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