalI-generated rightward segment of Tn5 (devoid of neo) to a part of a tetracycline resistance gene, tet, in a plasmid or downstream from a lac promoter in a plasmid resulted in significant levels of streptomycin resistance in an R . meliloti host, suggesting that the str gene product can function independent of neomycin phosphotransferase . A natural isolate of R . meliloti that does not express Tn5-associated streptomycin resistance has been identified . We have used the str of Tn5 as a genetic marker in Rhizobium spp.

Cell, 1984 Apr, 36(4), 1035 - 43
A Rhizobium meliloti symbiotic regulatory gene; Szeto WW et al.; We have characterized a Rhizobium meliloti regulatory gene required for the expression of two closely linked symbiotic operons, the nitrogenase operon (nifHDK genes) and the "P2" operon . This regulatory gene maps to a 1.8 kb region located 5.5 kb upstream of the nifHDK operon . The regulatory gene is required for the accumulation of nifHDK and P2 mRNA and for the derepression of an R . meliloti nifH-lacZ fusion plasmid during symbiotic growth . The nifH and P2 promoters can be activated in free-living cultures of R . meliloti containing plasmids that produce the Escherichia coli ntrC(glnG) or the Klebsiella pneumoniae nifA regulatory gene products constitutively . The R . meliloti regulatory gene hybridizes to E . coli ntrC(glnG) and, to a lesser extent, to K . pneumoniae nifA DNA . Our results suggest that the R . meliloti regulatory gene acts as a positive transcriptional activator and that it is related to the K . pneumoniae nif regulatory genes.

J Bacteriol, 1984 Apr, 158(1), 148 - 55
Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions; Noel KD et al.; Rhizobium phaseoli CFN42 DNA was mutated by random insertion of Tn5 from suicide plasmid pJB4JI to obtain independently arising strains that were defective in symbiosis with Phaseolus vulgaris but grew normally outside the plant . When these mutants were incubated with the plant, one did not initiate visible nodule tissue (Nod-), seven led to slow nodule development (Ndv), and two led to superficially normal early nodule development but lacked symbiotic nitrogenase activity (Sna-) . The Nod- mutant lacked the large transmissible indigenous plasmid pCFN42d that has homology to Klebsiella pneumoniae nitrogenase (nif) genes . The other mutants had normal plasmid content . In the two Sna- mutants and one Ndv mutant, Tn5 had inserted into plasmid pCFN42d outside the region of nif homology . The insertions of the other Ndv mutants were apparently in the chromosome . They were not in plasmids detected on agarose gels, and, in contrast to insertions on indigenous plasmids, they were transmitted in crosses to wild-type strain CFN42 at the same frequency as auxotrophic markers and with the same enhancement of transmission by conjugation plasmid R68.45 . In these Ndv mutants the Tn5 insertions were the same as or very closely linked to mutations causing the Ndv phenotype . However, in two mutants with Tn5 insertions on plasmid pCFN42d, an additional mutation on the same plasmid, rather than Tn5, was responsible for the Sna- or Ndv phenotype . When plasmid pJB4JI was transferred to two other R . phaseoli strains, analysis of symbiotic mutants was complicated by Tn5-containing deleted forms of pJB4JI that were stably maintained.

J Bacteriol, 1984 Mar, 157(3), 703 - 7
Rhizobium meliloti competitiveness and the alfalfa agglutinin; Handelsman J et al.; We have isolated two types of isolates having identical colony morphologies from stock cultures of two different Rhizobium meliloti strains . One isolate was agglutinated at a high-dilution titer (HA, highly agglutinable) of the alfalfa agglutinin and was sensitive to phage F20, and the other was agglutinated at a lower agglutinin titer (LA) and was sensitive to phage 16B . All LA isolates from the original slant produced nodules on alfalfa earlier than did HA strains from the original slant . When these HA and LA strains were mixed and used as the inoculum in both vermiculite and field soil in the laboratory, LA strains were always the predominant strains recovered from the nodules . LA strains were obtained from HA cells by selection for resistance to phage F20, and HA strains were obtained from LA cells by selection for resistance to phage 16B . All of the strains with the HA phenotype that were derived from LA strains by phage selection had the nodulation properties of the HA strains from the original slant . Two classes of strains with the LA phenotype were obtained from HA cells by phage selection . One was identical to the original LA strains from the slant, and the other had the nodulation properties of the HA strains . Thus, we have shown that some cell surface properties change the nodulation abilities of R . meliloti strains and, furthermore, that specific phages can be used to enrich for more competitive rhizobia.

Can J Microbiol, 1984 Mar, 30(3), 285 - 9
Role of rhizobitoxine in protecting soybean roots from Macrophomina phaseolina infection; Chakraborty U et al.; Bacterization of soybean seeds or roots with Rhizobium japonicum significantly reduced charcoal rot disease caused by Macrophomina phaseolina . Rhizobium japonicum inhibited the growth of M . phaseolina on both liquid and solid media . Replacement of nutrient medium with culture filtrate of R . japonicum significantly reduced mycelial growth of M . phaseolina . Whole culture extracts of R . japonicum yielded a toxic substance which was identified as rhizobitoxine after chromatographic, ultraviolet, and infrared spectrophotometric analyses . This compound also was detected in the roots of soybean inoculated with either R . japonicum alone or in combination of R . japonicum and M . phaseolina . Dosage response curves with rhizobitoxine showed it to be antifungal . The possible role of rhizobitoxine in protecting soybean roots from M . phaseolina infection is discussed.

J Virol Methods, 1984 Feb, 8(1-2), 155 - 60
Large scale preparation of Rhizobium meliloti bacteriophages by fermenter culture; Werquin M et al.; A simple method for preparation of Rhizobium meliloti bacteriophages was established employing fermenter culture . This technique allowed phage production to be checked by dissolved oxygen measure . Phage suspensions ranging from 5.10(12) to 1.2.10(13) PFU/ml were found after polyethylene glycol precipitation and centrifugation in CsCl.

J Mol Appl Genet, 1984, 2(4), 392 - 405
The nifH and nifDK promoter regions from Rhizobium japonicum share structural homologies with each other and with nitrogen-regulated promoters from other organisms; Adams TH et al.; In several species of Rhizobium the three genes which encode the nitrogenase complex are separated into two operons, nifH and nifDK . We have mapped the transcriptional promoter sites for these two operons from R . japonicum USDA strain 110 by S1 protection analyses using bacterial RNA isolated from soybean nodules . Transcription of the nifDK operon is initiated at a site located 46 nucleotides upstream of the proposed translation initiation codon . nifH transcription initiates predominantly at a site 152 nucleotides upstream of the proposed translation initiation codon . An additional minor start for nifH is found 35 nucleotides downstream of the major initiation site . The nucleotide sequences of these promoter sites are presented . Comparison of the major R . japonicum nif promoter sequences reveals a high degree of sequence homology with the conserved sequences clustered in the -11 to -15, -21 to -25, -27 to -31, and -38 to -41 regions . Conservation is also observed for the -11 to -15 and -21 to -25 regions with the R . meliloti nifHDK, a cowpea Rhizobium nifH, and even Klebsiella pneumoniae nif promoters . The -27 to -31 and -38 to -42 region homologies are not conserved with the other known nif promoters . These results are discussed in light of models for nif promoter activation.

Antonie Van Leeuwenhoek, 1984, 50(2), 155 - 60
Application of immunodiffusion to the identification of Rhizobium meliloti strains competing for nodulation on Medicago sativa; Sinha RC et al.; The immunodiffusion technique was successfully used to unambiguously recognize four strains of Rhizobium meliloti in a study of competition for nodulation with Medicago sativa cv . Apollo inoculated with two-, three- and four-strain mixtures . The serological reactions of all R . meliloti strains revealed no significant changes following plant passage indicating that the antigens involved in immunodiffusion were stable . R . meliloti 102F70 formed 50% or more of the nodules on M . sativa inoculated with two-, three- and four-strain mixtures . The remaining three strains were less competitive and produced similar proportions of nodules (14-20%) on plants inoculated with three- and four-strain mixtures . Cases of mixed-strain occupancy of nodules involving either two of three strains were detected in a sub-sample of nodules . The data also indicated considerable variation in the proportions of strains in the nodules of individual plants.

Antonie Van Leeuwenhoek, 1984, 50(5-6), 505 - 24
Hydrogen oxidation and nitrogen fixation in rhizobia, with special attention focused on strain ORS 571; de Vries W et al.; In this survey we describe the influence of hydrogen oxidation on the physiology of Rhizobium ORS 571 . The presence of hydrogen is required for the synthesis of hydrogenase . Carbon substrates do not repress the synthesis of hydrogenase . The respiratory system contains cytrochromes of the b- and c-type . Cytochrome alpha 600 is present after growth at high oxygen tensions . The nature of the terminal oxidases functioning at low oxygen tensions has not been established yet----H+/O values with endogenous substrates are between 6 and 7 . The results show the presence of two phosphorylation sites: site 1 (ATP/2e = 1.0) and site 2(ATP/2e = 1.33) . By measuring molar growth yields it has been demonstrated that carbon-limited, nitrogen-fixing cultures obtain additional ATP from hydrogen oxidation, and that site 2 of oxidative phosphorylation is passed during hydrogen oxidation . A method is described to calculate ATP/N2 values (the total amount of ATP used by nitrogenase during the fixation of 1 mol N2) and H2/N2 ratios (mol hydrogen formed per mol N2 fixed) in aerobic organisms . For Rhizobium ORS 571 the ATP/N2 value is about 40 and the H2/N2 ratio is between 5 and 7.5 . Cells obtained from oxygen-limited nitrogen-fixing cultures contain 30-40% poly-beta-hydroxybutyrate, which explains the high molar growth yields found . Hydrogen has not been detected in the effluent gas of these cultures, which may point to reoxidation of the hydrogen formed at nitrogen fixation . Calculations show that the effect of hydrogen reoxidation on the efficiency of nitrogen fixation (g N fixed X mol-1 substrate converted) is not very large and that the actual H2/N2 ratio is of much more importance . After addition of hydrogen to succinate-limited, ammonia-assimilating cultures, an initial increase of the Ysuccinate value (g dry wt X mol-1 succinate) is followed by a gradual decrease . This is accompanied by a large decrease of the YO2 value, and an increased permeability of the cytoplasmic membrane to protons . The results may be explained by a transition of the culture from an energy-limited state to a carbon-limited state.

Antonie Van Leeuwenhoek, 1984, 50(5-6), 489 - 503
Natural variation in symbiotic nitrogen-fixing Rhizobium and Frankia spp; Lie TA et al.; A description is given of the natural variation in nitrogen-fixing Rhizobium and Frankia spp . strains and the ability to form root nodules on compatible host plants . Arguments are given for the hypothesis that co-evolution has taken place through mutual interaction of host plants and indigenous Rhizobium and Frankia populations in the soil leading to most efficient symbiotic associations . The significance of root nodules as selective enrichment cultures of particular strains in natural and cultivated soils is exemplified by Rhizobium leguminosarum on various ecotypes of Pisum sativum and with Frankia sp . on various actinorhizal plants, in particular Alnus spp., in different geographic regions . The importance of a host-dependent distribution of Rhizobium and Frankia spp . for agriculture and forestry is discussed.

J Bacteriol, 1984 Jan, 157(1), 218 - 24
Induced plasmid-genome rearrangements in Rhizobium japonicum; Berry JO et al.; The P group resistance plasmids RP1 and RP4 were introduced into Rhizobium japonicum by polyethylene-glycol-induced transformation of spheroplasts . After cell wall regeneration, transformants were recovered by selecting for plasmid determinants . Plant nodulation, nitrogen fixation, serological, and bacterial genetics studies revealed that the transformants were derived from the parental strains and possessed the introduced plasmid genetic markers . Agarose gel electrophoresis, restriction enzyme analysis, and DNA hybridization studies showed that many of the transformant strains had undergone genome rearrangements . In the RP1 transformants, chromosomal DNA was found to have transposed into a large indigenous plasmid of R . japonicum, producing an even larger plasmid, and the introduced R plasmid DNA was found to be chromosomally integrated rather than replicating autonomously or integrated into the endogenous plasmid . Seemingly, a similar section of chromosomal DNA was involved in all the genomic rearrangements observed in the R . japonicum RP1 and RP4 transformant strains.

J Mol Appl Genet, 1984, 2(4), 406 - 21
Molecular cloning and functional characterization of Rhizobium leguminosarum structural nif-genes by site-directed transposon mutagenesis and expression in Escherichia coli minicells; Schetgens TM et al.; In order to study the structural organization and regulation of the expression of the nitrogenase gene cluster in Rhizobium leguminosarum PRE we selected relevant subfragments of the sym-plasmid from clone banks by homology with R . meliloti nif-genes . Site-directed Tn5 mutagenesis was applied to a nif DH-specific clone and subsequently the transposon insertions were transferred back into the wild-type rhizobial genome by homologous recombination . Phenotypic effects of Tn5 mutations in the region of the structural nif-genes were determined by measuring acetylene reduction in nodulated plants and by immunological analysis of bacteroid-specific proteins . The localization of Tn5 insertion sites was in accordance with observed consequences: two genotypically different Tn5-induced mutations within nif D caused repression of CI alpha and beta synthesis and a strong reduction of CII production, thus resulting in a Fix- phenotype . Expression of different cloned Rhizobium DNA inserts, bearing nif K, nif D, nif H, or nif DH, was achieved in Escherichia coli minicells dependent upon the presence of a strong upstream vector promoter sequence . Gene products were identified by immunoprecipitation with specific antisera . Endogenous rhizobial transcriptional start signals in one case (nif H) seemed to be recognized at a low rate by the E . coli system; in contrast, Rhizobium ribosome binding sites for all three structural nif-genes functioned normally in minicells . The approximate location of the coding regions for nif KDH genes was determined and found to be contiguous.

J Mol Appl Genet, 1984, 2(6), 589 - 99
Glutamine synthetase of Phaseolus vulgaris L.: organ-specific expression of a multigene family; Cullimore JV et al.; A recombinant plasmid (pcPvNGS-01) containing sequences related to glutamine synthetase (GS) has been identified from a cDNA library constructed from poly (A)+ RNA isolated from root nodules of Phaseolus vulgaris L . The identification of this recombinant relied on the observations that: (a) the clone hybridized strongly to purified GS mRNA; (b) in hybrid-select translation experiments, the clone selected mRNA that produced a polypeptide identical in molecular weight to purified GS subunits which was immunoprecipitated with anti-GS-antiserum; and (c) the translated nucleotide sequence of the cloned cDNA was homologous to a partial amino acid sequence of higher plant GS . The cloned cDNA hybridized to poly (A)+ RNA of different mobilities from leaves, roots, and nodules of P . vulgaris . In RNA "dot" blots washed at different stringencies, differences were observed both in the relative amounts of GS mRNA in different tissues and in the strength of their hybridization to the cDNA probe . The cloned probe hybridized to several fragments of restricted P . vulgaris DNA but not to DNA from Rhizobium phaseoli . These results suggest that GS is coded for by a small multigene family showing organ-specific expression.

Mol Gen Genet, 1984, 196(3), 413 - 20
High frequency mobilization of gram-negative bacterial replicons by the in vitro constructed Tn5-Mob transposon; Simon R; A DNA fragment of the broad host range plasmid RP4 carrying the cis-acting DNA recognition site for conjugative DNA transfer between bacterial cells (Mobsite) was cloned into the kanamycin-neomycin resistance transposon Tn5 . Using conventional transposon mutagenesis techniques the new transposon, called Tn5-Mob, can easily be inserted into the host DNA of gram-negative bacteria . A host replicon carrying Tn5-Mob is then mobilizable into any other gram-negative species if the transfer functions of plasmid RP4 are provided in trans . The potential of Tn5-Mob was demonstrated by mobilizing Rhizobium meliloti plasmids as well as the E . coli chromosome at high frequencies.

J Bacteriol, 1983 Dec, 156(3), 1356 - 8
Tryptophan auxotrophs of Rhizobium japonicum; Wells SE et al.; Eleven tryptophan-requiring mutants of Rhizobium japonicum I-110 ARS were isolated after nitrous acid mutagenesis and fell into five groups based on characterization by supplementation with intermediates and enzyme assays.

Cell, 1983 Dec, 35(2 Pt 1), 479 - 85
Structural relationships among Rhizobium meliloti symbiotic promoters; Better M et al.; Symbiotic nitrogen fixation by Rhizobium meliloti requires the developmentally specific expression of certain bacterial genes . One set of these genes encodes the subunits of nitrogenase, the enzyme responsible for the reduction of atmospheric dinitrogen to ammonia, and another set consists of closely linked genes also essential for nitrogen fixation . Examination of promoter and probable regulatory regions for these gene sets has revealed extensive DNA sequence conservation for more than 160 bp upstream of the respective transcription start points . Three such promoter regions have been identified in the nitrogen fixation (nif) gene cluster of R . meliloti strain 102F34 . Using one of these promoter regions as a hybridization probe, three additional sequences were found in the genome of this strain . The DNA of other R . meliloti strains and Rhizobium species were also examined for homology to the symbiotically regulated promoters of R . meliloti 102F34 . DNA sequences homologous to these R . meliloti promoters were found among diverse rhizobia, and in at least some cases were associated with nif genes.

J Bacteriol, 1983 Dec, 156(3), 1292 - 300
Suicide plasmid vehicles for insertion mutagenesis in Rhizobium meliloti and related bacteria; Selvaraj G et al.; We describe the construction and use of a set of plasmid vectors of the transposons Tn1, Tn5, and Tn9 that are suicidal in Rhizobium species and therefore suitable for mutagenesis with these three transposons . The vectors are composed of the p15A replicon which functions in Escherichia coli but not in Rhizobium species and a region encoding the N type of bacterial conjugation system which is very efficient in matings between E . coli and Rhizobium species . The usefulness of the vectors has been most extensively assessed in Rhizobium meliloti . It is likely that they will be useful for mutagenesis and genome manipulation in other bacteria as well.

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