Rhizobacter

J Bacteriol, 1991 Apr, 173(8), 2530 - 8
Identification and nucleotide sequence of Rhizobium meliloti insertion sequence ISRm3: similarity between the putative transposase encoded by ISRm3 and those encoded by Staphylococcus aureus IS256 and Thiobacillus ferrooxidans IST2; Wheatcroft R et al.; The insertion sequence ISRm3 was discovered simultaneously in different Rhizobium meliloti strains by probing Southern blots of total cellular DNA with 32P-labeled pTA2 . This plasmid is indigenous to strain IZ450 and fortuitously contained four copies of ISRm3 . By using an internal EcoRI fragment as a specific probe (pRWRm31), homology to ISRm3 was subsequently detected in over 90% of R . meliloti strains tested from different geographical locations around the world . The frequency of stable nonlethal ISRm3 transpositions was estimated to be 4 x 10(-5) per generation per cell in strain SU47 when grown in liquid culture . The entire nucleotide sequence of ISRm3 in R . meliloti 102F70 is 1,298 bp and has 30-bp terminal inverted repeats which are perfectly matched . Analysis of six copies of ISRm3 in two strains showed that a variable number of base pairs (usually eight or nine) were duplicated and formed direct repeats adjacent to the site of insertion . On one DNA strand, ISRm3 contains an open reading frame spanning 93% of its length . Comparison of the putative protein encoded with sequences derived from the EMBL and GenBank databases showed significant similarity between the putative transposases of ISRm3 from R . meliloti, IS256 from Staphylococcus aureus, and IST2 from Thiobacillus ferroxidans . These insertion sequences appear to be distantly related members of a distinct class.

Nature, 1991 Mar 14, 350(6314), 170 - 2
A haemoprotein with kinase activity encoded by the oxygen sensor of Rhizobium meliloti; Gilles-Gonzalez MA et al.; The expression of the nitrogen-fixation genes of Rhizobium meliloti is controlled by oxygen . These genes are induced when the free oxygen concentration is reduced to microaerobic levels . Two regulator proteins, FixL and FixJ, initiate the oxygen-response cascade, and the genes that encode them have been cloned . The fixL product seems to be a transmembrane sensor that modulates the activity of the fixJ product, a cytoplasmic regulator . FixL and FixJ are homologous to a family of bacterial two-component regulators, for which the mode of signal transduction is phosphorylation . We report here the purification of both FixJ and a soluble truncated FixL (FixL*), overproduced from a single plasmid construct . FixL* catalyses its own phosphorylation and the transfer of the gamma-phosphate of ATP to Fix J . The resulting FixJ-phosphate linkage is sensitive to base, as are the aspartyl phosphates of homologous systems . Visible spectra of purified FixL* show that it is an oxygen-binding haemoprotein . We propose that FixL senses oxygen through its haem moiety and transduces this signal by controlling the phosphorylation of FixJ.

Mol Microbiol, 1991 Mar, 5(3), 665 - 73
Involvement of fixLJ in the regulation of nitrogen fixation in Azorhizobium caulinodans; Kaminski PA et al.; A gene bank of Azorhizobium caulinodans DNA constructed in the bacteriophage lambda GEM11 was screened with Rhizobium meliloti fixL and fixJ genes as probes . One positive recombinant phage, ORS lambda L, was isolated . The nucleotide sequence of a 3.7 kb fragment was established . Two open reading frames of 1512bp and 613bp were identified as fixL and fixJ . Kanamycin cartridges were inserted into the cloned fixL and fixJ genes and recombined into the host genome . The resulting mutants were Nif- Fix-, suggesting that the two genes were required for symbiotic nitrogen fixation and for nitrogen fixation in the free-living state . Using pnifH-lacZ and pnifA-lacZ fusions, it was shown that the FixLJ products controlled the expression of nifH and nifA in bacteria grown in the free-living state.

J Bacteriol, 1991 Mar, 173(6), 2077 - 85
Expression of two Rhizobium meliloti flagellin genes and their contribution to the complex filament structure; Pleier E et al.; The complex flagellar filaments of Rhizobium meliloti are composed of two related (87% identical) flagellins that are encoded by closely linked, separately transcribed genes, flaA and flaB (E . Pleier and R . Schmitt, J . Bacteriol . 171:1467-1475, 1989) . To elucidate the role of the subunits, A and B, in assembling the complex filament, the wild-type alleles were replaced with defective ones containing a 2,249-bp deletion (accompanied by substitution of a kanamycin resistance cartridge), which eliminates 74% of flaA (3' end) and 85% of flaB (5' end) . The resulting nonmotile, filamentless mutant, RU11011, was tested for complementation with wild-type flaA, flaB, and flaA flaB genes provided on the multiple-copy vector pRK290 . Whereas flaA alone did not restore motility and filament production, both flaB and flaA flaB restored 20 to 30% of wild-type motility . Apparent causes of this reduced motility were fewer flagella per cell and/or shortened filaments sometimes ending in unusually thin, fragile structures . Tests with enzyme-linked antiflagellin antibodies indicated that flaA is expressed at higher levels than flaB and that multiple copies of flaA lead to reduced flagellin export . We conclude that the proximal portion of the complex filament is assembled from B subunits (not produced sufficiently to form full-length flagella) and that the distal portion is made from A subunits . Multiple copies of the strong flaA promoter may offset transcriptional controls that regulate the synthesis of flagellar structures required for flagellin export.

Proc Natl Acad Sci U S A, 1991 Mar 1, 88(5), 1923 - 7
Canonical ordered cosmid library of the symbiotic plasmid of Rhizobium species NGR234; Perret X et al.; Many of the bacterial genes involved in nodulation (nod) and nitrogen fixation (nif) are dispersed over the 500-kilobase plasmid pNGR234a of the broad host-range Rhizobium species NGR234 . As a first step toward generating a complete physical and genetic map of the plasmid, a full overlapping collection of cosmids was derived from a total genomic library . Clones were aligned by combining fingerprinting, hybridization, and pulsed-field gel electrophoresis data . Symbiotic loci were localized by probing a representative set of cosmids with both homologous and heterologous genes . nodABC, nodD1, nodD2, nodSU, nolB, and region II are widely dispersed over pNGR234a, while the two functional copies of nifKDH are separated by only 28 kilobases . Interestingly, sequences homologous to nodE, nodG, nodP, and nodQ have been assigned to another autonomously replicating element in Rhizobium species NGR234 . Similarly one copy of the structural dctA gene is located on the symbiotic plasmid (dctA1) while the other is on what we assume to be the chromosome.

Mol Gen Genet, 1991 Mar, 225(3), 514 - 20
Nucleotide sequence of the fixABC region of Azorhizobium caulinodans ORS571: similarity of the fixB product with eukaryotic flavoproteins, characterization of fixX, and identification of nifW; Arigoni F et al.; The nucleotide sequence of a 4.1 kb DNA fragment containing the fixABC region of Azorhizobium caulinodans was established . The three gene products were very similar to the corresponding polypeptides of Rhizobium meliloti . The C-terminal domains of both fixB products displayed a high degree of similarity with the alpha-subunits of rat and human electron transfer flavoproteins, suggesting a role for the FixB protein in a redox reaction . Two open reading frames (ORF) were found downstream of fixC . The first ORF was identified as fixX on the basis of sequence homology with fixX from several Rhizobium and Bradyrhizobium strains . The second ORF potentially encoded a 69 amino acid product and was found to be homologous to a DNA region in the Rhodobacter capsulatus nif cluster I . Insertion mutagenesis of the A . caulinodans fixX gene conferred a Nif- phenotype to bacteria growth in the free-living state and a Fix- phenotype in symbiotic association with the host plant Sesbania rostrata . A crude extract from the fixX mutant had no nitrogenase activity . Furthermore, data presented in this paper also indicate that the previously identified nifO gene located upstream of fixA was probably a homologue of the nifW gene of Klebsiella pneumoniae and Azotobacter vinelandii.

Mol Gen Mikrobiol Virusol, 1991 Mar, (3), 30 - 1
{Site-specific restriction endonuclease RME211 from Rhizobium meliloti}; Andreeva IS et al.; A new site-specific restriction endonuclease Rme211 from Rhizobium meliloti has been shown to recognize the following nucleotide sequence 5'-ATCGAT-3' in the double-stranded DNA . Thus, the enzyme is a true isoschizomer for restriction endonucleases Bsu151 and ClaI.

FEMS Microbiol Lett, 1991 Mar 1, 62(2-3), 325 - 31
Characterization of a soluble catalase-peroxidase hemoprotein b-590, previously identified as 'cytochrome alpha 1' from Bradyrhizobium japonicum bacteroids; Appleby CA et al.; The cytochrome "a1" or P-428, previously proposed to be a high affinity terminal oxidase in nitrogen-fixing bacteroids of Bradyrhizobium japonicum has been purified . The water-soluble native hemoprotein has an Mr of 136,000, lacks heme a and is a high-spin ferric protohemoprotein: It is slowly reduced with dithionite to give a species with an optical spectrum like that of hemoprotein b-590 (Escherichia coli; peak at 555 nm, shoulder at 590 nm), and which reacts slowly with CO . It has catalase and peroxidase activities, again resembling the E . coli b-590 . Neither hemoprotein forms a stable oxy complex under conditions in which dithionite-reduced horseradish peroxidase reacts with oxygen to form such a complex . The hemoprotein, which we name hemoprotein b-590 (Bradyrhizobium japonicum), may play a role in removal of peroxides generated during respiration in the bacteroids of several Rhizobium and Bradyrhizobium species . The high-affinity terminal oxidase under nitrogen-fixing conditions remains to be identified.

Nucleic Acids Res, 1991 Feb 25, 19(4), 921 - 7
Genetic and physical analysis of the nodD3 region of Rhizobium meliloti; Rushing BG et al.; The nodulation (nod) genes of the symbiont Rhizobium meliloti are transcriptionally controlled by protein activators in the nodD gene family . While NodD1 and NodD2 act in concert with small molecular weight inducers provided by the host legume plant, NodD3 is an inducer-independent activator of the nod promoters . We determined the sequence of the nodD3 gene, confirmed the expression of a 35 kDa protein in vitro, and determined the insertion points of five Tn5 insertions in the region of the nodD3 gene . We found the NodD3 amino acid sequence to be markedly diverged from the sequences of NodD1 and NodD2, which were more similar to the inducer-dependent NodD of another species, Rhizobium leguminosarum biovar viciae . The expression of nodD3 is not well understood, but involves at least SyrM, another positive activator related to the LysR-NodD family . One of the phenotypically mutant Tn5 insertions used in genetic studies of NodD3-dependent nod regulation lacks NodD3 protein as determined by Western blots, but another expresses about 50-60% of the wild type level . The location of these Tn5 insertions substantially upstream of the open reading frame for NodD3 suggests importance of relatively distant regulatory sequences for nodD3 expression . An insertion that did not cause a NodD3- phenotype is located in the extreme C-terminus of the protein coding region.

Gene, 1991 Feb 1, 98(1), 7 - 13
Construction of a mobilizable cloning vector for site-directed mutagenesis of gram-negative bacteria: application to Rhizobium leguminosarum; Kokotek W et al.; A mobilizable cloning vector was constructed from defined fragments to serve as a suicide plasmid for site-directed mutagenesis . The new vector, pKOK4, closely resembles plasmid pBR325 . However, the inverted duplication existing in the latter was not introduced . The useful cloning sites of pBR325 (EcoRI, HindIII, EcoRV, BamHI, SalI, PstI and PvuI) were retained and are located in one of the three resistance markers, ApR, CmR or TcR, respectively . Also, in pKOK4 the CmR gene retains its own promoter . The mob site of plasmid RP4 was introduced as a 760-bp fragment at a defined location . The mobilization frequency of pKOK4 within Escherichia coli strains is approx . 4 x 10(-2) per recipient cell . The size of pKOK4, deduced from the construction, is 6368 bp . We used pKOK4 for site-directed mutagenesis of hup-specific DNA from Rhizobium leguminosarum B10 . Integration of the vector could be distinguished reliably from marker exchange by screening for the antibiotic resistance(s) of the plasmid . This reduced the number of clones to be retested by colony and Southern hybridization to approx . 1% of the original number . Of these, almost 70% contained the desired marker exchange.

J Bacteriol, 1991 Feb, 173(3), 1344 - 6
High-frequency rearrangements in Rhizobium leguminosarum bv . phaseoli plasmids; Brom S et al.; High-frequency genomic rearrangements affecting the plasmids of Rhizobium leguminosarum bv . phaseoli CFN42 were analyzed . This strain contains six large plasmids ranging in size from 200 to 600 kb . In the absence of any selective pressure, we found 11 strains from 320 analyzed colonies that presented different kinds of plasmid-borne rearrangements, including sequence amplification, deletion, cointegration, and loss of plasmids . These data support the concept that the R . leguminosarum bv . phaseoli genome is a dynamic structure and imply that strains are mixtures of similar but not identical cells.

J Bacteriol, 1991 Feb, 173(3), 1250 - 8
Novel organization of the common nodulation genes in Rhizobium leguminosarum bv . phaseoli strains; Vazquez M et al.; Nodulation by Rhizobium, Bradyrhizobium, and Azorhizobium species in the roots of legumes and nonlegumes requires the proper expression of plant genes and of both common and specific bacterial nodulation genes . The common nodABC genes form an operon or are physically mapped together in all species studied thus far . Rhizobium leguminosarum bv . phaseoli strains are classified in two groups . The type I group has reiterated nifHDK genes and a narrow host range of nodulation . The type II group has a single copy of the nifHDK genes and a wide host range of nodulation . We have found by genetic and nucleotide sequence analysis that in type I strain CE-3, the functional common nodA gene is separated from the nodBC genes by 20 kb and thus is transcriptionally separated from the latter genes . This novel organization could be the result of a complex rearrangement, as we found zones of identity between the two separated nodA and nodBC regions . Moreover, this novel organization of the common nodABC genes seems to be a general characteristic of R . leguminosarum bv . phaseoli type I strains . Despite the separation, the coordination of the expression of these genes seems not to be altered.

Biochem J, 1991 Feb 1, 273 ( Pt 3), 511 - 6
Purification and properties of malonyl-CoA synthetase from Rhizobium japonicum; Kim YS et al.; A novel malonyl-CoA synthetase was found in Rhizobium japonicum bacteroid of the soybean nodule . The levels of the enzyme in the free-living cells grown on a variety of carbon sources including glucose were similar, indicating that this enzyme is not inducible . The malonyl-CoA synthetase from glucose-grown Rhizobium japonicum was purified to homogeneity . The Mr of the enzyme was determined to be 58,000 by gel filtration on a Sephacryl S-300 and by SDS/PAGE respectively, indicating a single polypeptide enzyme . N-Terminal amino acid of the enzyme was methionine but the enzyme preparation contained about 40% de-methionylated protein . The enzyme catalyses the formation of malonyl-CoA, AMP and PPi directly from malonate, CoA and ATP in the presence of Mg2+ . High substrate specificity on malonate and ATP was revealed, but Mn2+ could be substituted for Mg2+ without any difference in activity . Optimum pH was 7.9 . Kinetic constants, Km and Vmax, for malonate, CoA and ATP were 200 microM and 21.3 mumol/min per mg, 87 microM and 41.7 mumol/min per mg, and 33.3 microM and 29.4 mumol/min per mg respectively . Succinate inhibited the enzyme noncompetitively, whereas AMP and ADP inhibited competitively . Diethylpyrocarbonate and pyridoxal-5'-phosphate severely inhibited the enzyme, but iodoacetamide, p-chloromercuriphenylsulphonate, N-acetylimidazole and phenylglyoxal did not.

J Bacteriol, 1991 Feb, 173(4), 1502 - 8
Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors; Simon R et al.; On the basis of an RSF1010-derived broad-host-range vector, three different systems which enable positive detection and isolation of insertion sequence (IS) elements from gram-negative bacteria were constructed . Vectors pSUP104-pheS, pSUP104-rpsL, and pSUP104-sac were used successfully in a number of Rhizobium strains and in Xanthomonas campestris . More than 20 different IS elements were isolated and characterized . The 16 IS elements from Rhizobium meliloti were further used to characterize various R . meliloti strains by hybridization . The resulting hybridization patterns were different for every strain and gave a clear and definite IS fingerprint of each strain . These IS fingerprints can be used to identify and characterize R . meliloti strains rapidly and unequivocally, as they proved to be relatively stable . Some of the IS elements were found to be identical when the IS fingerprints from a given strain were compared . This method of IS fingerprinting can also establish whether IS elements are the same, related, or different.

J Bacteriol, 1991 Feb, 173(3), 1145 - 50
Aerobic growth and respiration of a delta-aminolevulinic acid synthase (hemA) mutant of Bradyrhizobium japonicum; Frustaci JM et al.; Oxygen-dependent growth of the Bradyrhizobium japonicum hemA mutant MLG1 (M.L . Guerinot and B.K . Chelm, Proc . Natl . Acad . Sci . USA 83:1837-1841, 1986) was demonstrated in cultured cells in the absence of exogenous delta-aminolevulinic acid (ALA), but growth of analogous mutants of Rhizobium meliloti or of Escherichia coli was not observed unless ALA was added to the yeast extract-containing media . No heme could be detected in extracts of strain MLG1 cells as measured by the absorption or by the peroxidase activity of the heme moiety, but the rates of growth and endogenous respiration of the mutant were essentially identical to those found in the parent strain . A role for ALA in the viability of strain MLG1 could not be ruled out since the ALA analog levulinic acid inhibited growth, but neither ALA synthase nor glutamate-dependent ALA synthesis activity was found in the mutant . The data show that the cytochromes normally discerned in wild-type B . japonicum cultured cells by absorption spectroscopy are not essential for aerobic growth or respiration.

Appl Environ Microbiol, 1991 Feb, 57(2), 426 - 33
Genetic structure of a soil population of nonsymbiotic Rhizobium leguminosarum; Segovia L et al.; The genetic structure of a population of nonsymbiotic Rhizobium leguminosarum strains was determined by the electrophoretic mobilities of eight metabolic enzymes . Nonsymbiotic strains were isolated from the rhizosphere of bean plants and characterized by growth on differential media and at different temperatures, intrinsic antibiotic resistance, the lack of homology to a nifH probe, and their inability to form nodules on bean roots . All the isolates clustered with R . leguminosarum bv . phaseoli reference strains and did not encompass any other Rhizobium taxa . Their rRNA operon restriction fragment length polymorphisms and the nucleotide sequence of a fragment of the 16S rRNA gene were also found to be identical to those of R . leguminosarum bv . phaseoli reference strains . When complemented with an R . leguminosarum bv . phaseoli symbiotic plasmid (p42d), the nonsymbiotic isolates were able to fix nitrogen in symbiosis with bean roots at levels similar to those of the parental strain . The symbiotic isolates were found at a relative frequency of 1 in 40 nonsymbiotic R . leguminosarum strains.

Biochim Biophys Acta, 1991 Jan 30, 1061(2), 197 - 205
Identification of an osmotically induced periplasmic glycine betaine-binding protein from Rhizobium meliloti; Le Rudulier D et al.; The effect of salt stress on glycine betaine-binding activity has been investigated in periplasmic fractions released from Rhizobium meliloti 102F34 by cold osmotic shock . Binding activity was monitored by three techniques: equilibrium dialysis, filter procedure, and detection of 14C ligand-protein binding by direct non-denaturing polyacrylamide gel electrophoresis (PAGE) followed by autoradiography . The three methods demonstrated the existence of a strong glycine betaine-binding activity, but only in periplasmic fractions from cells grown at high osmolarity . The non-denaturing PAGE of such periplasmic shock fluids mixed with {methyl-14C}glycine betaine showed only one radioactive band, indicating the involvement of one glycine betaine-binding protein . To determine the possible implication of this binding protein in glycine betaine uptake, transport activity was measured with cells submitted to cold osmotic shock . No significant decrease of transport activity was noticed . This lack of effect could be explained by the small quantity of periplasmic proteins released as judged by the low activity of phosphodiesterase, a periplasmic marker enzyme, observed in the shock fluid . The specificity of binding was analysed with different potential competitors: other betaines such as gamma-butyrobetaine, proline betaine, pipecolate betaine, trigonelline and homarine, or amino acids like glycine and proline, did not bind to the glycine betaine-binding protein, whereas glycine betaine aldehyde and choline were weak competitors . Optimum pH for binding was around 7.0, but approx . 90% of the glycine betaine-binding activity remained at pH 6.0 or 8.0 . The calculated binding affinity (KD) was 2.5 microM . Both glycine betaine-binding activity and affinity were not significantly modified whether or not the binding assays were done at high osmolarity . A 32 kDa osmotically inducible periplasmic protein, identified by SDS-PAGE, apparently corresponds to the glycine betaine-binding protein.

Carbohydr Res, 1991 Jan 15, 209, 203 - 9
Effect of the concentration of sodium chloride in the medium on the relative proportions of poly- and oligo-saccharides excreted by Rhizobium meliloti strain YE-2SL; Zevenhuizen LP et al.; Rhizobium meliloti mutant strains have been found which, in the presence of low concentrations of NaCl, produce a galactoglucan instead of the usual succinoglycan . When grown in a mannitol-glutamic acid-salts medium, the principal products secreted by R . meliloti YE-2S1 were comparable quantities of succinoglycan repeating-units and galactoglucan . As NaCl was added progressively to the culture medium, the repeating units nearly completely disappeared and the galactoglucan was gradually replaced by a succinoglycan.

Zentralbl Mikrobiol, 1991, 146(2), 99 - 101
Catechol degradation by immobilized Rhizobium sp; Gajendiran N et al.; Entrapped cells of Rhizobium sp . isolated from Lablab purpureus in calcium alginate degraded catechol (1,2-dihydroxybenzene) compared with the free cells . Agitation enhanced its rapid degradation . The versatility of Rhizobium sp . to degrade phenolic substances can be exploited in biotechnological application.

Mol Microbiol, 1991 Jan, 5(1), 89 - 95
Partial deletion of the Rhizobium phaseoli CFN23 symbiotic plasmid implies a concomitant amplification of plasmid DNA sequences; Soberon-Chavez G et al.; Rhizobium leguminosarum biovar phaseoli CFN23 loses its ability to nodulate beans at a high frequency because of a deletion of part of its symbiotic (pSym) plasmid (Soberon-Chavez et al., 1986) . We report here that at least 80 kb of pSym are deleted upon loss of the symbiotic phenotype; the deletion removes the nitrogenase structural nifHDK and the common nodABC genes . The size of the deleted pSym is not reduced, since it is accompanied by an amplification of other pSym plasmid sequences . This genetic rearrangement is similar to the deletion and amplification of yeast mitochondrial DNA leading to 'petite' mutations.

Mol Microbiol, 1991 Jan, 5(1), 157 - 62
Sequences downstream from the transcriptional start site are essential for microaerobic, but not symbiotic, expression of the Rhizobium meliloti nifHDK promoter; Wang YP et al.; Deletion analysis studies have been carried out on the nifHDK promoter (P1) of R . meliloti in an attempt to determine sequences involved in the expression of this promoter under both free-living microaerobic and symbiotic conditions . Deletion of a region downstream (+17 to +61) from the promoter element resulted in low levels of expression under free-living microaerobic conditions . However, wild-type levels of expression were obtained during symbiosis with Alfalfa plants . The sequences in this region were designated the "downstream sequences' . The pattern of expression observed when the downstream sequences were deleted was similar to that observed when a previously identified upstream activator sequence (UAS) was deleted . Only when both the downstream sequences and the UAS were deleted, did activity from the P1 promoter become significantly decreased during symbiosis . Expression studies of the P1 promoter in a nifA mutant background indicate that nifA is required for symbiotic expression of P1 which is enhanced by the presence of the downstream sequences.

Mol Gen Genet, 1991 Jan, 225(1), 38 - 48
The regulatory status of the fixL- and fixJ-like genes in Bradyrhizobium japonicum may be different from that in Rhizobium meliloti; Anthamatten D et al.; The cloning, sequencing and mutational analysis of the Bradyrhizobium japonicum symbiotic nitrogen fixation genes fixL and fixJ are reported here . The two genes were adjacent and probably formed an operon, fixLJ . The predicted FixL and FixJ proteins, members of the two-component sensor/regulator family, were homologous over almost their entire lengths to the corresponding Rhizobium meliloti proteins (approx . 50% identity) . Downstream of the B . japonicum fixJ gene was found an open reading frame with 138 codons (ORF138) whose product shared 36% homology with the N-terminal part of FixJ . Deletion and insertion mutations within fixL and fixJ led to a loss of approximately 90% wild-type symbiotic nitrogen fixation (Fix) activity, whereas an ORF138 mutant was Fix+ . In fixL, fixJ and ORF138 mutant backgrounds, the aerobic expression of the fixR-nifA operon was not affected . NifA itself did not regulate the expression of the fixJ gene . Thus, the B . japonicum FixL and FixJ proteins were neither involved in the regulation of aerobic nifA gene expression nor in the anaerobic NifA-dependent autoregulation of the fixRnifA operon, rather they appeared to control symbiotically important genes other than those whose expression was dependent on the NifA protein . The fixL and fixJ mutant strains were unable to grow anaerobically with nitrate as the terminal electron acceptor . Therefore, some of the FixJ-dependent genes in B . japonicum may be concerned with anaerobic respiration.

J Bacteriol, 1991 Jan, 173(2), 933 - 6
Visualization of bacterial flagella by video-enhanced light microscopy; Block SM et al.; We have imaged individual flagellar filaments of Escherichia coli, a motile Streptococcus sp., and Rhizobium meliloti by video-enhanced differential interference-contrast microscopy (Nomarski DIC) and computer-based image processing . This approach has advantages over existing methods in that filaments on living cells can be seen over their entire lengths.

J Bacteriol, 1991 Jan, 173(2), 851 - 9
Exopolysaccharide mutants of Rhizobium loti are fully effective on a determinate nodulating host but are ineffective on an indeterminate nodulating host; Hotter GS et al.; By Tn5 mutagenesis of Rhizobium loti PN184 (NZP2037 str-1) and selection for nonfluorescence of colonies on Calcofluor agar, eight independently generated expolysaccharide (EPS) mutants (three smooth and five rough) were isolated . The parent strain, PN184, was found to produce an acidic EPS . This EPS was produced . with reduced O acetylation, by the smooth EPS mutants but not by the rough EPS mutants . Lipopolysaccharide was isolated from all mutants and was identical to that of PN184 as defined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . All mutants were resistant to lysis by R . loti bacteriophage phi 2037/1 . Cosmids that complemented the mutations in the rough EPS mutants were isolated from a pLAFR1 gene library of NZP2037 by complementation of the nonfluorescent phenotype . The genes identified were shown to be unlinked and located on the chromosome . All mutants were fully effective when inoculated onto Lotus pedunculatus, a determinate nodulating host, but were ineffective, inducing the formation of very small nodules or tumorlike growths, when inoculated onto Leucaena leucocephala, an indeterminate nodulating host . These results, obtained in an isogenic Rhizobium background, support suggestions that acidic EPS is required for effective nodulation of indeterminate nodulating legumes but is not required for effective nodulation of determinate nodulating legumes.

J Bacteriol, 1991 Jan, 173(2), 664 - 77
The exoD gene of Rhizobium meliloti encodes a novel function needed for alfalfa nodule invasion; Reed JW et al.; During the symbiotic interaction between alfalfa and the nitrogen-fixing bacterium Rhizobium meliloti, the bacterium induces the formation of nodules on the plant roots and then invades these nodules . Among the bacterial genes required for nodule invasion are the exo genes, involved in production of an extracellular polysaccharide, and the ndv genes, needed for production of a periplasmic cyclic glucan . Mutations in the exoD gene result in altered exopolysaccharide production and in a nodule invasion defect . In this work we show that the stage of symbiotic arrest of exoD mutants is similar to that of other exo and ndv mutants . However, the effects of exoD mutations on exopolysaccharide production and growth on various media are different from the effects of other exo and ndv mutations . Finally, exoD mutations behave differently from other exo mutations in their ability to be suppressed or complemented extracellularly . The results suggest that exoD represents a new class of Rhizobium genes required for nodule invasion, distinct from the other exo genes and the ndv genes . We discuss models for the function of exoD.

J Bacteriol, 1991 Jan, 173(1), 365 - 71
Detection of alternative nitrogenases in aerobic gram-negative nitrogen-fixing bacteria; Fallik E et al.; Strains of aerobic, microaerobic, nonsymbiotic, and symbiotic dinitrogen-fixing bacteria were screened for the presence of alternative nitrogenase (N2ase) genes by DNA hybridization between genomic DNA and DNA encoding structural genes for components 1 of three different enzymes . A nifDK gene probe was used as a control to test for the presence of the commonly occurring Mo-Fe N2ase, a vnfDGK gene probe was used to show the presence of V-Fe N2ase, and an anfDGK probe was used to detect Fe N2ase . Hitherto, all three enzymes have been identified in Azotobacter vinelandii OP, and all but the Fe N2ase are present in Azotobacter chroococcum ATCC 4412 (MCD1) . Mo-Fe N2ase and V-Fe N2ase structural genes only were confirmed in this strain and in two other strains of A . chroococcum (ATCC 480 and ATCC 9043) . A similar pattern was observed with Azotobacter beijerinckii ATCC 19360 and Azotobacter nigricans ATCC 35009 . Genes for all three systems are apparently present in two strains of Azotobacter paspali (ATCC 23367 and ATCC 23833) and also in Azomonas agilis ATCC 7494 . There was no good evidence for the existence of any genes other than Mo-Fe N2ase structural genes in several Rhizobium meliloti strains, cowpea Rhizobium strain 32H1, or Bradyrhizobium japonicum . Nitrogenase and nitrogenase genes in Azorhizobium caulinodans behaved in an intermediate fashion, showing (i) the formation of ethane from acetylene under Mo starvation, a characteristic of alternative nitrogenases, and (ii) a surprising degree of cross-hybridization to the vnfDGK, but not the anfDGK, probe . vnfDGK- and anfDGK-like sequences were not detected in two saccharolytic Pseudomonas species or Azospirillum brasilense Sp7 . The occurrence of alternative N2ases seems restricted to members of the family Azotobacteraceae among the aerobic and microaerobic diazotrophs tested, suggesting that an ability to cope with O2 when fixing N2 may be an important factor influencing the distribution of alternative nitrogenases.

Arch Microbiol, 1991, 155(6), 543 - 8
NifA-NtrA regulatory system activates transcription of nfe, a gene locus involved in nodulation competitiveness of Rhizobium meliloti; Sanjuan J et al.; We have previously demonstrated that the Rhizobium meliloti large plasmid pRmeGR4b carries the gene locus nodule formation efficiency (nfe) which is responsible for nodulation efficiency and competitive ability of strain GR4 on alfalfa roots . In this study we report that expression of nfe-lacZ fusions in Escherichia coli is activated in the presence of the cloned nifA gene of R . meliloti . This activation was found to be oxygen sensitive and to require the E . coli ntrA gene product . In contrast to the R . meliloti nifA, the cloned nifA gene of Klebsiella pneumoniae was able to activate expression of nfe in aerobically grown cells of both E . coli and R . meliloti . Hybridization experiments did not show homology to nfe in four R . meliloti wild-type strains tested . These strains were uncompetitive when coinoculated with a GR4 derivative carrying plasmid pRmeGR4b, but were competitive when coinoculated with a GR4 derivative carrying a single transposon mutation into the nfe region . When nfe DNA was introduced into the four wild-type strains, a significant increase in the competitive ability of two of them was observed, as deduced from their respective percentages of alfalfa root nodule occupancy in two-strains coinoculation experiments.

Mol Gen Genet, 1991 Jan, 225(1), 1 - 10
Structural genes of glutamate 1-semialdehyde aminotransferase for porphyrin synthesis in a cyanobacterium and Escherichia coli; Grimm B et al.; In bacteria 5-aminolevulinate, the universal precursor in the biosynthesis of the porphyrin nucleus of hemes, chlorophylls and bilins is synthesised by two different pathways: in non-sulphur purple bacteria (Rhodobacter) or Rhizobium 5-aminolevulinate synthase condenses glycine and succinyl-CoA into 5-aminolevulinate as is the case in mammalian cells and yeast . In cyanobacteria, green and purple sulphur bacteria, as in chloroplasts of higher plants and algae a three step pathway converts glutamate into 5-aminolevulinate . The last step is the conversion of glutamate 1-semialdehyde into 5-aminolevulinate . Using a cDNA clone encoding glutamate 1-semialdehyde aminotransferase from barley, genes for this enzyme were cloned from Synechococcus PCC6301 and Escherichia coli and sequenced . The popC gene of E . coli, previously considered to encode 5-aminolevulinate synthase, appears to be a structural gene for glutamate 1-semialdehyde aminotransferase . Domains with identical amino acid sequences comprise 48% of the primary structure of the barley, cyanobacterial and putative E . coli glutamate 1-semialdehyde aminotransferases . The cyanobacterial and barley enzymes share 72% identical residues . The peptide containing a likely pyridoxamine phosphate binding lysine is conserved in all three protein sequences.

Genetics, 1991 Jan, 127(1), 5 - 20
Analysis of a 1600-kilobase Rhizobium meliloti megaplasmid using defined deletions generated in vivo; Charles TC et al.; A series of 120-600 kilobase deletions with defined endpoints were made in the 1600-kilobase Rhizobium meliloti megaplasmid pRmeSU47b, by homologous recombination between the IS50 elements of transposon insertions . Utilizing IS 50-mediated homologous recombination we also made defined reductions in deletion size and combined adjacent deletions . Deletion structure was confirmed by phage transduction and Southern hybridization analysis . Collectively these deletions span 1400 kilobases of pRmeSU47b, indicating that the majority of the plasmid is not essential for cell viability . This was further confirmed by the construction of a strain SU47 derivative which carries only 450 kilobases of the pRmeSU47b megaplasmid . Examination of the deletion mutants for phenotype revealed novel loci required for dulcitol, melibiose, raffinose, beta-hydroxybutyrate, acetoacetate, protocatechuate and quinate utilization . Previously unidentified loci required for effective root nodule development and exopolysaccharide synthesis were also found . Various deletion mutants were deficient in dicarboxylate transport, lactose utilization, and thiamine and exopolysaccharide biosynthesis, as predicted from earlier studies of this megaplasmid.

J Bacteriol, 1991 Jan, 173(2), 704 - 9
The genomes of the family Rhizobiaceae: size, stability, and rarely cutting restriction endonucleases; Sobral BW et al.; The lack of high-resolution genetic or physical maps for the family Rhizobiaceae limits our understanding of this agronomically important bacterial family . On the basis of statistical analyses of DNA sequences of the Rhizobiaceae and direct evaluation by pulsed-field agarose gel electrophoresis (PFE), five restriction endonucleases with AT-rich target sites were identified as the most rarely cutting: AseI (5'-ATTAAT-3'), DraI (5'-TTTAAA-3'), SpeI (5'-ACTAGT-3'), SspI (5'-AATAAT-3'), and XbaI (5'-TCTAGA-3') . We computed the sizes of the genomes of Bradyrhizobium japonicum USDA 424 and Rhizobium meliloti 1021 by adding the sizes of DNA fragments generated by SpeI digests . The genome sizes of R . meliloti 1021 and B . japonicum USDA 424 were 5,379 +/- 282.5 kb and 6,195 +/- 192.4 kb, respectively . We also compared the organization of the genomes of free-living and bacteroid forms of B . japonicum . No differences between the PFE-resolved genomic fingerprints of free-living and mature (35 days after inoculation) bacteroids of B . japonicum USDA 123 and USDA 122 were observed . Also, B . japonicum USDA 123 genomic fingerprints were unchanged after passage through nodules and after maintenance on a rich growth medium for 100 generations . We conclude that large-scale DNA rearrangements are not seen in mature bacteroids or during free-living growth on rich growth media under laboratory conditions.

J Bacteriol, 1991 Jan, 173(2), 426 - 34
Regulation of Rhizobium meliloti exo genes in free-living cells and in planta examined by using TnphoA fusions; Reuber TL et al.; The exo loci of Rhizobium meliloti are necessary for the production of an acidic exopolysaccharide, EPS I, that is needed for alfalfa nodule invasion by strain Rm1021 . We have isolated and characterized alkaline phosphatase fusions made with TnphoA in several exo loci of R . meliloti and used these fusions to examine the subcellular localization of exo gene products and the regulation of exo genes in free-living cells and in planta . In the course of this work, we isolated a new exo locus, exoT . We have obtained evidence that several of the exo loci may encode membrane proteins . The activity of TnphoA fusions in several exo loci is increased two- to fivefold in the presence of the regulatory mutations exoR95 and exoS96 . While examining the regulation of the exo gens by exoR95 and exoS96, we found that certain classes of exo mutations are lethal in an exoR95 or exoS96 background unless a plasmid complementing the exo mutation is present . This result has possible implications for the role of these exo loci in EPS I biosynthesis . We have developed a method for staining nodules specifically for the alkaline phosphatase activity present in the inducing bacteria and used this method to show that an exoF::TnphoA fusion is expressed mainly in the invasion zone of the nodule.

J Bacteriol, 1991 Jan, 173(1), 382 - 90
Aerobic inactivation of Rhizobium meliloti NifA in Escherichia coli is mediated by lon and two newly identified genes, snoB and snoC; Huala E et al.; The Rhizobium meliloti NifA protein is an oxygen-sensitive transcriptional regulator of nitrogen fixation genes . Regulation of NifA activity by oxygen occurs at the transcriptional level through fixLJ and at the posttranslational level through the sensitivity of NifA to oxygen . We have previously reported that the NifA protein is sensitive to oxygen in Escherichia coli as well as in R . meliloti . To investigate whether the posttranslational regulation of NifA is dependent on host factors conserved between R . meliloti and E . coli, we carried out a Tn5 mutagenesis of E . coli and isolated mutants with increased NifA activity under aerobic conditions . Fifteen insertion mutations occurred at three unlinked loci . One locus is the previously characterized lon gene; the other two loci, which we have named snoB and snoC, define previously uncharacterized E . coli genes . The products of snoC and lon affect the rate of NifA degradation, whereas the product of snoB may affect both NifA degradation and inactivation . A snoB lon double mutant showed a higher level of NifA accumulation than did a lon mutant, suggesting that the snoB product affects the ability of NifA to be degraded by a lon-independent pathway . The effects of a snoC mutation and lon mutation were not additive, suggesting that the snoC and lon products function in the same degradative pathway.

Folia Microbiol (Praha), 1991, 36(3), 271 - 6
Comparative response of Pisum sativum nodulated with indigenous soil Rhizobium populations and/or co-inoculated with a Rhizobium leguminosarum strain . I . Acetylene-reducing, dihydrogen- and carbon dioxide-evolving activities; Skrdleta V et al.; No significant differences in the acetylene-reducing activity and evolution of H2 and CO2 from nodulated roots of Pisum sativum inoculated with soil Rhizobium populations from two soils with different acidities (Ruzyne soil 7.6; Lukavec soil 4.9) were observed . Rhizobium population from Lukavec soil formed nodules, exhibiting a higher H2 evolution . Co-inoculation with the Hup+ strain 128C30 (7 x 10(7) cells per seedling) eliminated, to some extent, the effect of soil populations on physiological activity.

Folia Microbiol (Praha), 1991, 36(2), 164 - 8
Production of Tsr factor by Rhizobium meliloti; Jain V et al.; The root exudates of alfalfa (Medicago sativa) and mungbean (Vigna radiata) induced the Tsr (thick and short roots) factor production in Rhizobium meliloti . The factor caused a 30-40% reduction of root length in alfalfa seedlings . Pea root exudate had no Tsr induction activity . The flavonoid naringenin could replace the roots in inducing Tsr production . Naringenin-induced Tsr factor caused 70% shortening of main roots . The Tsr inducing property of naringenin was specific since quercetin and syringaldehyde had no such effect.

Acta Biochim Pol, 1991, 38(4), 423 - 35
The region for exopolysaccharide synthesis in Rhizobium leguminosarum biovar trifolii is located on the non-symbiotic plasmid; Skorupska A et al.; An Exo- mutant of Rhizobium leguminosarum biovar trifolii was isolated which did not produce acidic exopolysaccharide and induced defective, non-fixing nodules on clover plants . The nodules were defective at a late stage of development, they contained infection threads and bacteria were released into the host cells . Cosmid pARF136 capable of complementing the Exo- mutation was isolated from a cosmid bank made from total R . trifolii DNA . Hybridization between DNA of pARF136 and plasmids of R . trifolii strains separated by Eckhardt's technique suggested that the exo locus is located on a 300 kb megaplasmid, and nodDABC and nifKDH genes are located on another 180 kb pSym plasmid . A 5.4 kb BamH1 fragment of the recombinant cosmid pARF136 was able to restore exopolysaccharide synthesis in Exo- mutant of R . trifolii 93 but it did not complement the symbiotic defect.

Acta Microbiol Pol, 1991, 40(3-4), 265 - 8
Biological activity of rhizobial siderophore; Derylo M et al.; Non-nodulating mutant of Rhizobium leguminosarum biovar trifolli produces the phenolate type of siderophore consisting of 2,3-dihydroxybenzoic acid and threonine . The activity of this compound against the various bacteria was tested . Only, the growth of R . leguminosarum strains was stimulated by siderophore . The other species of Rhizobium, especially R . meliloti, were sensitive to this agent . The growth of R . meliloti was also inhibited by agrobactin and pseudobactin . This effect was reversed by ferric iron.

Nature, 1990 Dec 13, 348(6302), 644 - 7
ATP sulphurylase activity of the nodP and nodQ gene products of Rhizobium meliloti; Schwedock J et al.; The symbiotic bacterium Rhizobium meliloti stimulates alfalfa (Medicago sativa L.) roots to undergo morphogenesis and form nitrogen-fixing nodules . It has been proposed that the bacterial genes nodABC, common to all Rhizobium, are required for synthesis of an oligosaccharide factor, which is converted to a sulphated form (NodRm-1) by the products of the R . meliloti-specific genes nodH and nodQ1-5; NodRm-1 elicits host-specific plant responses . Previously we have shown that the nodP gene is homologous to a segment of the Escherichia coli genome; when we cloned this E . coli fragment we found that it mapped near 59 minutes, corresponding to the cysDNC locus . The genes cysD and cysN encode proteins that catalyse the synthesis of adenosine 5'-phosphosulphate, the first step in the activation of inorganic sulphate . Here we demonstrate that nodP and nodQ correspond to cysD and cysN, and that their proteins have ATP sulphurylase activity both in vivo and in vitro . We propose that nodP and nodQ synthesize an activated sulphate that is an intermediate in the formation of the alfalfa-specific sulphated nodRm-1 factor.

J Biol Chem, 1990 Dec 5, 265(34), 21122 - 7
Rhizobium leguminosarum exoB mutants are deficient in the synthesis of UDP-glucose 4'-epimerase; Canter Cremers HC et al.; Rhizobium leguminosarum bv . viciae Exo- mutant strains RBL5523,exo7::Tn5,RBL5523,exo8::Tn5 and RBL5523,exo52::Tn5 are affected in nodulation and in the syntheses of lipopolysaccharide, capsular polysaccharide, and exocellular polysaccharide . These mutants were complemented for nodulation and for the syntheses of these polysaccharides by plasmid pMP2603 . The gene in which these mutants are defective is functionally homologous to the exoB gene of Rhizobium meliloti . The repeating unit of the residual amounts of EPS still made by the exoB mutants of R . leguminosarum bv . viciae lacks galactose and the substituents attached to it . The R . leguminosarum bv . viciae and R . meliloti exoB mutants fail to synthesize active UDP-glucose 4'-epimerase.

Biochem J, 1990 Nov 1, 271(3), 713 - 20
3-Oxoacyl-(acyl-carrier protein) reductase from avocado (Persea americana) fruit mesocarp; Sheldon PS et al.; The NADPH-linked 3-oxoacyl-(acyl-carrier protein) (ACP) reductase (EC 1.1.1.100), also known as 'beta-ketoacyl-ACP reductase', has been purified from the mesocarp of mature avocado pears (Persea americana) . The enzyme is inactivated by low ionic strength and low temperature . On SDS/PAGE under reducing conditions, purified 3-oxoacyl-ACP reductase migrated as a single polypeptide giving a molecular mass of 28 kDa . Gel-filtration chromatography gave an apparent native molecular mass of 130 kDa, suggesting that the enzyme is tetrameric . The enzyme is inactivated by dilution, but some protection is afforded by the presence of NADPH . Kinetic constants have been determined using synthetic analogues as well as the natural ACP substrate . It exhibits a broad pH optimum around neutrality . Phenylglyoxal inactivates the enzyme, and partial protection is given by 1 mM-NADPH . Antibodies have been raised against the protein, which were used to localize it using immunogold electron microscopy . It is localized in plastids . N-Terminal amino-acid-sequence analysis was performed on the enzyme, and it shows close structural similarity with cytochrome f . Internal amino-acid-sequence data, derived from tryptic peptides, shows similarity with the putative gene products encoded by the nodG gene from the nitrogen-fixing bacterium Rhizobium meliloti and the gra III act III genes from Streptomyces spp.

J Bacteriol, 1990 Nov, 172(11), 6596 - 8
Rhizobium meliloti chromosomal loci required for suppression of exopolysaccharide mutations by lipopolysaccharide; Williams MN et al.; Mutants of alfalfa symbiont Rhizobium meliloti SU47 that fail to make extracellular polysaccharide (exo mutants) induce the formation of nodules that are devoid of bacteria and consequently do not fix nitrogen . This Fix- phenotype can be suppressed by an R . meliloti Rm41 gene that affects lipopolysaccharide structure . Here we describe mutations preventing suppression that map at two new chromosomal loci, lpsY and lpsX, present in both strains . Two other lps mutations isolated previously from SU47 also prevented suppression.

Infect Immun, 1990 Nov, 58(11), 3743 - 50
Cytokine induction by lipopolysaccharide (LPS) corresponds to lethal toxicity and is inhibited by nontoxic Rhodobacter capsulatus LPS; Loppnow H et al.; Many pathological effects of gram-negative bacteria are produced by their cell wall-derived lipopolysaccharides (LPSs) . Differing pathogenicity of gram-negative LPSs, however, may depend on their capacities to induce cytokines . Thus, we studied the lethal toxicity of four nonenterobacterial LPSs and compared it with their capacity to induce mononuclear cell (MNC)-derived interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) . Unstimulated MNC did not release these cytokines . LPS from the phototrophic strain Rhodobacter capsulatus 37b4 elaborated little toxicity in galactosamine-treated mice (10 micrograms of LPS per mouse was the 100% lethal dose {LD100}) and induced IL-1 and IL-6 release only at high concentrations (10 to 50 micrograms of LPS per ml) . R . capsulatus LPS failed to induce TNF activity even at the highest concentration tested (100 micrograms of LPS per ml) . In contrast, LPS derived from Pseudomonas diminuta NCTC 8545 or the nodulating species Bradyrhizobium lupini DSM 30140 and Rhizobium meliloti 10406 expressed lethal toxicity (LD100, 1,000, 100, and 10 ng per mouse, respectively) and induced IL-1 or IL-6 (10 to 100, 10, and 1 ng of LPS per ml, respectively) at concentrations 1,000- to 10,000-fold lower than effective levels of R . capsulatus LPS . LPSs from P . diminuta, B . lupini, and R . meliloti also stimulated TNF production and release . MNC accumulated cell-associated IL-1 activities under circumstances in which released activity was readily detected . The cells contained only scant IL-6 activity, indicating release of this mediator rather than intracellular accumulation . Antisera to the respective cytokines inactivated biological activities of the samples selectively . The R . capsulatus LPS inhibited cytokine induction by LPS from P . diminuta, B . lupini, and R . meliloti in coincubation experiments . These results show that the in vivo lethality of the LPSs tested correlates with the induction of monocyte-derived cytokines in vitro . The results of this study suggest that the different lethality of various LPSs from gram-negative bacteria may be due to the differential ability of these LPSs to induce cytokine production.

Mol Plant Microbe Interact, 1990 Nov-Dec, 3(6), 389 - 400
Characterization of two Azospirillum brasilense Sp7 plasmid genes homologous to Rhizobium meliloti nodPQ; Vieille C et al.; Bacteria belonging to the Azospirillum genus are nitrogen fixers that colonize the roots of grasses, but do not cause the formation of differentiated structures . Sequences from total DNA of several Azospirillum strains are homologous to restriction fragments containing Rhizobium meliloti nodulation genes . A 10-kilobase (kb) EcoRI fragment from A . brasilense Sp7, sharing homology with a 6.8-kb EcoRI fragment carrying nodGEFH and part of nodP of R . meliloti 41, was cloned in pUC18 to yield pAB502 . The nucleotide sequence of a 3.5-kb EcoRI-SmaI fragment of the pAB502 insert revealed 60% homology with R . meliloti nodP and nodQ genes . The nodP gene product shares no homology to any known protein sequence . The Azospirillum nodQ gene product shares homology with a family of initiation and elongation factors as does the R . meliloti nodQ gene product . Since the nodQ gene overlaps the nodP gene, the two genes might be cotranscribed . Azospirillum contains large plasmids, and the nodPQ genes were found on the 90-MDa plasmid (p90) . A translational nodP-lacZ fusion was constructed in the broad host range plasmid pGD926 . No beta-galactosidase activity was detected in Escherichia coli, but the fusion was functional in Azospirillum and constitutively expressed . Deletions and mutations of nodPQ did not modify growth, nitrogen fixation, or interaction with wheat seedlings.

Mol Microbiol, 1990 Nov, 4(11), 1975 - 84
Role of the nodD and syrM genes in the activation of the regulatory gene nodD3, and of the common and host-specific nod genes of Rhizobium meliloti; Maillet F et al.; To analyse the regulation of the nodulation (nod) genes of Rhizobium meliloti RCR2011 we have isolated lacZ gene fusions to a number of common, host-range and regulatory nod genes, using the mini-Mu-lac bacteriophage transposon MudII1734 . Common (nodA, nodC, nod region IIa) and host-range (nodE, nodG, nodH) genes were found to be regulated similarly . They were activated (i) by the regulatory nodD1 gene in the presence of flavones such as chrysoeriol, luteolin and 7,3',4'-trihydroxyflavone, (ii) by nodD2 in the presence of alfalfa root exudate but not with the NodD1-activating flavones, and (iii) by the regulatory genes syrM-nodD3 even in the absence of plant inducers . Thus common and host-range nod genes belong to the same regulon . In contrast to the nodD1 gene, the regulatory nodD3 gene was not expressed constitutively and exhibited a complex regulation . It required syrM for expression, was activated by nodD1 in the presence of luteolin and was positively autoregulated.

Mol Microbiol, 1990 Nov, 4(11), 1899 - 910
Identification and cloning of nodulation genes and host specificity determinants of the broad host-range Rhizobium leguminosarum biovar phaseoli strain CIAT899; Vargas C et al.; Rhizobium leguminosarum biovar phaseoli type II strain CIAT899 nodulates a wide range of hosts: Phaseolus vulgaris (beans), Leucaena esculenta (leucaena) and Macroptilium atropurpureum (siratro) . A nodulation region from the symbiotic plasmid has been isolated and characterized . This region, which is contained in the overlapping cosmid clones pCV38 and pCV117, is able to induce nodules in beans, leucaena and siratro roots when introduced in strains cured for the symbiotic plasmid, pSym . In addition, this cloned region extends the host range of Rhizobium meliloti and R . leguminosarum biovar (bv.) trifolii wild-type strains to nodulate beans . Analysis of constructed subclones indicates that a 6.4kb HindIII fragment contains the essential genes required for nodule induction on all three hosts . Rhizobium leguminosarum bv . phaseoli type I strain CE3 nodulates only beans . However, CE3 transconjugants harbouring plasmid pCV3802 (which hybridized to a nodD heterologous probe), were capable of eliciting nodules on leucaena and siratro roots . Our results suggest that the CIAT899 DNA region hybridizing with the R . meliloti nodD detector is involved in the extension of host specificity to promote nodule formation in P . vulgaris, L . esculenta and M . atropurpureum.

Plasmid, 1990 Nov, 24(3), 235 - 9
Isolation of DNA insertion elements from Rhizobium meliloti which are able to promote transcription of adjacent genes; Labes G et al.; In order to select insertion sequences able to promote transcription of flanking genes (ISp elements), three mobilizable RSF1010 derived vectors were constructed . Using promoterless antibiotic resistance genes, ISp elements ranging from 0.75 to 2.9 kb were isolated from Escherichia coli and Rhizobium meliloti . Restriction and hybridization experiments revealed that identical ISp elements could be isolated from different R . meliloti strains and that one of these is similar to an insertion sequence found previously in R . meliloti 2011.

J Biol Chem, 1990 Oct 25, 265(30), 18248 - 55
Glutamyl-tRNA synthetases of Bacillus subtilis 168T and of Bacillus stearothermophilus . Cloning and sequencing of the gltX genes and comparison with other aminoacyl-tRNA synthetases; Breton R et al.; The glutamyl-tRNA synthetase (GluRS) of Bacillus subtilis 168T aminoacylates with glutamate its homologous tRNA(Glu) and tRNA(Gln) in vivo and Escherichia coli tRNA(1Gln) in vitro (Lapointe, J., Duplain, L., and Proulx, M . (1986) J . Bacteriol . 165, 88-93) . The gltX gene encoding this enzyme was cloned and sequenced . It encodes a protein of 483 amino acids with a Mr of 55,671 . Alignment of the amino acid sequences of four bacterial GluRSs (from B . subtilis, Bacillus stearothermophilus, E . coli, and Rhizobium meliloti) gives 20% identity and reveals the presence of several short highly conserved motifs in the first two thirds of these proteins . Conserved motifs are found at corresponding positions in several other aminoacyl-tRNA synthetases . The only sequence similarity between the GluRSs of these Bacillus species and the E . coli glutaminyl-tRNA synthetase (GlnRS), which has no counterpart in the E . coli GluRS, is in a segment of 30 amino acids in the last third of these synthetases . In the three-dimensional structure of the E . coli tRNA(Gln).GlnRS.ATP complex, this conserved peptide is near the anticodon of tRNA(Gln) (Rould, M . A., Perona, J . J., Soll, D., and Steitz, T . A . (1989) Science 246, 1135-1142), suggesting that this region is involved in the specific interactions between these enzymes and the anticodon regions of their tRNA substrates.

Indian J Exp Biol, 1990 Oct, 28(10), 994 - 5
Binary conjugational transfer system of vectors pRK290 and pLAFRI is proficient both ways between Escherichia coli and Rhizobium; Singh RK et al.; Wide host range vector plasmids pRK290 and pLAFRI carrying genomic fragments of Rhizobium are transferable both ways between R . meliloti and R . leguminosarum cells on the one hand and to E . coli cells on the other, in triparental matings involving E . coli cells carrying pRK2013, the helper for Tra functions to the vector plasmids . The vector plasmids pRK290 and pLAFRI can be employed for recovering clones harbored by R . leguminosarum and R . meliloti by transfer to Rhizobium cells by direct matings of the library with them.

J Bacteriol, 1990 Oct, 172(10), 6066 - 76
Identification of a Caulobacter basal body structural gene and a cis-acting site required for activation of transcription; Dingwall A et al.; The genes that encode the components and regulatory proteins of the Caulobacter crescentus flagellum are transcribed at specific times in the cell cycle . One of these genes, flbN, is required early in the flagellar assembly process . The flbN gene was cloned and sequenced, and the time of transcription activation was determined . The derived amino acid sequence indicates that fibN encodes a 25-kilodalton protein with a cleavable leader peptide . The flbN-encoded protein has 30.8% identity with the protein encoded by the Salmonella typhimurium basal body L-ring gene, flgH . Site-directed mutagenesis and gel mobility shift assays identified a binding site at -100 from the transcription start site for a trans-acting protein, RF-2, that functions to partially activate flbN transcription at a defined time in the cell cycle . The RF-2 binding region is similar to a NifA binding site normally used in the activation of some sigma 54 promoters involved in nitrogen fixation in other bacteria . Transcription of a flbN-reporter gene fusion in an Escherichia coli background was dependent on the presence of a NifA transcription factor supplied by a plasmid-borne Rhizobium meliloti gene encoding NifA . A deletion or base changes in the RF-2 binding region eliminated expression of the flbN gene in E . coli even when a NifA protein was provided in trans, suggesting that a sigma 54 promoter with an upstream activator element is used by the C . crescentus flbN gene . A consensus sequence for a sigma 54 promoter was found at the appropriate distance 5' to one of two identified transcription start sites . Site-directed mutagenesis confirmed that a conserved nucleotide in this sigma 54 promoter consensus sequence was required for transcription . Deletion of the region 5' to the apparent sigma 54 promoter caused a complete loss of transcription activation . Transcription activation of flbN in C . crescentus involves the combination of several elements: the NifA-like site is required for full activation, and other sequence elements 5' to the promoter and 3' to the transcription start site are necessary for the correct time of transcription initiation.

Plant Cell, 1990 Oct, 2(10), 973 - 86
Functional analysis of the Sesbania rostrata leghemoglobin glb3 gene 5'-upstream region in transgenic Lotus corniculatus and Nicotiana tabacum plants; Szabados L et al.; Expression of the Sesbania rostrata leghemoglobin glb3 gene was analyzed in transgenic Lotus corniculatus and tobacco plants harboring chimeric glb3-uidA (gus) gene fusions to identify cis-acting elements involved in nodule-specific gene expression and general transcriptional control . A 1.9-kilobase fragment of the glb3 5'-upstream region was found to direct a high level of nodule-specific beta-glucuronidase (GUS) activity in L . corniculatus, restricted to the Rhizobium-infected cells of the nodules . The same fragment directed a low level of GUS activity in tobacco, restricted primarily to the roots and to phloem cells of the stem and petiole vascular system . A deletion analysis revealed that the region between coordinates -429 and -48 relative to the ATG was sufficient for nodule-specific expression . Replacement of the -161 to -48 region, containing the glb3 CAAT and TATA boxes, with the heterologous truncated promoters delta-p35S and delta-pnos resulted in a loss of nodule specificity and reduction of GUS activity in L . corniculatus but a significant increase in tobacco, primarily in the roots . The same fragment could not direct nodule-specific expression when fused to a heterologous enhancer in cis . This region contains DNA sequences required, but not sufficient, for nodule-specific expression in L . corniculatus that function poorly or may be involved in promoter silencing in tobacco . By fusing further upstream fragments to the delta-p35S and delta-pnos promoters, two positive regulatory regions were delimited between coordinates -1601 and -670, as well as -429 and -162 . The former region appears to function as a general enhancer because it significantly increased promoter activity in both orientations in L . corniculatus and tobacco . The latter region could enhance gene expression in both orientations in tobacco, but only in the correct orientation in L . corniculatus . These results show that efficient expression of the S . rostrata glb3 gene in nodules is mediated by an ATG-proximal, tissue-specific element, as well as further 5'-upstream positive elements; that the S . rostrata glb3 promoter is induced in a nodule-specific fashion in the heterologous legume L . corniculatus, suggesting a high degree of conservation of the relevant regulatory signals; and that the S . rostrata lb promoter is not silent in the nonlegume tobacco, but is expressed primarily in the roots.

Mol Microbiol, 1990 Oct, 4(10), 1727 - 35
Transcriptional analysis of the glnB-glnA region of Rhizobium leguminosarum biovar viciae; Chiurazzi M et al.; We report that the glnB and glnA genes of Rhizobium leguminosarum biovar viciae are preceded by promoters located upstream of each gene . We find the presence of a glnB-glnA and a glnA mRNA whose intracellular concentration changes two- to three-fold when R . leguminosarum is grown on different nitrogen sources . Primer extension analysis shows unique transcriptional initiation sites upstream of glnB and glnA . The glnB promoter is rpoN(ntrA)-dependent, while the glnA promoter does not contain a typical consensus sequence for previously described promoters . In Klebsiella pneumoniae the glnB promoter requires active ntrC and ntrA genes and a DNA fragment containing 53 nucleotides upstream of the transcription initiation site shows full promoter activity, thus indicating that no NtrC binding sites are necessary for this activation in the glnB upstream region.

Carbohydr Res, 1990 Sep 5, 204, 103 - 7
Structure of the extracellular polysaccharide secreted by Rhizobium leguminosarum var . phaseoli CIAT 899; Gil-Serrano A et al.; The structure of the extracellular polysaccharide secreted by Rhizobium leguminosarum var . phaseoli CIAT 899 has been studied by methylation analysis . 1H-n.m.r . spectroscopy, and partial acid hydrolysis . The repeating unit is an octasaccharide made up of D-glucose, D-galactose, pyruvic acid, and acetic acid in the molar ratios 6:2:1.5:1.5 . Half of the terminal Gal groups are 4,6-substituted by pyruvic acid acetal groups and the other half by O-acetyl groups at position 3 . Also, one of the 3-linked glucosyl residues carries a pyruvic acid 4,6-acetal group and one of the 4-linked glucosyl residues is acetylated at position 6.

J Bacteriol, 1990 Sep, 172(9), 5394 - 401
A biovar-specific signal of Rhizobium leguminosarum bv . viciae induces increased nodulation gene-inducing activity in root exudate of Vicia sativa subsp . nigra; van Brussel AA et al.; Flavonoids in root exudate of leguminous plants activate the transcription of Rhizobium genes involved in the formation of root nodules (nod genes) . We report that inoculation with the homologous symbiont R . leguminosarum bv . viciae results in an increased nod gene-inducing activity (Ini) in root exudate of V . sativa subsp . nigra, whereas inoculation with heterologous Rhizobium strains results in exudates with nod gene-inducing activity comparable to that of uninfected plants . Ini can be demonstrated by using either of the isogenic indicator strains containing an inducible nod promoter fused to the Escherichia coli lacZ reporter gene and the regulatory nodD gene of R . leguminosarum bv . viciae, R . leguminosarum bv . trifolii, or R . meliloti . The presence of genes nodDABCEL of R . leguminosarum bv . viciae appeared to be essential for induction of Ini . Mutation of the genes nodI and nodJ causes a delay of Ini, whereas gene nodF appears to be required for both the timely appearance and the maximum level of Ini activity . The nodE gene is responsible for the biovar specificity of induction of Ini by Rhizobium spp . Ini is caused by a soluble heat-stable factor of rhizobial origin . This Rhizobium-produced Ini factor has an apparent molecular weight between 1,000 and 10,000 and does not originate from flavonoid precursors.

Mol Microbiol, 1990 Sep, 4(9), 1425 - 31
Exopolysaccharide production in Rhizobium and its role in invasion; Gray JX et al.; A complex interaction between rhizobia and specific legume plants results in the formation of nitrogen-fixing root nodules . The necessity for signal exchange and a chemically based recognition system between the symbiotic partners has been appreciated for some time, but the details are only gradually being elucidated . The two basic nodule ontogenies exhibit different requirements for Rhizobium exopolysaccharides . These surface exopolysaccharide molecules of Rhizobium are synthesized at a membrane complex, which is regulated by both transcriptional and post-translational controls . The acidic exopolysaccharide probably plays both a passive and an active role during the invasion process.

J Bacteriol, 1990 Sep, 172(9), 5486 - 9
Subcellular localization of the Rhizobium leguminosarum nodI gene product; Schlaman HR et al.; By the use of antibodies raised against a fusion protein of lacZ'-nodI (produced in Escherichia coli) which specifically react with NodI protein, it was shown that in wild-type Rhizobium leguminosarum biovar viciae NodI protein (i) is recovered with the cytoplasmic membrane fraction and (ii) is translated as part of the nodABCIJ operon . In addition, it was found that the bacterial chromosomal background strongly influences the expression of several nod genes.

J Bacteriol, 1990 Sep, 172(9), 5245 - 53
Functional and evolutionary relatedness of genes for exopolysaccharide synthesis in Rhizobium meliloti and Rhizobium sp . strain NGR234; Zhan HJ et al.; Rhizobium meliloti SU47 and Rhizobium sp . strain NGR234 produce distinct exopolysaccharides that have some similarities in structure . R . meliloti has a narrow host range, whereas Rhizobium strain NGR234 has a very broad host range . In cross-species complementation and hybridization experiments, we found that several of the genes required for the production of the two polysaccharides were functionally interchangeable and similar in evolutionary origin . NGR234 exoC and exoY corresponded to R . meliloti exoB and exoF, respectively . NGR234 exoD was found to be an operon that included genes equivalent to exoM, exoA, and exoL in R . meliloti . Complementation of R . meliloti exoP, -N, and -G by NGR234 R'3222 indicated that additional equivalent genes remain to be found on the R-prime . We were not able to complement NGR234 exoB with R . meliloti DNA . In addition to functional and evolutionary equivalence of individual genes, the general organization of the exo regions was similar between the two species . It is likely that the same ancestral genes were used in the evolution of both exopolysaccharide biosynthetic pathways and probably of pathways in other species as well.

J Bacteriol, 1990 Sep, 172(9), 5450 - 8
Rhizobium meliloti lipopolysaccharide and exopolysaccharide can have the same function in the plant-bacterium interaction; Putnoky P et al.; A fix region of Rhizobium meliloti 41 involved both in symbiotic nodule development and in the adsorption of bacteriophage 16-3 was delimited by directed Tn5 mutagenesis . Mutations in this DNA region were assigned to four complementation units and were mapped close to the pyr-2 and pyr-29 chromosomal markers . Phage inactivation studies with bacterial cell envelope preparations and crude lipopolysaccharides (LPS) as well as preliminary characterization of LPS in the mutants indicated that these genes are involved in the synthesis of a strain-specific LPS . Mutations in this DNA region resulted in a Fix- phenotype in AK631, an exopolysaccharide (EPS)-deficient derivative of R . meliloti 41; however, they did not influence the symbiotic efficiency of the parent strain . An exo region able to restore the EPS production of AK631 was isolated and shown to be homologous to the exoB region of R . meliloti SU47 . By generating double mutants, we demonstrated that exo and lps genes determine similar functions in the course of nodule development, suggesting that EPS and LPS may provide equivalent information for the host plant.

J Bacteriol, 1990 Sep, 172(9), 5173 - 9
Cloning, characterization, and complementation of lesions causing acid sensitivity in Tn5-induced mutants of Rhizobium meliloti WSM419; Goss TJ et al.; Four Tn5-induced mutants of Rhizobium meliloti WSM419 were unable to grow or maintain intracellular pH at an external pH of 5.6 . Restriction analysis of DNA fragments carrying Tn5 and flanking sequences cloned from these mutants indicated that all four cloned mutations are unique and that the two strains (TG1-6 and TG1-11) carry Tn5 insertions which are within 4.4 kilobases of each other on a single EcoRI fragment . Southern analysis of total mutant DNA indicated a single copy of Tn5 in each mutant . A limited cosmid gene bank of wild-type WSM419 DNA was probed for homology to mutant DNA cloned from the acid-sensitive mutants . Dot hybridization experiments identified one cosmid element within this bank carrying wild-type DNA sequences corresponding to DNA implicated in acid tolerance . This cosmid was able to complement defects in growth and intracellular pH maintenance in TG1-11 but not TG1-6.

J Bacteriol, 1990 Sep, 172(9), 5440 - 4
Isolation and characterization of the constitutive acyl carrier protein from Rhizobium meliloti; Platt MW et al.; Rhizobium species produce an inducible acyl carrier protein (ACP), encoded by the nodF gene, that somehow functions in an exchange of cell signals between bacteria and specific plant hosts, leading to nodulation of plant roots and symbiotic nitrogen fixation, as well as a constitutive ACP needed for the synthesis of essential cell lipids . The periplasmic cyclic glucans of Rhizobium spp . are also involved in specific rhizobium-plant interaction . These glucans are strongly similar to the periplasmic membrane-derived oligosaccharides (MDO) of Escherichia coli . E . coli ACP is an essential component of a membrane-bound transglucosylase needed for the biosynthesis of MDO, raising the possibility that either or both of the rhizobial ACPs might have a similar function . We have now isolated the constitutive ACP of R . meliloti and determined its primary structure . We have also examined its function, together with those of ACPs from E . coli, Rhodobacter sphaeroides, and spinach, in the MDO transglucosylase system and as substrate for the E . coli ACP acylase enzyme . All four ACPs act as acceptors of acyl residues, but only the E . coli ACP functions in the transglucosylase system.

Mol Plant Microbe Interact, 1990 Sep-Oct, 3(5), 317 - 26
nodSU, two new nod genes of the broad host range Rhizobium strain NGR234 encode host-specific nodulation of the tropical tree Leucaena leucocephala; Lewin A et al.; Rhizobium species strain NGR234 nodulates at least 35 diverse genera of legumes as well as the nonlegume Parasponia andersonii . Most nodulation genes are located on the 500-kilobase pair symbiotic plasmid, pNGR234a . Previously, three plasmid-borne host range determinants (HsnI, HsnII, and HsnIII) were identified by their ability to extend the nodulation capacity of heterologous rhizobia to include Vigna unguiculata . In this study, we show that HsnII contains two new nod-box linked hsn genes, nodS and nodU.nodS controls nodulation of the tropical tree Leucaena leucocephala, while the nodSU genes regulate nodulation of the pasture legume Desmodium intortum and the grain legume V . unguiculata . Regulation of the nod-box upstream of nodSU by the flavonoid naringenin was shown using a fusion with a promoterless lacZ gene . Determination of the nucleotide sequence of the nodS gene did not reveal homology with any gene in the EMBL library, although Bradyrhizobium japonicum USDA110 contains both nodS and nodU (M . Gottfert, S . Hitz, and H . Hennecke, Molecular Plant-Microbe Interactions 3:308-316, 1990) . We suggest that broad host range in NGR234 is controlled in part by a nodD gene which interacts with a wide range of flavonoids, and in part by host-specific nod genes such as nodS.

Mol Plant Microbe Interact, 1990 Sep-Oct, 3(5), 308 - 16
Identification of nodS and nodU, two inducible genes inserted between the Bradyrhizobium japonicum nodYABC and nodIJ genes; Gottfert M et al.; The so-called common nodulation (nod) gene cluster of Bradyrhizobium japonicum is characterized by a unique composition of genes that are arranged in the following order: nodY, nodA, nodB, nodC, nodS, nodU, nodI, nodJ . As reported here, the identification of the two new genes nodS and nodU resulted from the DNA sequencing of a 4.5-kilobase nodC-downstream region covering nodS, nodU, nodI, and nodJ . The predicted NodS, NodU, NodI, and NodJ proteins had the following respective amino acid (aa) lengths and molecular weights (Mr): 209 aa, Mr 23,405; 569 aa, Mr 62,068; 306 aa, Mr 34,127; and 262 aa, Mr 28,194 . The 3' end of nodC overlapped the 5' end of nodS by 71 nucleotides . Using translational fusions of lacZ to nodC, nodS, and nodU, the expression of these genes was shown to be inducible by the isoflavone daidzein and depended on transcription from a DNA region farther upstream . These data and the adjacent location of all genes suggested the existence of a nodYABCSUIJ operon . The nodI and nodJ gene products shared about 70% sequence similarity with the corresponding Rhizobium leguminosarum bv . viciae proteins; NodI belongs to the family of ATP-binding proteins that are constituents of bacterial binding protein-dependent transport systems . By interspecies hybridization, DNA regions homologous to nodSU were detected in other strains of Bradyrhizobium . Likewise, nodS- and nodU-like genes were identified in Rhizobium sp . strain NGR234 (A . Lewin, E . Cervantes, W . Chee-Hoong, and W . J . Broughton, Molecular Plant-Microbe Interactions 3:317-326, 1990) in which nodS confers host specificity for Leucaena leucocephala.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1990 Sep, 172(9), 5254 - 9
Two genes that regulate exopolysaccharide production in Rhizobium meliloti; Zhan HJ et al.; We describe a new Rhizobium meliloti gene, exoX, that regulates the synthesis of the exopolysaccharide, succinoglycan, exoX resembled the psi gene of R . leguminosarum bv . phaseoli and the exoX gene of Rhizobium sp . strain NGR234 in its ability to inhibit exopolysaccharide synthesis when present in multiple copies, exoX did not appear to regulate the expression of exoP . The effect of exoX was counterbalanced by another R . meliloti gene, exoF . exoF is equivalent to Rhizobium sp . strain NGR234 exoY and resembles R . leguminosarum bv . phaseoli pss2 in its mutant phenotype and in portions of its deduced amino acid sequence . The effect of exoF on the succinoglycan-inhibiting activity of exoX depended on the relative copy numbers of the two genes . exoX-lacZ fusions manifested threefold-higher beta-galactosidase activities in exoF backgrounds than in the wild-type background . exoX mutants produced increased levels of succinoglycan . However, the exoF gene was required for succinoglycan synthesis even in an exoX mutant background . exoF did not affect the expression of exoP . Strains containing multicopy exoX formed non-nitrogen-fixing nodules on alfalfa that resembled nodules formed by exo mutants defective in succinoglycan synthesis . exoX mutants formed nitrogen-fixing nodules, indicating that, if the inhibition of succinoglycan synthesis within the nodule is necessary for nitrogen fixation, then exoX is not required for this inhibition . We present indirect evidence that succinoglycan synthesis within the nodule is not necessary for bacteroid function.

J Bacteriol, 1990 Sep, 172(9), 5326 - 34
Overexpression of a Streptomyces viridochromogenes gene (glnII) encoding a glutamine synthetase similar to those of eucaryotes confers resistance against the antibiotic phosphinothricyl-alanyl-alanine; Behrmann I et al.; Phosphinothricyl-alanyl-alanine (PTT), also known as bialaphos, contains phosphinothricin, a potent inhibitor of glutamine synthetase (GS) . A 2.75-kilobase NcoI fragment of the Streptomyces viridochromogenes PTT-resistant mutant ES2 cloned on a multicopy vector mediated PTT resistance to S . lividans and to S . viridochromogenes . Nucleotide sequence analysis of the 2.75-kb NcoI fragment revealed the presence of three open reading frames . Open reading frame 3 was termed glnII since significant similarity was found between its deduced amino acid sequence and those from GS of eucaryotes and GSII of members of the family Rhizobiaceae . Subcloning experiments showed that PTT resistance is mediated by overexpression of glnII encoding a 37.3-kilodalton protein of 343 amino acids . A three- to fourfold increase in gamma-glutamyltransferase activity could be observed in S . lividans transformants carrying the glnII gene on a multicopy plasmid . For S . viridochromogenes it was shown that PTT resistance conferred by the 2.75-kb NcoI fragment was dependent on its multicopy state . GS activity encoded by glnII was found to be heat labile . Southern hybridization with seven different Streptomyces strains suggested that they all carry two types of GS genes, glnA and glnII.

J Bacteriol, 1990 Aug, 172(8), 4295 - 306
Correlation between ultrastructural differentiation of bacteroids and nitrogen fixation in alfalfa nodules; Vasse J et al.; Bacteroid differentiation was examined in developing and mature alfalfa nodules elicited by wild-type or Fix- mutant strains of Rhizobium meliloti . Ultrastructural studies of wild-type nodules distinguished five steps in bacteroid differentiation (types 1 to 5), each being restricted to a well-defined histological region of the nodule . Correlative studies between nodule development, bacteroid differentiation, and acetylene reduction showed that nitrogenase activity was always associated with the differentiation of the distal zone III of the nodule . In this region, the invaded cells were filled with heterogeneous type 4 bacteroids, the cytoplasm of which displayed an alternation of areas enriched with ribosomes or with DNA fibrils . Cytological studies of complementary halves of transversally sectioned mature nodules confirmed that type 4 bacteroids were always observed in the half of the nodule expressing nitrogenase activity, while the presence of type 5 bacteroids could never be correlated with acetylene reduction . Bacteria with a transposon Tn5 insertion in pSym fix genes elicited the development of Fix- nodules in which bacteroids could not develop into the last two ultrastructural types . The use of mutant strains deleted of DNA fragments bearing functional reiterated pSym fix genes and complemented with recombinant plasmids, each carrying one of these fragments, strengthened the correlation between the occurrence of type 4 bacteroids and acetylene reduction . A new nomenclature is proposed to distinguish the histological areas in alfalfa nodules which account for and are correlated with the multiple stages of bacteroid development.

FEMS Microbiol Rev, 1990 Aug, 6(4), 399 - 428
FNR and its role in oxygen-regulated gene expression in Escherichia coli; Spiro S et al.; Bacteria which can grow in different environments have developed regulatory systems which allow them to exploit specific habitats to their best advantage . In the facultative anaerobe Escherichia coli two transcriptional regulators controlling independent networks of oxygen-regulated gene expression have been identified . One is a two-component sensor-regulator system (ArcB-A), which represses a wide variety of aerobic enzymes under anaerobic conditions . The other is FNR, the transcriptional regulator which is essential for expressing anaerobic respiratory processes . The purpose of this review is to summarize what is known about FNR . The fnr gene was initially defined by the isolation of some pleiotropic mutants which characteristically lacked the ability to use fumarate and nitrate as reducible substrates for supporting anaerobic growth and several other anaerobic respiratory functions . Its role as a transcriptional regulator emerged from genetic and molecular studies in which its homology with CRP (the cyclic AMP receptor protein which mediates catabolite repression) was established and has since been particularly important in identifying the structural basis of its regulatory specificities . FNR is a member of a growing family of CRP-related regulatory proteins which have a DNA-binding domain based on the helix-turn-helix structural motif, and a characteristic beta-roll that is involved in nucleotide-binding in CRP . The FNR protein has been isolated in a monomeric form (Mr 30,000) which exhibits a high but as yet non-specific affinity for DNA . Nevertheless, the DNA-recognition site and important residues conferring the functional specificity of FNR have been defined by site-directed mutagenesis . A consensus for the sequences that are recognized by FNR in the promoter regions of FNR-regulated genes, has likewise been identified . The basic features of the genes and operons regulated by FNR are reviewed, and examples in which FNR functions negatively as an anaerobic repressor as well as positively as an anaerobic activator, are included . Less is known about the way in which FNR senses anoxia and is thereby transformed into its 'active' form, but it seems likely that cysteine residues and possibly a metal ion are involved . Four of the five cysteine residues of FNR are clustered in an essential N-terminal 'domain' which is conserved in FNR and the HlyX protein of Actinobacillus pleuropneumoniae, but not in CRP or the FixK protein of Rhizobium meliloti . The relationships between FNR and other oxygen-related systems in E . coli are discussed, as well as parallel systems in other organisms.(ABSTRACT TRUNCATED AT 400 WORDS)

J Bacteriol, 1990 Aug, 172(8), 4505 - 9
Rhizobitoxine inhibition of hydrogenase synthesis in free-living Bradyrhizobium japonicum; Minamisawa K et al.; Rhizobitoxine produced by Bradyrhizobium species strongly prevented derepression of hydrogenase expression in free-living Bradyrhizobium japonicum, although the toxin had no effect on the activity of cells which had already synthesized hydrogenase protein . Dihydrorhizobitoxine, a structural analog of rhizobitoxine, proved to be a less potent inhibitor of hydrogenase derepression . Rhizobitoxine did not cause cell death at a concentration sufficient to eliminate hydrogenase expression . The large subunit of hydrogenase was not detectable with antibody after derepression in the presence of rhizobitoxine . The general pattern of proteins synthesized from 14C-labeled amino acids during derepression was not significantly different in the presence or absence of rhizobitoxine . These results indicated that rhizobitoxine inhibited hydrogenase synthesis in free-living B . japonicum . Cystathionine and methionine strongly prevented the inhibition of hydrogenase derepression by rhizobitoxine, suggesting that the inhibition involves the level of sulfur-containing amino acids in the cell.

J Bacteriol, 1990 Aug, 172(8), 4399 - 406
Complementation of Escherichia coli sigma 54 (NtrA)-dependent formate hydrogenlyase activity by a cloned Thiobacillus ferrooxidans ntrA gene; Berger DK et al.; The ntrA gene of Thiobacillus ferrooxidans was cloned by complementation of an Escherichia coli ntrA mutant that was unable to produce gas via the sigma 54 (NtrA)-dependent formate hydrogenlyase pathway . Analysis of the DNA sequence showed that the T . ferrooxidans ntrA gene coded for a protein of 475 amino acids (calculated Mr, 52,972) . The T . ferrooxidans NtrA protein had 49, 44, 33, and 18% amino acid similarity with the NtrA proteins of Klebsiella pneumoniae, Azotobacter vinelandii, Rhizobium meliloti, and Rhodobacter capsulatus, respectively . The ability of the T . ferrooxidans NtrA protein to direct transcription from sigma 54-dependent promoters was demonstrated in E . coli by using fdhF-lacZ and nifH-lacZ fusions . An open reading frame coding for a protein of 241 amino acids (calculated Mr, 27,023) was situated 12 base pairs upstream of the T . ferrooxidans ntrA gene . Comparison of this protein with the product of the open reading frame ORF1, located upstream of the R . meliloti ntrA gene, showed that the two proteins had 55% amino acid similarity . The cloned T . ferrooxidans ntrA gene was expressed in E . coli from a promoter located within the ORF1 coding region.

Mol Gen Genet, 1990 Aug, 223(1), 138 - 47
An Fnr-like protein encoded in Rhizobium leguminosarum biovar viciae shows structural and functional homology to Rhizobium meliloti FixK; Colonna-Romano S et al.; A 1.9 kb DNA region of Rhizobium leguminosarum biovar viciae strain VF39 capable of promoting microaerobic and symbiotic induction of the Rhizobium meliloti fixN gene was identified by heterologous complementation . Sequence analysis of this DNA region revealed the presence of two complete open reading frames, orf240 and orf114 . The deduced amino acid sequence of orf240 showed significant homology to Escherichia coli Fnr and R . meliloti FixK . The major difference between ORF240 and FixK is the presence of 21 N-terminal amino acids in ORF240 that have no counterpart in FixK . A similar protein domain is also present in E . coli Fnr and is essential for the oxygen-regulated activity of this protein . Analysis of the nucleotide sequence upstream of orf240 revealed a motif similar to the NtrA-dependent promoter consensus sequence, as well as two DNA regions resembling the Fnr consensus binding sequence . A Tn5-generated mutant in orf240 lost the ability to induce the R . meliloti fixN-lacZ fusion . Interestingly, this mutant was still capable of nitrogen fixation but showed reduced nitrogenase activity.

Plant Cell, 1990 Aug, 2(8), 687 - 700
Sequential induction of nodulin gene expression in the developing pea nodule; Scheres B et al.; A set of cDNA clones have been characterized that represent early nodulin mRNAs from pea root nodules . By RNA transfer blot analyses, the different early nodulin mRNAs were found to vary in time course of appearance during the development of the indeterminate pea root nodule . In situ hybridization studies demonstrated that the transcripts were located in different zones, representing subsequent steps in development of the central tissue of the root nodule . ENOD12 transcripts were present in every cell of the invasion zone, whereas ENOD5, ENOD3, and ENOD14 transcripts were restricted to the infected cells in successive but partially overlapping zones of the central tissue . We conclude that the corresponding nodulin genes are expressed at subsequent developmental stages . The amino acid sequence derived from the nucleotide sequences of the cDNAs, in combination with the localization data, showed that ENOD5 is an arabinogalactan-like protein involved in the infection process, whereas ENOD3 and ENOD14 have a cysteine cluster suggesting that these are metal-binding proteins . Furthermore, we showed that there is a clear difference in the way Rhizobium induced the infection-related early nodulin genes ENOD5 and ENOD12 . A factor acting over a long distance induced the ENOD12 gene, whereas a factor acting over a short distance activated the ENOD5 gene.

J Bacteriol, 1990 Aug, 172(8), 4255 - 62
Rhizobium meliloti Fix L is an oxygen sensor and regulates R . meliloti nifA and fixK genes differently in Escherichia coli; de Philip P et al.; In Rhizobium meliloti, nif and fix genes, involved in nitrogen fixation during symbiosis with alfalfa, are under the control of two transcriptional regulators encoded by nifA and fixK . Expression of nifA and fixK is under the control of FixL/J, a two-component regulatory system . We showed, using Escherichia coli as a heterologous host, that FixL/J controls nifA and fixK expression in response to microaerobiosis . Furthermore, expression of the sensor gene fixL and of the activator gene fixJ under the control of two different promoters allowed us to show that FixL mediates microaerobic induction of nifA when the level of FixJ is low and aerobic repression of nifA when the level of FixJ is high . Similarly, activation of fixK occurred in microaerobiosis when the FixJ level was low in the presence of FixL . In contrast to nifA, fixK expression was not affected by FixL in aerated cultures when the level of FixJ was high . We conclude that R . meliloti FixL senses oxygen in the heterologous host E . coli consistent with the microaerobic induction of nifA and fixK in R . meliloti and that nifA and fixK promoters are differentially activated by FixJ in response to the oxygen signal.

Nucleic Acids Res, 1990 Jul 25, 18(14), 4149 - 55
Study of Vitreoscilla globin (vgb) gene expression and promoter activity in E . coli through transcriptional fusion; Dikshit KL et al.; Bacterial hemoglobin (VtHb) is produced by the gram-negative bacterium, Vitreoscilla, in large quantity in response to hypoxic environmental conditions . The vgb gene coding for VtHb has been cloned in E . coli where it is expressed strongly by its natural promoter . The expression of the vgb gene in Vitreoscilla is transcriptionally regulated by oxygen . When E . coli cells were shifted from 20% to 5% oxygen, vgb specific transcript increased . In E . coli cells with plasmids carrying transcriptional fusions of the vgb gene promoter to either CAT (chloramphenicol acetyl transferase) or xylE (catechol-2,3-dioxygenase) genes, the promoter activity depended on the oxygen level . The concentration of CAT and xylE gene products in cells grown under 5% oxygen was 5-7 times that of aerobically (20% oxygen) grown cells . When the vgb gene promoter was deleted, VtHb was not produced under any conditions . When the promoter was replaced by the E . coli tac promoter, hypoxic oxygen did not affect the level of expression of vgb, but adding IPTG did increase the expression of this gene . These results indicate that the vgb gene promoter is transcriptionally regulated by oxygen even in E . coli, and that microaerobiosis is sufficient to induce vgb expression . The size of S1 nuclease-resistant hybrids, prepared using RNA transcripts protected with restriction enzyme fragments containing the promoter proximal region of vgb, was the same for both Vitreoscilla and E . coli, further evidence that the same promoter is used in both organisms . Transcriptional fusion of the vgb gene promoter to the xylE reporter gene on the broad host range plasmid, pKD-49, was used to demonstrate that the vgb promoter can be expressed in other gram-negative organisms, including Pseudomonas, Azotobacter, and Rhizobium.

J Bacteriol, 1990 Jul, 172(7), 3695 - 700
DNA sequence and translational product of a new nodulation-regulatory locus: syrM has sequence similarity to NodD proteins; Barnett MJ et al.; Rhizobium meliloti nodulation (nod) genes are expressed when activated by trans-acting proteins in the NodD family . The nodD1 and nodD2 gene products activate nod promoters when cells are exposed to plant-synthesized signal molecules . Alternatively, the same nod promoters are activated by the nodD3 gene when nodD3 is carried in trans along with a closely linked global regulatory locus, syrM (symbiotic regulator) (J . T . Mulligan and S . R . Long, Genetics 122:7-18, 1989) . In this article we report the nucleotide sequence of a 2.6-kilobase SphI fragment from R . meliloti SU47 containing syrM . Expression from this locus was confirmed by using in vitro transcription-translation assays . The open reading frame encoded a protein of either 33 or 36 kilodaltons whose sequence shows similarity to NodD regulatory proteins.

J Bacteriol, 1990 Jul, 172(7), 3888 - 97
Rhizobium meliloti and Rhizobium leguminosarum dctD gene products bind to tandem sites in an activation sequence located upstream of sigma 54-dependent dctA promoters; Ledebur H et al.; Free-living rhizobia transport external C4-dicarboxylates to use as sole carbon sources, and uptake of these compounds is essential for nitrogen fixation by rhizobial bacteroids . In both Rhizobium leguminosarum and Rhizobium meliloti, the genes dctB and dctD are believed to form an ntrB/ntrC-like two-component system which regulates the synthesis of a C4-dicarboxylate transport protein encoded by dctA . Here we confirm the identity of sigma 54-dependent promoters previously hypothesized for the R . leguminosarum and R . meliloti dctA genes and demonstrate that repeated, partial dyad symmetry elements located about 75 base pairs upstream of each promoter are essential for fully regulated transcription . Furthermore, we show that both repeats bound dctD protein and that together they resulted in succinate-sensitive transcription when placed upstream of another sigma 54 consensus promoter, that of R . meliloti nifH.

Plant Cell, 1990 Jul, 2(7), 633 - 41
Nonlegume hemoglobin genes retain organ-specific expression in heterologous transgenic plants; Bogusz D et al.; Hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa have been isolated {Landsmann et al . (1986) . Nature 324, 166-168; Bogusz et al . (1988) . Nature 331, 178-180} . The promoters of these genes have been linked to a beta-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus . Both promoters directed root-specific expression in transgenic tobacco . When transgenic Lotus plants were nodulated by Rhizobium loti, both promoter constructs showed a high level of nodule-specific expression confined to the central bacteroid-containing portion of the nodule corresponding to the expression seen for the endogenous Lotus leghemoglobin gene . The T . tomentosa promoter was also expressed at a low level in the vascular tissue of the Lotus roots . The hemoglobin promoters from both nonlegumes, including the non-nodulating species, must contain conserved cis-acting DNA signals that are responsible for nodule-specific expression in legumes . We have identified sequence motifs postulated previously as the nodule-specific regulatory elements of the soybean leghemoglobin genes {Stougaard et al . (1987) . EMBO J . 6, 3565-3569}.

Appl Environ Microbiol, 1990 Jul, 56(7), 2080 - 6
Excessive excretion of cyclic beta-(1,2)-glucan by Rhizobium trifolii TA-1; Breedveld MW et al.; At 25 degrees C, the optimal temperature for growth of Rhizobium trifolii TA-1, extracellular and capsular polysaccharide (EPS and CPS) were the main carbohydrate products synthesized in mannitol-rich medium (10 g of mannitol and 1 g of glutamic acid per liter) . In the same medium at 33 degrees C, EPS and CPS production was inhibited, and up to 3.9 g of cyclic beta-(1,2)-glucan was produced during an incubation period of 20 days with a total biomass of 0.55 g of protein . In a medium containing 50 g of mannitol and 10 g of glutamic acid per liter, high cell densities (3.95 g of protein) were obtained at 25 degrees C . This biomass excreted 10.9 g of cyclic beta-(1,2)-glucan within 10 days . Concomitantly, 4.8 g of EPS were synthesized, while CPS production was strongly suppressed . The excreted cyclic beta-(1,2)-glucans were neutral and had degrees of polymerization ranging from 17 to 25, with a degree of polymerization of 19 as the major glucan cycle.

J Bacteriol, 1990 Jul, 172(7), 3559 - 68
Rhizobium meliloti suhR suppresses the phenotype of an Escherichia coli RNA polymerase sigma 32 mutant; Bent AF et al.; sigma 32, the product of the Escherichia coli rpoH locus, is an alternative RNA polymerase sigma factor utilized to express heat shock genes upon a sudden rise in temperature . E . coli K165 {rpoH165(Am) supC(Ts)} is temperature sensitive for growth and does not induce heat shock protein synthesis . We have isolated a locus from Rhizobium meliloti called suhR that allows E . coli K165 to grow at high temperature and induce heat shock protein synthesis . R . meliloti suhR mutants were viable and symbiotically effective . suhR was found to have no DNA or derived amino acid sequence similarity to the genes of previously sequenced sigma factors or other data base entries, although a helix-turn-helix DNA-binding protein motif is present . suhR did not restore the phenotypic defects of delta rpoH E . coli; suppression of the E . coli K165 phenotype is thus likely to involve E . coli sigma 32 . Western immunoblots showed that suhR caused an approximately twofold elevation of sigma 32 levels in K165; RNA blots indicated that rpoH mRNA level and stability were not altered . Stabilization of sigma 32 protein and increased rpoH mRNA translation are thus the most probable mechanisms of suppression.

Appl Environ Microbiol, 1990 Jul, 56(7), 2262 - 4
Production and epitope analysis of monoclonal antibodies against a Rhizobium leguminosarum biovar trifolii strain; Wright SF; Heat-treated cells of Rhizobium leguminosarum biovar trifolii strain 162X95 were used to produce monoclonal antibodies (MAbs) . The fusion produced three cross-reactive MAbs and eight MAbs specific for the immunizing strain and a group of five other R . trifolii strains from the same geographic region where 162X95 was isolated (California) . Seven MAbs were analyzed by competitive enzyme-linked immunosorbent assay to determine the number of different epitopes detectable on strain 162X95 . The results indicated that six MAbs reacted with the same or overlapping epitopes, and the seventh MAb gave inconclusive results.

Biochem Biophys Res Commun, 1990 Jun 15, 169(2), 692 - 9
A model of nitrogen flow by malonamate in Rhizobium japonicum-soybean symbiosis; Kim YS et al.; Two types of novel malonamidases were found in soybean nodules . One (E1) catalyzes the formation of malonamate from malonate and its hydrolysis to ammonia, whereas the other (E2) acts mainly on the hydrolysis of malonamate . E1 and E2 were found in bacteroids, but only E2 was found in the plant cytosol of the nodule . The substrate requirements of E1 and E2 were highly specific for malonate and malonamate, respectively . From these and other results reported previously, we propose that malonamate plays an important role as a nitrogen carrier in the Rhizobium legume symbiosis.

Mol Gen Genet, 1990 Jun, 222(1), 81 - 6
Either of two nod gene loci can complement the nodulation defect of a nod deletion mutant of Rhizobium leguminosarum bv viciae; Downie JA et al.; A deletion mutant of Rhizobium leguminosarum biovar viciae lacking the host-specific nodulation (nod) gene region (nodFEL nodMNT and nodO) but retaining the other nod genes (nodD nodABCIJ) was unable to nodulate peas or Vicia hirsuta, although it did induce root hair deformation . The mutant appeared to be blocked in its ability to induce infection threads and could be rescued for nodulation of V . hirsuta in mixed inoculation experiments with an exopolysaccharide deficient mutant (which is also Nod-) . The nodulation deficiency of the deletion mutant strain could be partially restored by plasmids carrying the nodFE, nodFEL or nodFELMNT genes but not by nodLMN . Surprisingly, the mutant strain could also be complemented with a plasmid that did not carry any of the nodFELMNT genes but which did carry the nodO gene on a 30 kb cloned region of DNA . Using appropriate mutations it was established that nodO is essential for nodulation in the absence of nodFE . Thus, either of two independent nod gene regions can complement the deletion mutant for nodulation of V . hirsuta . Similar observations were made for pea nodulation except that nodL was required in addition to nodO for nodulation in the absence of the nodFE genes . These observations show that nodulation can occur via either of two pathways encoded by non-homologous genes.

Mol Microbiol, 1990 Jun, 4(6), 921 - 32
Analysis of three nodD genes in Rhizobium leguminosarum biovar phaseoli; nodD1 is preceded by noIE, a gene whose product is secreted from the cytoplasm; Davis EO et al.; In a strain of Rhizobium leguminosarum biovar phaseoli, three copies of the regulatory nodulation gene nodD were identified on the Sym plasmid and sequenced . Two were closely linked to each other and the third was near, but not adjacent, to the nodABC genes . Each of these nodD genes could correct the Nod- defect of a nodD mutant strain of R . leguminosarum biovar viciae on peas . A truncated form of nodD2 could also correct this mutant, indicating that the C-terminus of NodD2 is not needed for inducing activity . Upstream of nodD1 and in the same operon is a newly described gene, noIE, whose product appears to be exported into the periplasm . Close to nodD2 is another gene, noIP, with no known counterpart in other rhizobia . Both noIP and noIE-nodD1 are preceded by 'nod-box' sequences and, in the former case, there appear to be two tandemly repeated nod-box sequences . Mutations in each of the nodD genes and in the noIE and noIP genes did not abolish nodulation or nitrogen fixation on beans.

Mol Microbiol, 1990 Jun, 4(6), 933 - 41
Regulatory functions of the three nodD genes of Rhizobium leguminosarum biovar phaseoli; Davis EO et al.; The three nodD genes of a strain of Rhizobium leguminosarum biovar phaseoli were cloned to study their effects on transcription of themselves and of the nodC genes of biovars phaseoli and viciae . Efficient transcription of nodD1 required nodD1 and was enhanced by exposure of the cells to bean exudate consistent with the presence of a nod-box preceding the noIE-nodD1 operon . Transcription of nodD2 and nodD3 was constitutive . nodC of R . leguminosarum biovar phaseoli was activated by each of the nodD genes of that biovar in the absence of inducers but expression was enhanced in cells grown with bean exudate or the flavonoids genistein or naringenin . A mutant of nodD2, lacking 60 bp at its 3' end, activated nodC in the presence of inducer, but was defective in regulating certain of the nodD genes . The nodC gene of R . leguminosarum biovar viciae responded differently to the various nodD genes of R . leguminosarum biovar phaseoli than did the nodC of the latter biovar.

J Bacteriol, 1990 Jun, 172(6), 3318 - 27
The Rhizobium meliloti trpE(G) gene is regulated by attenuation, and its product, anthranilate synthase, is regulated by feedback inhibition; Bae YM et al.; In Rhizobium meliloti, the genes involved in biosynthesis of the amino acid tryptophan are found at three separate chromosomal locations . Of the three gene clusters, trpE(G), trpDC, and trpFBA, only the trpE(G) gene is regulated by the end product of the pathway, tryptophan . We found that trpE(G) mRNA contains a leader transcript that terminates at a stem-loop structure in a putative transcription attenuator . The level of this leader transcript was constant regardless of the amount of tryptophan in the growth medium . However, the level of full-length trpE(G) mRNA decreased as the amount of tryptophan increased . The beta-galactosidase activity of an R . meliloti strain carrying a trpL'-'lacZ fusion remained constant at different tryptophan concentrations, but the beta-galactosidase activity of the same strain carrying a trpE(G)'-'lacZ fusion decreased as the tryptophan concentration increased . These data indicate that transcription of the R . meliloti trpE(G) gene is regulated only by attenuation . We also found that the product of the trpE(G) gene, anthranilate synthase, is feedback inhibited by tryptophan.

Carbohydr Res, 1990 May 1, 198(2), 305 - 12
Structural studies of a novel exopolysaccharide produced by a mutant of Rhizobium meliloti strain Rm1021; Her GR et al.; The structure of a novel expolysaccharide obtained from a mutant of Rhizobium meliloti strain Rm1021 was elucidated by a combination of enzymic, chemical, and spectroscopic methods . The polysaccharide is composed of a disaccharide repeating-unit, beta-D-Glcp-(1----3)-alpha-D-Galp-(1----3), having a 6-O-acetyl group attached to most D-glucose residues and a 4,6-O-(1-carboxyethylidene) group attached to every D-galactose residue.

J Bacteriol, 1990 May, 172(5), 2769 - 73
Effects of alfalfa nod gene-inducing flavonoids on nodABC transcription in Rhizobium meliloti strains containing different nodD genes; Hartwig UA et al.; Transcription of the nodulation genes nodABC in Rhizobium meliloti requires a plant flavonoid signal and nodD, a family of bacterial regulatory genes (nodD1, nodD2, and nodD3) . Results from this study show that all previously identified nod gene inducers released by alfalfa seeds and roots induced nodABC-lacZ transcription in R . meliloti containing extra copies of nodD1, but only 4,4'-dihydroxy-2'-methoxychalcone gave high levels of induction with extra copies of nodD2 . While mixtures of the methoxychalcone and luteolin showed a positive synergism with extra NodD1 protein, they apparently competed for binding to the NodD2 protein.

Mutat Res, 1990 May, 235(3), 165 - 9
A constitutive O6-methylguanine-DNA methyltransferase of Rhizobium meliloti; Kaufman A et al.; We have identified a DNA methyltransferase activity of the nitrogen-fixing bacterium, Rhizobium meliloti, that repairs O6-methylguanine lesions . Repair of the O6-methylguanine residue results in transfer of the methyl group to a cysteine residue of a 28,000-dalton protein . The O6-methyltransferase activity is expressed constitutively and R . meliloti does not exhibit an adaptive response to alkylating agents.

J Bacteriol, 1990 May, 172(5), 2413 - 20
Rhizobium meliloti glutamate synthase: cloning and initial characterization of the glt locus; Lewis TA et al.; The genetic locus glt, encoding glutamate synthase from Rhizobium meliloti 1021, was selected from a pLAFR1 clone bank by complementation of the R . meliloti 41 Glt- mutant AK330 . A fragment of cloned DNA complementing this mutant also served to complement the Escherichia coli glt null mutant PA340 . Complementation studies using these mutants suggested that glutamate synthase expression requires two complementation groups present at this locus . Genomic Southern analysis using a probe of the R . meliloti 1021 glt region showed a close resemblance between R . meliloti 1021, 41, and 102f34 at glt, whereas R . meliloti 104A14 showed many differences in restriction fragment length polymorphism patterns at this locus . R . meliloti 102f34, but not the other strains, showed an additional region with sequence similarity to glt . Insertion alleles containing transposable kanamycin resistance elements were constructed and used to derive Glt- mutants of R . meliloti 1021 and 102f34 . These mutants were unable to assimilate ammonia and were Nod+ Fix+ on alfalfa seedlings . The mutants also showed poor or no growth on nitrogen sources such as glutamate, aspartate, arginine, and histidine, which are utilized by the wild-type parental strains . Strains that remained auxotrophic but grew nearly as well as the wild type on these nitrogen sources were readily isolated from populations of glt insertion mutants, indicating that degradation of these amino acids is negatively regulated in R . meliloti as a result of disruptions of glt.

Mol Gen Genet, 1990 May, 221(3), 363 - 70
Differential expression of hydrogen uptake (hup) genes in vegetative and symbiotic cells of Rhizobium leguminosarum; Palacios JM et al.; The genetic determinants responsible for H2-uptake (hup genes) in Rhizobium leguminosarum are organized in six transcriptional units, designated regions hupI to hupVI, with region hupI coding for the hydrogenase structural genes (Leyva et al . 1990) . Regulation of the expression of hup genes from R . leguminosarum was examined by using hup-lacZ fusions and mRNA dot-blot analysis . None of the six hup regions is transcribed in vegetative cells grown under normal aerobic conditions, whereas all six regions are transcribed in pea bacteroids . Additionally, exposure of cell cultures to low oxygen tensions specifically induces the expression of regions hupV and hupVI . By studying the expression of hupV- and hupVI-lacZ fusions in R . meliloti mutants it was determined that the microaerobic induction of these two regions is dependent on the regulatory fixLJ system, and that this control is exerted through fixK . Such expression was also shown to be nifA and ntrA independent . The functions of the hupV and hupVI gene products are unknown . The possibility that they play a regulatory role in hup gene expression is unlikely, since pea bacteroids from R . leguminosarum Hup- mutants carrying Tn5 insertions in regions hupV and hupVI contained normal levels of mRNA transcripts corresponding to the remaining hup regions.

J Bacteriol, 1990 May, 172(5), 2622 - 32
The symbiotic defect of Rhizobium meliloti exopolysaccharide mutants is suppressed by lpsZ+, a gene involved in lipopolysaccharide biosynthesis; Williams MN et al.; exo mutants of Rhizobium meliloti SU47, which fail to secrete acidic extracellular polysaccharide (EPS), induce Fix- nodules on alfalfa . However, mutants of R . meliloti Rm41 carrying the same exo lesions induce normal Fix+ nodules . We show that such induction is due to a gene from strain Rm41, which we call lpsZ+, that is missing in strain SU47 . lpsZ+ does not restore EPS production but instead alters the composition and structure of lipopolysaccharide . In both SU47 and Rm41, either lpsZ+ or exo+ is sufficient for normal nodulation . This suggests that in R . meliloti EPS and lipopolysaccharide can perform the same function in nodule development.

J Bacteriol, 1990 May, 172(5), 2469 - 76
Genetic map of Rhizobium meliloti megaplasmid pRmeSU47b; Charles TC et al.; A circular linkage map of the Rhizobium meliloti megaplasmid pRmeSU47b was constructed . The map consists of transposon insertions carrying alternating antibiotic resistance markers linked by phi M12 transduction . Data from conjugation experiments utilizing donor strains carrying Tn5-oriT insertions in the megaplasmid supported the proposed genetic map . In addition, the positions of previously identified Fix, exopolysaccharide synthetic, thiamine synthetic, and C4-dicarboxylate transport loci on the megaplasmid map were determined . By converting cotransduction frequencies to physical distance, we calculated the replicon to be 1,600 kilobases in size, which compares favorably with previous physical estimates.

Mol Plant Microbe Interact, 1990 May-Jun, 3(3), 174 - 81
Analysis of the C4-dicarboxylate transport genes of Rhizobium meliloti: nucleotide sequence and deduced products of dctA, dctB, and dctD; Watson RJ; Rhizobium meliloti transports succinate, fumarate, malate, and aspartate by means of the dicarboxylate transport system, which is encoded by dct genes located on the exo megaplasmid . Analysis of these genes using Tn5 insertion mutagenesis revealed three complementation groups within a 5.9-kb HindIII fragment . The sequence of this fragment and the sites of Tn5 insertion were determined . Three genes, dctA, dctB, and dctD, were identified as the only three open-reading frames in locations consistent with the complementation data . The dctA gene is preceded by the sequence CTGGCACG-N4-TTGCT, which is characteristic of promoters requiring the ntrA-encoded protein for activation . The dctA-encoded protein is highly hydrophobic and contains eight potential transmembrane helices, indicating that it is probably the structural component of the transport system responsible for movement of dicarboxylates from the periplasm across the inner membrane . The dctB and dctD genes are transcribed in the opposite direction to dctA . They encode proteins with homology to the R . leguminosarum bv . viceae dicarboxylate transport proteins regulating expression of dctA and to other proteins comprising two-component regulatory systems . The dctB-encoded protein includes a putative periplasmic N-terminal domain that senses the presence of dicarboxylates and a C-terminal cytoplasmic domain that activates the dctD-encoded protein . The C-terminus of the dctD-encoded protein shows homology to several DNA-binding proteins, indicating that it is probably the domain which binds DNA in the dctA promoter region to regulate dctA transcription . All the R . meliloti mutants altered in dctA, dctB, and dctD were complemented by the dct region from R . l . bv . viceae.

J Bacteriol, 1990 May, 172(5), 2614 - 21
Rhizobium meliloti adenylate cyclase is related to eucaryotic adenylate and guanylate cyclases; Beuve A et al.; A gene from Rhizobium meliloti coding for an adenylate cyclase was sequenced, and the deduced protein sequence was compared with those of other known adenylate cyclases . No similarity could be detected with the procaryotic counterparts . However, striking similarity was found with the catalytic region of Saccharomyces cerevisiae adenylate cyclase, the cytoplasmic domains of bovine adenylate cyclase, and two mammalian guanylate cyclases . The gene was fused to the enteric beta-galactosidase, and the chimeric protein was purified by affinity chromatography . This fusion protein was found to direct the synthesis of cyclic AMP in vitro . This activity was strongly inhibited by the presence of GTP, but no cyclic GMP synthesis could be detected in conditions permitting cyclic AMP synthesis.

Nature, 1990 Apr 19, 344(6268), 781 - 4
Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal; Lerouge P et al.; Rhizobia are symbiotic bacteria that elicit the formation on leguminous plants of specialized organs, root nodules, in which they fix nitrogen . In various Rhizobium species, such as R . leguminosarum and R . meliloti, common and host-specific nodulation (nod) genes have been identified which determine infection and nodulation of specific hosts . Common nodABC genes as well as host-specific nodH and nodQ genes were shown recently, using bioassays, to be involved in the production of extracellular Nod signals . Using R . meliloti strains overproducing symbiotic Nod factors, we have purified the major alfalfa-specific signal, NodRm-1, by gel permeation, ion exchange and C18 reverse-phase high performance liquid chromatography . From mass spectrometry, nuclear magnetic resonance, (35)S-labelling and chemical modification studies, NodRm-1 was shown to be a sulphated beta-1,4-tetrasaccharide of D-glucosamine (Mr 1,102) in which three amino groups were acetylated and one was acylated with a C16 bis-unsaturated fatty acid . This purified Nod signal specifically elicited root hair deformation on the homologous host when added in nanomolar concentration.

J Bacteriol, 1990 Apr, 172(4), 1804 - 13
Immunochemical analysis of lipopolysaccharides from free-living and endosymbiotic forms of Rhizobium leguminosarum; Sindhu SS et al.; Rhizobium leguminosarum B556 and 8002 differ only with respect to carrying symbiotic plasmids with specificity for Pisum or Phaseolus hosts, respectively . Protease-treated samples derived from free-living cultures of both strains revealed a ladder of lipopolysaccharide (LPS-1) bands after periodate-silver staining of sodium dodecyl sulfate-polyacrylamide gels . These bands were arranged as doublets . After Western (immuno-) blotting, all LPS-1 bands reacted with monoclonal antibody JIM 21, whereas monoclonal antibody MAC 57 reacted only with the upper (slower-migrating) band and monoclonal antibody MAC 114 reacted only with the lower band of each doublet pair . Preparations obtained from bacteroids of Pisum or Phaseolus nodules showed significant differences in the size distribution and antigenicity of LPS . In bacteroids from Phaseolus sp., JIM 21 and MAC 57 each stained a ladder of LPS-1 bands on sodium dodecyl sulfate-polyacrylamide gels which corresponded in mobility to the upper band of each doublet pair seen in free-living cultures . MAC 114 did not react with the LPS from Phaseolus sp.-derived bacteroids . In bacteroids from Pisum sp., only fast-migrating (lower-molecular-weight) forms of LPS-1 could be visualized on gels, but both upper and lower bands of each doublet were still present and could be stained by the appropriate monoclonal antibody, MAC 57 or MAC 114, respectively . Similarly, bacteroids from R . leguminosarum 3841, which nodulates Pisum species, differed with respect to the structure and antigenicity of their LPS-1 from bacteroids of a related strain, B625, which nodulates Phaseolus species . Physiological factors were investigated that could account for these differences between the structures of LPS-1 from free-living cultures of B556 and 8002 and that from bacteroids . The following modifications in growth conditions each tended to reduce the expression of MAC 114 antigen and enhance the expression of MAC 57 antigen: succinate rather than glucose as the carbon source; microaerobic (2.5%, vol/vol) oxygen concentrations; and acidic (pH 5 to 6) culture medium . When all three of these conditions were combined, the LPS-1 that resulted was very similar to that in bacteroids from Pisum nodules . However, it was not possible to reproduce the LPS-1 pattern observed for bacteroids from Phaseolus nodules, which maintained a ladder of LPS bands reacting with MAC 57 antibody.

Mol Microbiol, 1990 Apr, 4(4), 567 - 74
Two plasmids other than the nodulation plasmid are necessary for formation of nitrogen-fixing nodules by Rhizobium leguminosarum; Hynes MF et al.; A system which allows direct selection for curing of plasmids in Gram-negative bacteria was used to generate derivatives of Rhizobium leguminosarum VF39 cured of each of six plasmids present in this strain . Phenotypes could be correlated with the absence of five of the six plasmids . The smallest plasmid, pRleVF39a, carries genes for the production of a melanin-like pigment as has been previously reported . Plasmid pRleVF39d carries nodulation and nitrogen fixation genes . Curing of the plasmids pRleVF39c and pRleVF39e gave rise to strains which formed Fix- nodules on peas, lentils, and faba beans . The nodules formed by the strains cured of pRleVF39c contained few, if any, bacteria . Analysis of washed cells by SDS-PAGE showed that this strain is defective in lipopolysaccharide (LPS) production; the defect could be complemented by introducing plasmids from several other R . leguminosarum strains, and by the R . leguminosarum biovar phaseoli LPS gene clones pCos126 and pDel27 . The nodules formed by the strain cured of pRleVF39e had a reduced symbiotic zone, an enlarged senescence zone, and an abundance of starch granules . This strain grew at a much slower rate than the wild type, was unable to grow on minimal medium, and no longer produced melanin . These defects could be complemented by at least one other Rhizobium plasmid, pRle336e, a plasmid of strain 336 which is distinct from the nodulation plasmid (pRle336c) and the plasmid (pRle336d) which could complement the LPS defect associated with the loss of pRleVF39c . This demonstrates that genes necessary for symbiosis can be carried on at least three different plasmids in R . leguminosarum.

Plant Mol Biol, 1990 Apr, 14(4), 467 - 75
Identification and cDNA cloning of a new nodule-specific gene, Nms-25 (nodulin-25) of Medicago sativa; Kiss GB et al.; A new nodule-specific gene, Nms-25 (nodulin-25), was identified in cDNA clones isolated from a nodule-specific cDNA library of Medicago sativa . The first transcript of this gene appeared 9 days after inoculation of the roots with Rhizobium meliloti . The time of expression and the quantity of the transcripts of the Nms-25 gene was similar to that of leghemoglobin genes suggesting a similar regulation . A protein of 246 amino acids could be deduced from a full-length cDNA clone . The first 24 amino acids at the N-terminal end of this protein formed a signal sequence which might direct membrane transport into the peribacteroid space . Using different predictive methods the signal sequence cleaved protein was tentatively predicted to be a water-soluble enzyme, but not hydrolase.

Int J Biol Macromol, 1990 Apr, 12(2), 67 - 70
Genetic analyses of Rhizobium meliloti exopolysaccharides; Glazebrook J et al.; We have recently obtained strong genetic evidence that the acidic Calcofluor-binding exopolysaccharide (EPS I) of Rhizobium meliloti Rm1021 is required for nodule invasion and possibly for later events in nodule development . Thirteen loci on the second megaplasmid have been identified that are required for, or affect, the synthesis of EPS I . Mutations in certain of these loci completely abolish the production of EPS I and result in mutants that form empty Fix- nodules . exoH mutants fail to succinylate their EPS I and form empty Fix- nodules . We have identified two unlinked regulatory loci, exoR and exoS, whose products play negative roles in the regulation of expression of the exo genes . We have recently discovered that R . meliloti has a latent capacity to synthesize a second exopolysaccharide (EPS II) that can substitute for the role(s) of EPS I in nodulation of alfalfa but not of other hosts . Possible roles for Rhizobium exopolysaccharides in nodulation are discussed.

Plant Mol Biol, 1990 Apr, 14(4), 549 - 60
Expression of glutamine synthetase genes in roots and nodules of Phaseolus vulgaris following changes in the ammonium supply and infection with various Rhizobium mutants; Cock JM et al.; In this paper we have examined whether the four glutamine synthetase (gln) genes, expressed in roots and nodules of Phaseolus vulgaris are substrate-inducible by ammonium . Manipulation of the ammonium pool in roots, through addition and removal of exogenous ammonium, did not elicit any changes in the abundances of the four mRNAs thus suggesting that the gln genes in roots of this legume are neither substrate-inducible by ammonium nor derepressed during nitrogen starvation . In nodules the effect of the ammonium supply on expression of the gln genes has been examined by growing nodules under argon/oxygen atmospheres, or with a number of Fix- Rhizobium mutants, and following addition of exogenous ammonium . The results of these experiments suggest that the expression of the gln-gamma gene, which is strongly induced during nodule development, is primarily under a developmental control . However nitrogen fixation appears to have a quantitative effect on expression of gln-gamma as the abundance of this mRNA is about 2 to 4-fold higher under nitrogen-fixing conditions . This effect could not be mimicked by addition of exogenous ammonium and moreover is not specific to the gln-gamma gene as mRNA from a leghaemoglobin gene was similarly affected . Taken together these results have failed to find an effect of ammonium on specifically inducing the expression of glutamine synthetase genes in roots and nodules of P . vulgaris.

J Biol Chem, 1990 Mar 5, 265(7), 3844 - 50
Use of saturation mutagenesis to localize probable functional domains in the NahR protein, a LysR-type transcription activator; Schell MA et al.; The NahR protein of the Pseudomonas naphthalene degradation plasmid NAH7 encodes a 300-residue transcription activator which is very similar to the NodD transcription activator of Rhizobium and other proteins in the LysR activator family . NahR binds to conserved sequences upstream (nucleotides -80 to -47) of the nah and sal promoters and activates transcription of genes for naphthalene catabolism in response to the inducer salicylate . Transformation of an Escherichia coli gal deletion strain (containing a sal promoter-galK fusion plasmid) with hydroxylamine-treated nahR DNA and selection on galactose/salicylate plates allowed isolation of 30 unique activation-deficient nahR alleles which fell into two classes: class I, defective in both activation and specific binding to the NahR activation site of the sal promoter; and class II, defective in activation, but with wild-type DNA binding activity . DNA sequence analysis showed that the amino acid substitutions eliminating DNA binding activity were mostly clustered in an NH2-terminal helix-turn-helix motif (residues 23-45) or a COOH-terminal domain (residues 239-291) . Similar analysis of class II mutants identified a domain (residues 126-206) possibly involved in inducer binding and/or transcription activation functions . The partial trans-dominance of many mutant alleles and the size of NahR-specific DNA binding activity measured by gel filtration suggest that the active NahR protein may be a tetramer.

J Bacteriol, 1990 Mar, 172(3), 1647 - 55
Genetic organization of the hydrogen uptake (hup) cluster from Rhizobium leguminosarum; Leyva A et al.; In symbiosis with peas, Rhizobium leguminosarum UPM791 induces the synthesis of a hydrogen uptake (Hup) system that recycles hydrogen generated in nodules by nitrogenase . A cosmid (pAL618) containing hup genes from this strain on a 20-kilobase-pair (kb) DNA insert has previously been isolated in our laboratory (A . Leyva, J . M . Palacios, T . Mozo, and T.Ruiz-Argueso, J . Bacteriol . 169:4929-4934, 1987) . Here we show that cosmid pAL618 contains all of the genetic information required to confer high levels of hydrogenase activity on the naturally Hup- strains R . leguminosarum UML2 and Rhizobium phaseoli CFN42, and we also describe in detail the organization of hup genes on pAL618 . To study hup gene organization, site-directed transposon mutagenesis and complementation analysis were carried out . According to the Hup phenotype associated with the transposon insertions, hup genes were found to span a 15-kilobase-pair region within pAL618 insert DNA . Complementation analysis revealed that Hup- mutants fell into six distinct complementation groups that define six transcriptional units, designated regions hupI to hupVI . Region hupI was subcloned and expressed in Escherichia coli cells under the control of a bacteriophage T7 promoter . A polypeptide of ca . 65 kilodaltons that was cross-reactive with antiserum against the large subunit of Bradyrhizobium japonicum hydrogenase was detected both in E . coli cells carrying the cloned hupI region and in pea bacteroids from strain UPM791, indicating that region hupI codes for structural genes of R . leguminosarum hydrogenase.

J Bacteriol, 1990 Mar, 172(3), 1409 - 17
Symbiotic pseudorevertants of Rhizobium meliloti ndv mutants; Dylan T et al.; Nodule development (ndv) mutants of Rhizobium meliloti cannot invade alfalfa to establish a nitrogen-fixing symbiosis and instead induce the formation of small, white, unoccupied nodules on alfalfa roots . Such mutants also fail to produce the unusual cyclic oligosaccharide beta-(1----2)-glucan and show defects in several aspects of vegetative growth and function . Here we show that ndv mutants are severely reduced, although not totally deficient, in the ability to attach to and initiate infection threads on alfalfa seedlings, and we demonstrate that the symbiotic deficiency can be separated from the rest of the mutant phenotype by isolating second-site pseudorevertants . Pseudorevertants selected for restoration of motility, a vegetative property, regained a substantial amount of attachment capability but only slight infection thread initiation and symbiotic ability . Such strains also regained partial tolerance to growth at low osmolarity, even though they did not recover the ability to synthesize periplasmic beta-(1----2)-glucan . Pseudorevertants selected on alfalfa for restoration of symbiosis were unrestored for beta-(1----2)-glucan production or any other vegetative property and regained little or no attachment or infection thread initiation capability . We take these data to indicate that wild-type R . meliloti normally has considerable excess capability for both attachment and infection thread initiation and that the symbiotic block in ndv mutants lies further along the developmental pathway than either of these processes, probably at the level of infection thread extension . Further, the fact that neither type of pseudorevertant recovered the ability to produce periplasmic beta-(1----2)-glucan raises the possibility that this oligosaccharide is not directly required for nodule development.

Gene, 1990 Mar 1, 87(1), 31 - 6
The nifH promoter region of Rhizobium leguminosarum: nucleotide sequence and promoter elements controlling activation by NifA protein; Roelvink PW et al.; The nucleotide (nt) sequence of the Rhizobium leguminosarum nifH promoter region contains a consensus promoter, a consensus upstream activator sequence (UAS), a pseudo (psi) promoter and a psi UAS . We mapped the transcription start point for the consensus promoter sequence by primer extension . This promoter differs from the consensus in one of the four supposedly invariant nt and can be activated by the Klebsiella pneumoniae nifA product in Escherichia coli . Under these conditions the psi promoter and psi UAS do not function . A low-copy-number plasmid construct containing the psi UAS as well as the consensus UAS delayed the onset of symbiotic nitrogen fixation in nodules induced on Pisum sativum . Studies of high-copy-number nifH promoter constructs showed that partial deletion of the consensus UAS does not alter the ability to inhibit nitrogen fixation by titration of NifA suggesting that NifA can also complex with RNA polymerase containing the alternative sigma-factor RpoN.

Genes Dev, 1990 Mar, 4(3), 344 - 56
Temporal and spatial regulation of the symbiotic genes of Rhizobium meliloti in planta revealed by transposon Tn5-gusA; Sharma SB et al.; Tn5-gusA promoter/probe transposons have been constructed that fuse the Escherichia coli gusA reporter gene transcriptionally or translationally with a target promoter . These have been used to monitor expression of Rhizobium meliloti symbiotic genes within alfalfa nodules . Fusions in all 11 nod genes studied show the same pattern of expression: first on the root surface, then throughout the developing nodule, then mainly in the nodule meristem, falling off progressively through the central region, and then disappearing . In contrast, fusions in all five nif genes studied, all four fix genes, and syrM show a second, different pattern: expression beginning later, first throughout the nodule except for the meristem, strongest just behind the meristem, and falling off progressively through the central region . Novel features revealed by these studies include nod expression in the meristem, regulated in planta expression of control genes nodD1 and nodD3, disappearance of nod expression late in organogenesis, and properties of syrM.

J Bacteriol, 1990 Mar, 172(3), 1676 - 80
Isolation of Rhizobium phaseoli Tn5-induced mutants with altered expression of cytochrome terminal oxidases o and aa3; Soberon M et al.; Two Rhizobium phaseoli mutants affected in cytochrome expression were obtained by Tn5-mob mutagenesis of the wild-type strain (CE3) . Mutant strain CFN031 expressed sevenfold less cytochrome o in culture, expressed cytochrome aa3 under microaerophilic culture conditions, in contrast to strain CE3, and was affected in its vegetative growth properties and proliferation inside plant host cells . Mutant CFN037 expressed cytochrome aa3 under microaerophilic culture conditions, while bacteroid development and nitrogen fixation occurred earlier than in strain CE3.

J Bacteriol, 1990 Mar, 172(3), 1400 - 8
Hypoosmotic adaptation in Rhizobium meliloti requires beta-(1----2)-glucan; Dylan T et al.; beta-(1----2)-Glucan, an unusual cyclic oligosaccharide, can be isolated from the periplasm of bacteria belonging to the family Rhizobiaceae . Data presented here suggest that the periplasmic beta-(1----2)-glucan of Rhizobium meliloti plays a major role in osmotic adaptation . First, growth of R . meliloti in a low-osmolarity medium causes a large accumulation of periplasmic beta-(1----2)-glucan . Second, mutations in the ndv genes, which prevent this accumulation of beta-(1----2)-glucan, reduce cell growth rates under low-osmolarity conditions and cause several other phenotypic changes indicative of an altered or stressed surface . Third, growth of the ndv mutants can be restored by raising the osmolarity of the medium with the addition of a variety of ionic or nonionic compounds . The phenotypic changes associated with the cell surface of the mutants can also be substantially suppressed by increasing the medium osmolarity . On the basis of these data and general considerations about the periplasmic space in gram-negative bacteria, we suggest a mechanism of hypoosmotic adaptation in R . meliloti in which beta-(1----2)-glucan plays an essential role.

J Biol Chem, 1990 Feb 15, 265(5), 2843 - 51
The ndvB locus of Rhizobium meliloti encodes a 319-kDa protein involved in the production of beta-(1----2)-glucan; Ielpi L et al.; The ndvB locus of Rhizobium meliloti was sequenced and found to encode a 319-kDa protein involved in the production of beta-(1----2)-glucan . Transposon Tn5 mutagenesis revealed that a large portion of the downstream half of this gene is not essential for symbiosis but is required for optimal production of beta-(1----2)-glucan . A high molecular weight inner membrane protein, believed to be the ndvB gene product, was absent from two different upstream ndvB::Tn5 mutants . This protein could be labeled in vitro with UDP-{U-14C}glucose in the wild type but not in the symbiotically defective mutants . Inner membrane preparations from the symbiotically competent downstream mutants labeled less well than did those from wild type with UDP-{U-14C} glucose and did not show distinct bands after polyacrylamide gel electrophoresis and fluorography, suggesting that C-terminal truncations of NdvB might affect the stability of this molecule . These downstream mutants had reduced amounts of periplasmic beta-(1----2)-glucan and exhibited several vegetative defects seen also in the upstream mutants . These included alterations in phage and antibiotic sensitivity, in motility, and in growth in low osmolarity media . Bacteroids produced by two of the downstream mutants were morphologically abnormal, indicating that ndvB is involved not only in invasion but also in bacteroid development.

J Bacteriol, 1990 Feb, 172(2), 670 - 7
A Rhizobium leguminosarum mutant defective in symbiotic iron acquisition; Nadler KD et al.; Iron acquisition by symbiotic Rhizobium spp . is essential for nitrogen fixation in the legume root nodule symbiosis . Rhizobium leguminosarum 116, an ineffective mutant strain with a defect in iron acquisition, was isolated after nitrosoguanidine mutagenesis of the effective strain 1062 . The pop-1 mutation in strain 116 imparted to it a complex phenotype, characteristic of iron deficiency: the accumulation of porphyrins (precursors of hemes) so that colonies emitted a characteristic pinkish-red fluorescence when excited by UV light, reduced levels of cytochromes b and c, and wild-type growth on high-iron media but low or no growth in low-iron broth and on solid media supplemented with the iron scavenger dipyridyl . Several iron(III)-solubilizing agents, such as citrate, hydroxyquinoline, and dihydroxybenzoate, stimulated growth of 116 on low-iron solid medium; anthranilic acid, the R . leguminosarum siderophore, inhibited low-iron growth of 116 . The initial rate of 55Fe uptake by suspensions of iron-starved 116 cells was 10-fold less than that of iron-starved wild-type cells . Electron microscopic observations revealed no morphological abnormalities in the small, white nodules induced by 116 . Nodule cortical cells were filled with vesicles containing apparently normal bacteroids . No premature degeneration of bacteroids or of plant cell organelles was evident . We mapped pop-1 by R plasmid-mediated conjugation and recombination to the ade-27-rib-2 region of the R . leguminosarum chromosome . No segregation of pop-1 and the symbiotic defect was observed among the recombinants from these crosses . Cosmid pKN1, a pLAFR1 derivative containing a 24-kilobase-pair fragment of R . leguminosarum DNA, conferred on 116 the ability to grow on dipyridyl medium and to fix nitrogen symbiotically . These results indicate that the insert cloned in pKN1 encodes an element of the iron acquisition system of R . leguminosarum that is essential for symbiotic nitrogen fixation.

Mol Microbiol, 1990 Feb, 4(2), 245 - 52
Molecular characterization of the nodulation gene, nodT, from two biovars of Rhizobium leguminosarum; Surin BP et al.; DNA sequencing of the nodIJ region from Rhizobium leguminosarum biovar trifolii revealed the nodT gene immediately downstream of nodJ . DNA hybridizations using a nodT-specific probe showed that nodT is present in several R . leguminosarum strains . Interestingly, a flavonoid-inducible nodT gene homologue in R . leguminosarum bv . viciae is not in the nodABCIJ operon but is located downstream of nodMN . The sequence of the nodT gene from bv . viciae was determined and a comparison of the predicted amino-acid sequences of the two nodT genes shows them to be conserved; the predicted protein sequences appear to have a potential transit sequence typical of outer-membrane proteins . Mutations affecting nodT in either biovar had no observed effect on nodulation of the legumes tested.

EMBO J, 1990 Feb, 9(2), 349 - 54
The Rhizobium nodulation gene nodO encodes a Ca2(+)-binding protein that is exported without N-terminal cleavage and is homologous to haemolysin and related proteins; Economou A et al.; Nodulation and host-specific recognition of legumes such as peas and Vicia spp . are encoded by the nodulation (nod) genes of Rhizobium leguminosarum biovar viciae . One of these genes, nodO, has been shown to encode an exported protein that contains a multiple tandem repeat of a nine amino acid domain . This domain was found to be homologous to repeated sequences in a group of bacterial exported proteins that includes haemolysin, cyclolysin, leukotoxin and two proteases . These proteins are secreted by a mechanism that does not involve an N-terminal signal peptide . The NodO protein is present in the growth medium of Rhizobium bacteria induced for nod gene expression, and partial protein sequencing of the purified protein showed that there is no N-terminal cleavage of the exported protein . It has been suggested that the internally repeated domain of haemolysin may be involved in Ca2(+)-mediated binding to erythrocytes and we show that the NodO protein can bind 45Ca2+ . It is proposed that the NodO protein may interact directly with plant root cells in a Ca2(+)-dependent way, thereby mediating an early stage in the recognition that occurs between Rhizobium and its host legume.

J Bacteriol, 1990 Feb, 172(2), 901 - 11
Rhizobium meliloti nodD genes mediate host-specific activation of nodABC; Honma MA et al.; To differentiate among the roles of the three nodD genes of Rhizobium meliloti 1021, we studied the activation of a nodC-lacZ fusion by each of the three nodD genes in response to root exudates from several R . meliloti host plants and in response to the flavone luteolin . We found (i) that the nodD1 and nodD2 products (NodD1 and NodD2) responded differently to root exudates from a variety of hosts, (ii) that NodD1 but not NodD2 responded to luteolin, (iii) that NodD2 functioned synergistically with NodD1 or NodD3, (iv) that NodD2 interfered with NodD1-mediated activation of nodC-lacZ in response to luteolin, and (v) that a region adjacent to and upstream of nodD2 was required for NodD2-mediated activation of nodC-lacZ . We also studied the ability of each of the three R . meliloti nodD genes to complement nodD mutations in R . trifolii and Rhizobium sp . strain NGR234 . We found (i) that nodD1 complemented an R . trifolii nodD mutation but not a Rhizobium sp . strain NGR234 nodD1 mutation and (ii) that R . meliloti nodD2 or nodD3 plus R . meliloti syrM complemented the nodD mutations in both R . trifolii and Rhizobium sp . strain NGR234 . Finally, we determined the nucleotide sequence of the R . meliloti nodD2 gene and found that R . meliloti NodD1 and NodD2 are highly homologous except in the C-terminal region . Our results support the hypothesis that R . meliloti utilizes the three copies of nodD to optimize the interaction with each of its legume hosts.

J Bacteriol, 1990 Feb, 172(2), 548 - 55
Expression of Rhizobium leguminosarum CFN42 genes for lipopolysaccharide in strains derived from different R . leguminosarum soil isolates; Brink BA et al.; Two mutant derivatives of Rhizobium leguminosarum ANU843 defective in lipopolysaccharide (LPS) were isolated . The LPS of both mutants lacked O antigen and some sugar residues of the LPS core oligosaccharides . Genetic regions previously cloned from another Rhizobium leguminosarum wild-type isolate, strain CFN42, were used to complement these mutants . One mutant was complemented to give LPS that was apparently identical to the LPS of strain ANU843 in antigenicity, electrophoretic mobility, and sugar composition . The other mutant was complemented by a second CFN42 lps genetic region . In this case the resulting LPS contained O-antigen sugars characteristic of donor strain CFN42 and reacted weakly with antiserum against CFN42 cells, but did not react detectably with antiserum against ANU843 cells . Therefore, one of the CFN42 lps genetic regions specifies a function that is conserved between the two R . leguminosarum wild-type isolates, whereas the other region, at least in part, specifies a strain-specific LPS structure . Transfer of these two genetic regions into wild-type strains derived from R . leguminosarum ANU843 and 128C53 gave results consistent with this conclusion . The mutants derived from strain ANU843 elicited incompletely developed clover nodules that exhibited low bacterial populations and very low nitrogenase activity . Both mutants elicited normally developed, nitrogen-fixing clover nodules when they carried CFN42 lps DNA that permitted synthesis of O-antigen-containing LPS, regardless of whether the O antigen was the one originally made by strain ANU843.

Plant Mol Biol, 1990 Feb, 14(2), 207 - 16
An altered constitutive peptide in sym 5 mutants of Pisum sativum L; Fearn JC et al.; Mutational analysis of Pisum sativum L . was used to search for constitutive proteins that might function in nodule formation . The sym 5 locus is a mutational hot spot, represented by seven independently derived mutant lines with decreased nodulation . Comparison of two-dimensional polyacrylamide gels of in vitro-translated root RNA showed a consistent difference in the migrational pattern of one peptide . In the nodulating parental cultivar 'Sparkle', a 66 kDa peptide had a pI of 5.9 . In four of the five tested sym 5 mutants, the 66 kDa peptide had a more acidic pI of 5.8 . This 66 kDa peptide is found in lateral root, tap root, and shoot . Its expression was independent of rhizobial inoculation, root temperature, or light.

Cell, 1990 Jan 26, 60(2), 281 - 94
The ENOD12 gene product is involved in the infection process during the pea-Rhizobium interaction; Scheres B et al.; The pea cDNA clone pPsENOD12 represents a gene involved in the infection process during Pisum sativum L.-Rhizobium leguminosarum bv . viciae symbiosis . The ENOD12 protein is composed of pentapeptides containing two hydroxyprolines . The expression of the ENOD12 gene is induced in cells through which the infection thread is migrating, but also in cells that do not yet contain an infection thread . Soluble compounds from Rhizobium are involved in eliciting ENOD12 gene expression . Rhizobium common and host-specific nodulation genes are essential for the production of these compounds . Two ENOD12 genes are expressed in nodules and in stem tissue of uninoculated plants . The gene represented by the cloned ENOD12 mRNA is also expressed in flowers, but a different transcription start may be used.

J Gen Microbiol, 1990 Jan, 136 ( Pt 1), 157 - 63
Uptake of glycine betaine and its analogues by bacteroids of Rhizobium meliloti; Fougere F et al.; Bacteroids isolated from alfalfa nodules induced by Rhizobium meliloti 102F34 transported glycine betaine at a constant rate for up to 30 min . Addition of sodium salts greatly increased the uptake activity, whereas other salts or non-electrolytes had less effect . The apparent Km for glycine betaine uptake was 8.3 microM and V was about 0.84 nmol min-1 (mg protein)-1 in the presence of 200 mM-NaCl which gave maximum stimulation of the transport . Supplementing bacteroid suspensions with various energy-yielding substrates, or ATP, did not increase glycine betaine uptake rates . The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), and the respiratory inhibitor potassium cyanide strongly inhibited glycine betaine uptake, but arsenate was totally inactive . Glycine betaine transport showed considerable structural specificity: choline, proline betaine, gamma-butyrobetaine and trigonelline did not competitively inhibit the system, although choline and proline betaine were transported by bacteroids . Both a high-affinity activity and a low-affinity activity were found for choline uptake . These osmoprotective compounds might have a significant role in the maintenance of nitrogenase activity in bacteroids subjected to salt stress.

J Gen Microbiol, 1990 Jan, 136 ( Pt 1), 105 - 13
Nodule formation in soybeans by exopolysaccharide mutants of Rhizobium fredii USDA 191; Ko YH et al.; Production of exopolysaccharides by Rhizobium has been linked with efficient invasion and nodulation of leguminous plant roots by the bacteria . Exopolysaccharide-deficient (exo) mutants of Rhizobium fredii USDA 191 were isolated following Tn5-insertion mutagenesis . Five phenotypically unique exo mutants were investigated for exopolysaccharide synthesis and their ability to nodulate soybeans . The exopolysaccharides produced by these mutants were analysed for polysaccharide composition by column chromatography and thin-layer chromatography . Two mutants designed exo-3 and exo-5 were deficient in both neutral glucan and exopolysaccharide synthesis, but each induced some functional nodules on Glycine max (Peking) . The remaining three mutants (exo-1, exo-2 and exo-4) synthesized neutral glucans at levels higher or lower than those in wild-type and exhibited partial exopolysaccharide deficiencies . The data imply that neither exopolysaccharides nor neutral glucans are essential for the induction of determinate nodules by R . fredii.

Dev Genet, 1990, 11(3), 182 - 96
Chimeric genes and transgenic plants are used to study the regulation of genes involved in symbiotic plant-microbe interactions (nodulin genes); de Bruijn FJ et al.; Nodulin genes are plant genes specifically activated during the formation of nitrogen-fixing nodules on leguminous plants . These genes are interesting to study since they are not only induced in a specific developmental fashion by signals coming directly or indirectly from the rhizobial symbiont, but are also expressed in a tissue-specific manner . By examining the expression of chimeric nodulin-reporter genes in transgenic legume plants it has been shown that nodule specific expression is mediated by DNA sequences present in the 5 upstream region of several nodulin genes . Here we summarize the available data on these cis-acting elements and the trans-acting factors interacting with them . We also review experiments designed to identify rhizobial "signals" which may play a role in nodule specific gene expression.

Yi Chuan Xue Bao, 1990, 17(5), 405 - 10
{Construction of Frankia genomic libraries and isolation of clones homologous to nodulation genes from Rhizobium leguminosarum}; Cui YH et al.; High molecular genomic DNAs were isolated by using the lysozyme plus achromopeptidase system from Frankia strains At4, Ccol and Hr16, the root nodule endophytes of Alnus, Casuarina and Hippophae respectively, and used to construct genomic libraries in pLAFR1, a broad host range cosmid vector within many gram-negative hosts . The genomic libraries were screened by in situ colony hybridization to identify clones homologous to common nodulation genes of Rhizobium leguminosarum, based on the sequence homology of EcoRI-digested Frankia total DNA to nodABC from Rhizobium meliloti . Several clones showing relatively strong hybridization were found, the recombinant plasmid was isolated, and their homology with Rhizobium nodulation genes was confirmed by spot hybridization . Further work on these positive clones is now underway.

Prikl Biokhim Mikrobiol, 1990 Jan-Feb, 26(1), 117 - 20
{Construction of the gene library of symbiotic nitrogen-fixing Rhizobium lupini}; Ivanushkin AG et al.; The gene bank of the symbiotic nitrogen-fixing bacterium Rhizobium lupini (effective strain 359a) was constructed on plasmid pAYC31 that was used to transform Escherichia coli C6000 . The bank contains 6600 clones . Restriction analysis showed that the size of the mean insertion fragment in the plasmid in 6.5 kb.

J Bacteriol, 1990 Jan, 172(1), 193 - 203
Two genes that regulate exopolysaccharide production in Rhizobium sp . strain NGR234: DNA sequences and resultant phenotypes; Gray JX et al.; Two closely linked genes involved in the regulation of exopolysaccharide (EPS) production in Rhizobium sp . strain NGR234, exoX and exoY, were sequenced, and their corresponding phenotypes were investigated . Inhibition of EPS synthesis occurred in wild-type strains when extra copies of exoX were introduced, but only when exoY had been deleted or mutated or was present at a lower copy number . Normal EPS synthesis occurred in Rhizobium sp . when both exoX and exoY were introduced on the same replicon . Surprisingly, the presence of multiple copies of exoY in exoY:: Tn5 mutants of NGR234 adversely affected cellular growth . This was apparent when exoY was introduced into exoY mutants on IncP1 vectors, where the copy number was approximately 10, but was not apparent when present on much larger R-prime plasmids with lower copy numbers (approximately 3 per cell) . Multiple copies of exoX did not adversely affect cellular growth of any strain . The exoX gene appeared analogous, in size and phenotype, to a previously described Rhizobium leguminosarum biovar phaseoli EPS gene, psi (D . Borthakur and A.W.B . Johnston, Mol . Gen . Genet . 207:149-154, 1987), and the deduced ExoX and Psi shared strikingly similar secondary structures . Despite this, ExoX and Psi showed little homology at the primary amino acid level, except for a central region of 18 amino acids . The interaction of ExoX and ExoY could form the basis of a sensitive regulatory system for EPS acids . The interaction of ExoX and ExoY could form the basis of a sensitive regulatory system for EPS biosynthesis . The presence of a multicopy exoX in Rhizobium meliloti and R . fredii similarly abolished EPS biosynthesis in these species.

Biosystems, 1990, 24(3), 223 - 38
Density dependent diffusional model of an infective population; Lara-Ochoa F et al.; In this work, we present a density-dependent diffusional model which, coupled to three different types of growth, permitted us to study the infective potential of a bacteria species . The results show that those species with strong internal competency have the higher colonizing capacity in terms of invasion speed . Here, we also advanced a model for the static spatial inhomogeneous distribution that some species establish after migration . It is proposed that the origin of these patterns is the result of a balance between the dispersal tendency and the attractive behavior . The results obtained were compared with the observed behavior of Rhizobium spp . during infection of leguminous roots . A possible explanation of the observed morphologies of nodule development in different legumes is suggested.

Mol Plant Microbe Interact, 1990 Jan-Feb, 3(1), 18 - 27
Cloning and characterization of Rhizobium meliloti loci required for symbiotic root nodule invasion; Hoying JB et al.; An immunological assay of root nodule polypeptides was used to analyze the nodules induced by 25 symbiotically defective Rhizobium meliloti mutants . Differences in polypeptide accumulation in these nodules were used to divide the mutants into three subsets . One subset, containing two mutant strains, was further analyzed . Nodules induced by these mutant strains lack both infection threads and bacteria . The kinetics of nodule formation by these mutant strains, by an exoB mutant, and by mixed mutant inocula suggest that the gene products required for nodule invasion may also influence nodule meristem induction . One of the two mutants characterized in this study contains a transposon Tn5 insertion in the ndvB locus, which probably results in the loss of beta-glucan synthesis . The second mutant contains a transposon in a previously uncharacterized locus . RNA analysis suggests that the newly identified locus is transcribed in free-living cultures of ndvB and exoB strains, as well as in the parental R . meliloti strain . Southern blot analysis suggests that at least a portion of this locus is duplicated . This duplication may explain the apparently leaky phenotype of the mutant strain.

Arch Microbiol, 1990, 153(6), 596 - 9
Choline derivatives increase two different acid phosphatases in Rhizobium meliloti and Pseudomonas aeruginosa; Lucchini AE et al.; In Pseudomonas aeruginosa and Rhizobium meliloti several choline derivatives, utilized as the sole carbon and nitrogen source, increase acid phosphatase activity . The enzyme activity of both bacteria could be released into the surrounding medium by EDTA-lysozyme treatment . The R . meliloti acid phosphatase activity of crude periplasmic extracts measured with p-nitrophenylphosphate was not inhibited by the presence of 5 mM choline, betaine, trimethylammonium or phosphorylcholine . The activity could not be detected using phosphorylethanolamine or phosphorylcholine as substrates . Among several phosphoesters tested only pyridoxal-5-phosphate was hydrolyzed at a considerable rate . In 7.5% polyacrylamide slab gel electrophoresis (non-denaturing conditions) of crude extracts obtained from bacteria grown in the presence of serine, glutamate, aspartate or dimethylglycine a phosphatase activity with identical mobility could be detected with alpha-naphthylphosphate or pyridoxal-5-phosphate were used as substrates . In conclusion, although the choline metabolites are capable of increasing acid phosphatase activities in R . meliloti and in P . aeruginosa, there are two different enzymes involved, apparently in different metabolisms.

EMBO J, 1990 Jan, 9(1), 1 - 7
The early nodulin transcript ENOD2 is located in the nodule parenchyma (inner cortex) of pea and soybean root nodules; van de Wiel C et al.; A pea cDNA clone homologous to the soybean early nodulin clone pGmENOD2 that most probably encodes a cell wall protein was isolated . The derived amino acid sequence of the pea ENOD2 protein shows that it contains the same repeating pentapeptides, ProProHisGluLys and ProProGluTyrGln, as the soybean ENOD2 protein . By in situ hybridization the expression of the ENOD2 gene was shown to occur only in the inner cortex of the indeterminate pea nodule . The transcription of the pea ENOD2 gene starts when the inner cortical cells develop from the nodule meristem . In the determinate soybean nodule the ENOD2 gene is expressed in the inner cortex as well as in cells surrounding the vascular bundle that connects the nodule with the root central cylinder . The term 'nodule inner cortex' is misleading, as there is no direct homology with the root inner cortex . Therefore, we propose to consider this tissue as nodule parenchyma . A possible role of ENOD2 in a major function of the nodule parenchyma, namely creating an oxygen barrier for the central tissue with the Rhizobium containing cells, is discussed.

Gene, 1989 Dec 21, 85(1), 135 - 44
Genetic analysis and regulation of the Rhizobium meliloti genes controlling C4-dicarboxylic acid transport; Wang YP et al.; The genes controlling the transport of C4-dicarboxylic acids from Rhizobium meliloti have been cloned and analysed . The nucleotide sequence of the control region of the structural dctA and the regulatory dctBD genes has been determined . Comparison with the Rhizobium leguminosarum dct genes revealed a high degree of homology . Gene fusions to the enteric lacZY reporter gene were constructed and the expression of the dctA and dctBD genes studied under various physiological conditions . In free-living cells, the regulatory dctBD genes are absolutely required for the expression of the dctA gene . In the root nodule environment, a dctA::lacZY gene fusion was found to be expressed in an R . meliloti strain mutated in both the dctB and dctD genes, but not in a strain mutated in the dctB gene alone . The presence of the conserved upstream NifA-binding sites on the dctA promoter sequence, coupled with the fact that the dctA::lacZY gene fusion is not expressed in root nodules formed by a nifA mutant strain of R . meliloti, supports the suggestion that NifA may be involved in the symbiotic expression of dctA in the absence of the regulatory dctBD genes . Under micro-aerobic conditions, however, NifA induction alone is not sufficient for expression of the dctA promoter, even though the NifA-dependent nifHDK promoter is highly expressed under these conditions.

Carbohydr Res, 1989 Dec 21, 195(1), 101 - 10
The structures of the lipopolysaccharide core components from Rhizobium leguminosarum biovar phaseoli CE3 and two of its symbiotic mutants, CE109 and CE309; Carlson RW et al.; The structures for the core regions of the lipopolysaccharides (LPSs) from R . leguminosarum bv . phaseoli CE3 and two symbiotic mutants were determined by g.l.c.-m.s., proton nuclear magnetic resonance spectroscopy (n.m.r.), fast-atom-bombardment mass spectrometry (f.a.b.-m.s.), and by comparison with known structures from the LPS of R . leguminosarum bv . trifolii ANU843 . The core oligosaccharides were separated into two components, P2-2 and P2-3, by gel-filtration chromatography using Bio-Gel P2 . The P2-2 oligosaccharide from CE3 is a tetrasaccharide consisting of 3-deoxy-D-manno-2-octulosonic acid (Kdo), mannose, galactose and galacturonic acid . The mannosyl residue is alpha-linked to O-4 of Kdo, and the galactosyl and galactosyluronic residues are alpha-linked to O-4 and O-6, respectively, of the mannosyl residue . The P2-2 oligosaccharide from mutant CE109 is missing the galactosyluronic residue, while that from mutant CE309 is missing both the galactosyl and galactosyluronic residues . The P2-3 oligosaccharide from CE3 LPS is a trisaccharide consisting of two galactosyluronic residues alpha-linked to the O-4 and O-7 of Kdo . Fraction P2-3 from mutant CE309 has the same structure as CE3 P2-3 . Fraction P2-3 from mutant CE109 contains galacturonic acid and Kdo, but its structure differs from that of CE3 P2-3.

Biochem Biophys Res Commun, 1989 Dec 15, 165(2), 659 - 66
Fast-growing root nodule bacteria produce a novel polyamine, aminobutylhomospermidine; Fujihara S et al.; Polyamines in various root nodule bacteria including Bradyrhizobium japonicum, Rhizobium fredii, R . leguminosarum, R . meliloti and R . loti were identified by capillary gas chromatography . Homospermidine was the polyamine present in highest concentration in all the rhizobia tested . In addition to putrescine and homospermidine, fast-growing type of rhizobial cells contained a novel polyamine, aminobutylhomospermidine, NH2(CH2)4NH(CH2)4NH(CH2)4NH2 . The unusual tetraamine was not found in the cells of slow-growing type of rhizobia throughout their growth period, indicating a difference in polyamine metabolism between fast-growing type and slow-growing type of root nodule bacteria.

Gene, 1989 Dec 14, 84(2), 467 - 71
Construction of a lacZ-kanamycin-resistance cassette, useful for site-directed mutagenesis and as a promoter probe; Kokotek W et al.; A lacZ gene without a promoter, but containing its ribosome-binding site, was cloned next to the kanamycin-resistance (KmR) gene of plasmid pUC4K, yielding a lacZ-KmR cassette . From the resulting plasmid, pKOK5, the lacZ-KmR cassette was recloned by means of BamHI into plasmid pKOK4, a mobilizable derivative of pBR322 which mediates ampicillin, chloramphenicol and tetracycline resistance . The lacZ-KmR cassette can be excised from pKOK5 or pKOK6 by digestion with BamHI, SalI or PstI . It can be used for insertion mutagenesis by ligation of the cassette to target DNA that has been linearized by one of these enzymes . Insertions can be selected by the KmR phenotype and mapped by digestion, e.g., with PstI and SalI . The orientation of the inserted cassette can be determined by digestion, e.g., with EcoRI or HindIII . Within the lacZ-KmR cassette, the transcription of the lacZ and the KmR genes are directed towards each other, and the two genes are separated by the bidirectionally active terminator from phage fd . In Escherichia coli, no transcription emanating from the cassette was detected . Transcription within DNA mutagenized by the cassette can be monitored by the promoterless lacZ gene . The lacZ-KmR cassette is currently used by us for the site-directed mutagenesis of hydrogen uptake gene-specific DNA from Rhizobium leguminosarum B10.

J Bacteriol, 1989 Dec, 171(12), 6764 - 70
nodO, a new nod gene of the Rhizobium leguminosarum biovar viciae sym plasmid pRL1JI, encodes a secreted protein; de Maagd RA et al.; The region of the Rhizobium leguminosarum biovar viciae Sym plasmid pRL1JI, responsible for the production and secretion of a previously described 50-kilodalton protein (R . A . de Maagd, C . A . Wijffelman, E . Pees, and B . J . J . Lugtenberg, J . Bacteriol . 170:4424-4427, 1988), was cloned and its nucleotide sequence was determined . A new nod gene, nodO, preceded by a poorly conserved nod box, was identified and its transcriptional start site was determined . Comparison of its predicted protein product with the N-terminal amino acid sequence of the isolated secreted protein showed that nodO is the structural gene of this protein, although the nucleotide sequence predicted a protein only 30,002 daltons in size . This comparison also showed that the secreted protein is not the product of N-terminal processing of a larger precursor . A conventional N-terminal signal sequence was not detected in the NodO protein . The NodO protein has significant homology with a part (residues 720 to 920) of the hemolysin protein (HlyA) of Escherichia coli . Analysis of the transcriptional regulation of the nodO gene revealed that, in contrast with other nod promoters in this species, activity of the nodO promoter is greatly enhanced in the presence of multiple copies of the nodD gene.

Mol Microbiol, 1989 Dec, 3(12), 1753 - 64
Molecular linkage of the nif/fix and nod gene regions in Rhizobium leguminosarum biovar trifolii; Iismaa SE et al.; Nucleotide sequence analysis of a 2.5kb region downstream of the nifA gene from Rhizobium leguminosarum biovar trifolii has resulted in linkage, at the DNA sequence level, of the nifEN, nifHDK, fixABCX, nifA gene cluster with the nodEF, nodD, nodABCIJ genes . Four genes have been identified within this intervening region . Immediately 3' to the nifA gene is the nifB gene and the nifB-linked ferredoxin-encoding fdxN gene . Downstream of fdxN in R . leguminosarum bv . trifolii and in Rhizobium meliloti, we have identified an open reading frame which has not been described previously and which we propose to designate fixU . Downstream of fixU in R . leguminosarum bv . trifolii is a nod gene, nodT, which is contiguous with nodJ (B . Surin et al., manuscript in preparation) . As a result of this study, the linkage relationships of 22 symbiotic genes spanning a 24 kb region of the symbiotic plasmid from R . leguminosarum bv . trifolii are now known.

Mol Gen Genet, 1989 Dec, 220(1), 81 - 7
Nucleotide sequence of the gene encoding the nitrogenase iron protein (nifH) of Azospirillum brasilense and identification of a region controlling nifH transcription; Fani R et al.; The DNA sequence was determined for the Azospirillum brasilense nifH gene and part of the nifD gene . The nifH gene is 885 bp long and encodes 293 amino acid residues . The region upstream of the nifH open reading frame contains a putative promoter whose sequence shows perfect homology with promoters of other diazotrophic bacteria and two putative upstream activator sequences . Experiments with the promoter-probe vector pAF300 showed that this region promotes transcription in response to the nitrogen and oxygen availability of the cell . The amino acid sequence was deduced from the DNA nucleotide sequence of nifH; the polypeptide contains the four cysteine residues highly conserved among other nifH products and an arginine residue at position 101 which could be the site of the modification occurring during the "switch-off" of nitrogenase . The codon usage appears to be very biased reflecting the high G + C content of the Azospirillum nifH gene . In a comparison of the amino acid sequence with the other 18 known nifH gene products, the A . brasilense nifH product showed the highest level of homology with fast-growing Rhizobia suggesting interesting evolutionary implications.

Gene, 1989 Nov 30, 83(2), 243 - 9
Expression of the adenyl cyclase-encoding gene (cya) of Rhizobium meliloti F34: existence of two cya genes?
O'Regan M, Kiely B, O'Gara F.
To gain insight into the role of cyclic AMP (cAMP) in Gram-negative soil bacteria, we have studied the expression of an adenyl cyclase-encoding gene 'cya' of Rhizobium meliloti F34 . In both Escherichia coli and Bradyrhizobium japonicum, the gene is expressed from a promoter(s) contained on a 2.6-kb fragment of the cloned insert, which may indicate the presence of a functional 'cya' promoter or the coincidental presence of sequences which can function as promoters in these two species . The study of 'cya'-lac fusion activity in R . meliloti indicated that the 'cya' gene is not expressed at detectable levels and, thus, may not contribute to the modulation of cAMP levels under the growth conditions employed . R . meliloti strains carrying defined genomic mutations at the 'cya' locus were still capable of the synthesis of near wild-type levels of cAMP . These results suggest that the cloned 'cya' gene is not the key determinant responsible for cAMP synthesis under the culture conditions employed.

J Biol Chem, 1989 Nov 15, 264(32), 18939 - 43
Cloning and expression of rat cDNA encoding corticosteroid 11 beta-dehydrogenase; Agarwal AK et al.; Corticosteroid 11 beta-dehydrogenase (11-DH) catalyzes the conversion of cortisol to the inactive metabolite cortisone . Absence of 11-DH activity leads to a potentially fatal form of childhood hypertension termed apparent mineralocorticoid excess . As a first step in elucidating the molecular basis of this disorder, we isolated and characterized a rat cDNA clone encoding 11-DH . This clone hybridized to a single mRNA species in liver, kidney, and testis RNA but not to RNA from heart . The insert was 1265 base pairs long and included an 861-base pair open reading frame encoding 287 amino acids . A search of sequence databases revealed that 11-DH is identical in about 27% of amino acid residues to ribitol dehydrogenase from Klebsiella and to the product of the nodG gene from the nitrogen-fixing bacterium, Rhizobium meliloti, thus defining a new superfamily of genes encoding dehydrogenases . The 11-DH cDNA was expressed by transfection into Chinese hamster ovary cells under the control of an SV40 promoter . The expressed enzyme mediated both 11 beta-dehydrogenation and the reverse 11-oxoreduction reaction . Southern blot analysis of rat and human DNA suggested that additional genes related to 11-DH exist in both species.

J Mol Biol, 1989 Nov 5, 210(1), 65 - 77
In vivo studies on the interaction of RNA polymerase-sigma 54 with the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters . The role of NifA in the formation of an open promoter complex; Morett E et al.; Transcription from the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters requires the positive control protein NifA and the alternative sigma factor sigma 54, encoded by the rpoN gene . Transcription from the K . pneumoniae nifH promoter is fully dependent upon NifA bound at the upstream activator sequence (UAS) whereas the R . meliloti nifH promoter can be efficiently activated in the absence of this sequence and can also be activated by a mutant form of NifA unable to bind the UAS . The in vivo interaction of RNA polymerase-sigma 54 with these promoters was examined using dimethyl sulphate footprinting . The R . meliloti nifH promoter but not the K . pneumoniae nifH promoter showed sigma 54-dependent methylation protection of guanine residues at -14, -25 and -26, the most conserved nucleotides characteristic of sigma 54-dependent promoters . A mutant derivative of the K . pneumoniae nifH promoter bearing transitions at positions from -15 to -17 showed sigma 54-dependent methylation protection of guanines -13, -24 and -25 . The enhanced interaction of the RNA polymerase-sigma 54 with this mutant promoter correlates with its increased level of activation by a form of NifA unable to bind the UAS . Use of in vivo KMnO4 footprinting to detect single-stranded pyrimidine residues and in vivo methylation protection demonstrated that the sigma 54-dependent protection observed in the R . meliloti and mutant K . pneumoniae nifH promoter results from the formation of a closed promoter complex . The isomerization of the pre-existing closed complex to an open promoter form, as judged by the local denaturation of promoter DNA which rendered sequences from +5 to -10 reactive towards KMnO4, was shown to be fully dependent on NifA . We propose a model in which the fidelity of activation of sigma 54-dependent promoters relies on a weak activator-independent interaction of RNA polymerase-sigma 54 with the promoter . A specific interaction of the appropriate activator with its respective UAS is then required for the positive control protein to facilitate open complex formation.

Appl Environ Microbiol, 1989 Nov, 55(11), 2755 - 61
Quantitative comparison of the laboratory and field competitiveness of Rhizobium leguminosarum biovar phaseoli; Beattie GA et al.; Rhizobium leguminosarum bv . phaseoli KIM5s outcompeted strain CE3 in bean (Phaseolus vulgaris L.) root nodulation when plants were grown at any of three field sites, each with a different soil type and indigenous population, or in the laboratory in a sterilized sand, a sterilized peat-vermiculite mixture, or a nonsterile field soil . A mathematical model describing nodulation competitiveness was empirically derived to evaluate the relative competitiveness of the two strains under these conditions . This model relates the proportional representation of the two strains in the inoculum to the proportional representation of nodules occupied by each strain or both strains and provides a measure of competitiveness, which is referred to as the competitiveness index . Statistical comparisons of competitiveness indices showed that the relative competitiveness of KIM5s and CE3 remained constant when the two strains were applied in a constant ratio over a range of inoculum concentrations, from 10(3) to 10(7) cells per seed, and when they were applied in various ratios to six P . vulgaris cultivars . Furthermore, the relative competitiveness of KIM5s and CE3 in the laboratory did not differ significantly from their relative competitiveness at the three field sites studied . Thus, a study of the basis for nodulation competitiveness of KIM5s and CE3 in the laboratory has the potential to provide an understanding of competitiveness both in the laboratory and in the field.

Antonie Van Leeuwenhoek, 1989 Nov, 56(4), 357 - 60
Galactosyl residue in exopolysaccharide from Rhizobium meliloti JJ-1 exposed to manganese in furanoid; Appanna VD; Exopolysaccharide (EPS) elaborated by Rhizobium meliloti JJ-1 in a manganese supplemented medium was isolated . Periodate oxidation, reduction with sodium borohydride, followed by hydrolysis and subsequent capillary gas liquid chromatography of the derived alditol acetates revealed that D-galactose in this complex biopolymer is in furanoid form . This observation was further confirmed by 13carbon nuclear magnetic resonance (13C NMR).

Mol Microbiol, 1989 Nov, 3(11), 1649 - 51
The nodL gene from Rhizobium leguminosarum is homologous to the acetyl transferases encoded by lacA and cysE; Downie JA; The predicted protein sequence of the nodL gene from Rhizobium leguminosarum was screened against translations of the GenBank DNA sequence database . A very strong homology was found to lacA, which encodes thiogalactoside transferase; homology between NodL and the cysE gene product (serine acetyl transferase) was also found . Comparison of the conserved regions of the three protein sequences indicated a domain that may be an active site of the enzymes.

Mol Microbiol, 1989 Nov, 3(11), 1567 - 77
Single and multiple mutations affecting properties of the regulatory gene nodD of Rhizobium; Burn JE et al.; nodD of Rhizobium leguminosarum has two regulatory properties: it autoregulates and, in cells grown with specific flavonoids, activates other nod genes . We isolated mutations in nodD affecting one or both properties . Those abolishing autoregulation and nod gene induction were at the 5' end of nodD, as were those which only affected autoregulation . Mutations affecting nod gene activation are at the 3' end of nodD . Eleven mutations in this region of nodD were isolated: some had little effect on the regulatory properties; other reduced activation of other nod genes . 265 bps were removed from the 3' end of nodD: this abolished nodD function . Doubly mutant derivatives of nodD were constructed by making nodD genes with a mutation that conferred the ability to activate transcription of nod genes in the absence of inducers (class IV) plus another that abolished autoregulation and/or flavonoid-dependent nod gene activation . The behaviour of such double mutants was complex; e.g . in one case, a doubly mutant nodD gene containing the class IV mutation, coupled to one of those that (alone) abolished autoregulation and nod gene induction, was similar in behaviour to the wild type . In other cases, double mutants were similar to one of the parentals.

FEMS Microbiol Lett, 1989 Nov, 53(1-2), 211 - 7
Polysaccharides production by Rhizobium phaseoli and the typing of their excreted anionic polysaccharides; Zevenhuizen LP et al.; The pattern of polysaccharide production amongst strains of Rhizobium phaseoli appear very varied: some strains produce anionic exopolysaccharides (EPS) as major polysaccharides (EPS) as major polymer without any other product, but most strains exhibit greater polysaccharide diversity . Apart from EPS they excrete capsular polysaccharides (CPS) and accumulate poly-beta-hydroxybutyric acid (PHB) and/or glycogen in their cells . The latter can then be used as C-sources for further synthesis of EPS and CPS . Some strains are only very poor producers or do not produce at all . Nine strains of R . phaseoli have been analysed and shown to possess the K-36 type of polysaccharide (EPS), as do strains of R . leguminosarum (6 strains) and R . trifolii (9 strains) . Three strains of R . phaseoli have been found to possess the K-87 type of polysaccharide and types K-38 and K-44 polysaccharides have only been found in their own type strains.

Plant Mol Biol, 1989 Nov, 13(5), 481 - 90
The isolation and characterization of a cDNA clone encoding Lupinus angustifolius root nodule glutamine synthetase; Grant MR et al.; Glutamine synthetase, purified from Lupinus angustifolius legume nodules, was carboxymethylated and succinylated prior to chemical or enzymatic cleavage . Peptides were purified and sequenced . An oligonucleotide probe was constructed for the sequence MPGQW . This probe was used to identify a glutamine synthetase cDNA clone, pGS5, from a lupin nodule cDNA library constructed in pBR322 . pGS5 was sequenced (1043 bp) and computer-assisted homology searching revealed a high degree of conservation between this lupin partial cDNA clone and other plant glutamine synthetases at both the amino acid (greater than 90%) and nucleotide (greater than 80%) level . Northern and Southern analyses using pGS5 supported the conclusion that a multigene glutamine synthetase family exists in lupin which is differentially expressed in both an organ-specific and temporal manner . Western and Northern blot analyses indicated the accumulation of a glutamine synthetase specific mRNA species during nodule development corresponded to the appearance of a novel glutamine synthetase polypeptide between 8 and 10 days after rhizobial inoculation.

J Mol Evol, 1989 Nov, 29(5), 422 - 8
Glutamine synthetase II in Rhizobium: reexamination of the proposed horizontal transfer of DNA from eukaryotes to prokaryotes; Shatters RG et al.; We have determined the DNA sequence of a Rhizobium meliloti gene that encodes glutamine synthetase II (GSII) . The deduced amino acid sequence was compared to that of Bradyrhizobium japonicum GSII and those of various plant and mammalian glutamine synthetases (GS) in order to evaluate a proposal that the gene for this enzyme was recently transferred from plants to their symbiotic bacteria . There is 83.6% identity between the R . meliloti and B . japonicum proteins . The bacterial GSII proteins average 42.5% identity with the plant GS proteins and 41.8% identity with their mammalian counterparts . The plant proteins average 53.7% identity with the mammalian proteins . Thus, the GS proteins are highly conserved and the divergence of these proteins is proportional to the phylogenetic divergence of the organisms from which the sequences were determined . No transfer of genes across large taxonomic gaps is needed to explain the presence of GSII in these bacteria.

J Mol Recognit, 1989 Nov, 2(3), 114 - 21
Model-building of Fnr and FixK DNA-binding domains suggests a basis for specific DNA recognition; Cherfils J et al.; The DNA-binding C-terminal domains of the regulatory proteins Fnr from Escherichia coli and FixK from Rhizobium meliloti have been modelled on the basis of their homologies to the CAP protein from E . coli . Residues Glu181, Thr182 and Arg185 of CAP, which are exposed residues of the DNA-recognition helix alpha F, are conserved in Fnr and FixK . However, Arg180 and Gly184 are substituted by Val and Ser respectively in Fnr . We propose that this valine makes a Van der Waals' contact with the first thymine in the Fnr consensus TTGA-N6-TCAA, and that the serine contributes to the binding by displacing a thymine-bound water molecule . The corresponding residues in FixK, Ile and Ser allow the same interactions with a thymine . Therefore we predict that FixK may recognize the same sites as Fnr . This is supported experimentally by showing that Fnr can substitute for FixK in activating the fixN gene in E . coli.

J Bacteriol, 1989 Nov, 171(11), 6161 - 8
Genes involved in lipopolysaccharide production and symbiosis are clustered on the chromosome of Rhizobium leguminosarum biovar viciae VF39; Priefer UB; Four mutants of Rhizobium leguminosarum biovar viciae VF39 altered in lipopolysaccharide (LPS) synthesis were isolated upon random Tn5 mutagenesis . These mutants produced matt colonies on TY medium and showed autoagglutination and loss of motility . On sodium dodecyl sulfate-polyacrylamide gels, they lacked a slow-migrating carbohydrate band, corresponding to the complete LPS (LPSI) . All four mutants formed small white nodules on Vicia hirsuta . These nodules were infected but showed no nitrogen-fixing activity and senesced prematurely . Three of the mutants were complemented by a wild-type cosmid to synthesis of normal LPS and induction of nitrogen-fixing nodules . By hybridization and in vivo cloning experiments, the mutations were mapped within different EcoRI fragments which could be localized on the VF39 chromosome . Cross-complementation analyses revealed that the three mutants were affected in different transcriptional units . The results indicate that a cluster of genes necessary for LPSI production and symbiotic efficiency is located within a defined region of 20 kilobases on the R . leguminosarum bv . viciae chromosome.

J Bacteriol, 1989 Oct, 171(10), 5492 - 502
DNA footprint analysis of the transcriptional activator proteins NodD1 and NodD3 on inducible nod gene promoters; Fisher RF et al.; The Rhizobium meliloti nodD1 and nodD3 gene products (NodD1 and NodD3) are members of the lysR-nodD gene regulator family . They are functionally distinct in that NodD1 transcriptionally activates other nod genes in the presence of a flavonoid inducer such as luteolin, while NodD3 is capable of activating nod gene expression at high levels in the absence of inducer . NodD1 and NodD3 are DNA-binding proteins which interact with DNA sequences situated upstream of the transcription initiation sites of at least three sets of inducible nod genes . We report the footprinting of NodD1- and NodD3-DNA complexes with both DNase I and the 1,10-phenanthroline-copper ion reagent . NodD1 and NodD3 both interacted with the nodABC, nodFE, and nodH promoters and protected from cleavage an extensive piece of DNA, including the nod box, from approximately -20 to -75 from the transcription start site for each of the three promoters . The constitutively activating protein NodD3 displayed an additional hypersensitive cleavage site in its footprint compared with NodD1.

J Bacteriol, 1989 Oct, 171(10), 5244 - 53
Conservation between coding and regulatory elements of Rhizobium meliloti and Rhizobium leguminosarum dct genes; Jiang J et al.; Complementation of Rhizobium leguminosarum dct mutants with a cosmid bank yielded Rhizobium meliloti homologs of the dctA, dctB, and dctD genes . The genes dctB and dctD are thought to form a two-component system which responds to the presence of C4-dicarboxylates to regulate expression of a transport protein encoded by dctA . DNA sequence analysis showed that dct coding and intergenic regions, including putative binding sites for the dctD protein and sigma 54-RNA polymerase, were highly conserved between these two Rhizobium species . Mutation of R . meliloti dctD showed that it was not essential for symbiotic nitrogen fixation but was needed for growth on succinate and the expression of a dctA-lacZ fusion gene in free-living cells . Hybridization of R . meliloti genomic DNA with probes representing the central portion of dctD potentially identified more than 20 similar regulatory genes, all of which are likely to depend upon the alternative sigma factor encoded by rpoN and stimulate transcription in a manner very similar to ntrC activation of glnA in enteric bacteria.

Mol Microbiol, 1989 Oct, 3(10), 1441 - 7
Nucleotide sequence of the regulatory nifA gene of Rhizobium leguminosarum PRE: transcriptional control sites and expression in Escherichia coli; Roelvink PW et al.; We report the sequence of the regulatory nifA gene of Rhizobium leguminosarum PRE . The transcription initiation and termination sites of nifA were mapped and a potential promoter and a rho-independent terminator identified . The nifA gene has two possible translation start sites, both of which are used in an Escherichia coli background, resulting in proteins with apparent molecular weights of 58 kD and 57 kD; initiation at the second site is preferred over initiation at the first . The nifA-nifB intergenic region contains an rpoN-dependent promoter for the nifB gene but no consensus upstream activator sequence (UAS) . A potential DNA-binding domain, consisting of two alpha-helices separated by a four-amino-acid linker, is located at the C-terminal end of the NifA amino acid sequence.

EMBO J, 1989 Oct, 8(10), 2811 - 8
Genetic analysis and cellular localization of the Rhizobium host specificity-determining NodE protein; Spaink HP et al.; The nucleotide sequence of the nodE gene of Rhizobium trifolii strain ANU843 was determined . Like the nodE gene of R . leguminosarum strain 248 it encodes a protein with a predicted mol . wt of 42.0 kd . The predicted NodE proteins of R.trifolii and R.leguminosarum have a homology of 78% . Using antibodies raised against the NodE protein of R.trifolii it was shown that the NodE products of R.leguminosarum and R.trifolii are localized in the cytoplasmic membrane . Furthermore, these NodE proteins are predicted to contain at least two non-polar transbilayer alpha-helices . The nodE genes of R.trifolii and R.leguminosarum were separated from the nodF genes that precede them in the respective nodFE operons . Using the resulting clones, in which NodE was produced under control of the flavonoid-inducible nodABCIJ promoter of R.leguminosarum, it was shown that the NodE product is the main factor that distinguishes the host range of nodulation of R.trifolii and R.leguminosarum . Hybrid nodE genes, which consist of a 5' part of the R.leguminosarum nodE gene and a 3' part of the R.trifolii gene, were constructed . From the properties of these hybrid genes it was concluded that a central region of 185 amino acids at the most, containing only 44 non-identical amino acids, determines the difference in the host-specific characteristics of the two NodE proteins.

Mol Gen Genet, 1989 Oct, 219(1-2), 289 - 98
Common nodABC genes in Nod locus 1 of Azorhizobium caulinodans: nucleotide sequence and plant-inducible expression; Goethals K et al.; Azorhizobium caulinodans strain ORS571 induces nitrogen-fixing nodules on roots and stem-located root primordia of Sesbania rostrata . Two essential Nod loci have been previously identified in the bacterial genome, one of which (Nod locus 1) shows weak homology with the common nodC gene of Rhizobium meliloti . Here we present the nucleotide sequence of this region and show that it contains three contiguous open reading frames (ORFA, ORFB and ORFC) that are related to the nodABC genes of Rhizobium and Bradyrhizobium species . ORFC is followed by a fourth (ORF4) and probably a fifth (ORF5) open reading frame . ORF4 may be analogous to the nodI gene of R . leguminosarum, whereas ORF5 could be similar to the rhizobial nodF genes . Coordinated expression of this set of five genes seems likely from the sequence organization . There is no typical nod promoter consensus sequence (nod box) in the region upstream of the first gene (ORFA) and there is no nodD-like gene . LacZ fusions constructed with ORFA, ORFB, ORFC, and ORF4 showed inducible beta-galactosidase expression in the presence of S . rostrata seedlings as well as around stem-located root primordia . Among a series of phenolic compounds tested, the flavanone naringenin was the most efficient inducer of the expression of this ORS571 nod gene cluster.

Biochem Cell Biol, 1989 Oct, 67(10), 674 - 9
Purification and partial amino acid sequence of a glutamyl-tRNA synthetase from Rhizobium meliloti; Laberge S et al.; A glutamyl-tRNA synthetase has been purified to homogeneity from Rhizobium meliloti, using reversed-phase chromatography as the last step . Amino acid sequencing of the amino-terminal region of the enzyme indicates that it contains a single polypeptide, whose molecular weight is about 54,000, as judged by SDS-gel electrophoresis . The primary structures of the amino-terminus region and of an internal peptide obtained by cleavage of the enzyme with CNBr have similarities of 58 and 48% with regions of the glutamyl-tRNA synthase of Escherichia coli; these are thought to be involved in the binding of ATP and tRNA, respectively . The small amount of glutamyl-tRNA synthetase present in R . meliloti is consistent with the metabolic regulation of the biosynthesis of many aminoacyl-tRNA synthetases.

J Bacteriol, 1989 Oct, 171(10), 5561 - 6
Restriction fragment length polymorphism analysis of Rhizobium galegae strains; Kaijalainen S et al.; Total DNA of various Rhizobium galegae strains representing different geographical origins, and taxonomic divergence was digested with three restriction enzymes separately, Southern blotted, and hybridized with six heterologous probes . The sequence divergences for different pairwise comparisons were calculated from proportions of conserved hybridizing fragments . The unweighted pair group method was used to group the strains . The symbiotic common nod and nifHDK probes used were highly conserved and grouped the strains according to the host plant, Galega orientalis or G . officinalis . The grouping derived from combined data of the constitutive hemA, glnA, ntrC, and recA probes was similar to that obtained in total DNA-DNA hybridization experiments . The constitutive probes grouped the strains in a different order than did the symbiotic probes, a result that may reflect interstrain transfer of symbiotic sequences in the course of evolution.

J Bacteriol, 1989 Oct, 171(10), 5551 - 60
Identification and sequence analysis of the Rhizobium meliloti dctA gene encoding the C4-dicarboxylate carrier; Engelke T et al.; Transposon Tn5-induced C4-dicarboxylate transport mutants of Rhizobium meliloti 2011 which could be complemented by cosmid pRmSC121 were subdivided into two classes . Class I mutants (RMS37 and RMS938) were defective in symbiotic C4-dicarboxylate transport and in nitrogen fixation . They were mutated in the structural gene dctA, which codes for the C4-dicarboxylate carrier . Class II mutants (RMS11, RMS16, RMS17, RMS24, and RMS31) expressed reduced activity in symbiotic C4-dicarboxylate transport and in nitrogen fixation . These mutants were mutated in regulatory dct genes which do not play an essential role in the symbiotic state . Thin sections of alfalfa nodules induced by the wild type and class I and class II mutants were analyzed by light microscopy . Class mutants induced typical Fix- nodules, showing a large senescent zone, whereas nodules induced by class II mutants only differed in an enhanced content of starch granules compared with wild-type nodules . Class I mutants could be complemented by a 2.1-kilobase SalI-HindIII subfragment of cosmid pRmSC121 . DNA sequencing of this fragment resulted in the identification of an open reading frame, which was designated dctA because Tn5 insertion sites of the class I mutants mapped within this coding region . The dctA gene was preceded by a nif consensus promoter and an upstream NifA-binding element . Upstream of the dctA promoter, the 5' end of the R . meliloti dctB gene could be localized . The amino acid sequence of the N-terminal part of the R . meliloti DctB protein shared 49% homology with the corresponding part of the R . leguminosarum DctB protein . The DctA protein consisted of 441 or 453 amino acids due to two possible ATG start codons, with calculated molecular masses of 46.1 and 47.6 kilodaltons, respectively . The hydrophobicity plot suggests that DctA is a membrane protein with several membrane passages . The amino acid sequences of the R . meliloti and the R . leguminosarum DctA proteins were highly conserved (82%).

J Bacteriol, 1989 Oct, 171(10), 5458 - 66
Aromatic aminotransferase activity and indoleacetic acid production in Rhizobium meliloti; Kittell BL et al.; Bacterial indoleacetic acid (IAA) production, which has been proposed to play a role in the Rhizobium-legume symbiosis, is a poorly understood process . Previous data have suggested that IAA biosynthesis in Rhizobium meliloti can occur through an indolepyruvate intermediate derived from tryptophan by an aminotransferase activity . To further examine this biosynthetic pathway, the aromatic aminotransferase (AAT) activity of Rhizobium meliloti 102F34 (F34) was characterized . At least four proteins were detected on nondenaturing gels of F34 protein extracts that exhibited AAT activity . All four of these AATs were constitutively produced and utilized the aromatic amino acids tryptophan, phenylalanine, and tyrosine as amino substrates . Two AATs were also capable of using aspartate . Plasmids from an F34 gene bank were identified that coded for the synthesis of at least three of these proteins, and the respective gene sequences were localized by transposon mutagenesis . Selected transposon insertions were recombined into the F34 genome to produce strains defective in two of these proteins (AAT1 and AAT2) . Characterization of the mutants revealed that neither was essential for the biosynthesis of IAA in the absence of exogenous tryptophan, but that both contributed to IAA biosynthesis when high levels of exogenous tryptophan were present . AAT1 and AAT2 were also not required for the production of a minimal level of aromatic amino acids, but both were able to scavenge nitrogen from the aromatic amino acids during nitrogen deprivation . Neither AAT1 nor AAT2 was essential for symbiosis with alfalfa.

J Bacteriol, 1989 Sep, 171(9), 5031 - 8
The predicted protein product of a pathogenicity locus from Pseudomonas syringae pv . phaseolicola is homologous to a highly conserved domain of several procaryotic regulatory proteins; Grimm C et al.; A ca . 20-kilobase (kb) region (hrp) that controls the interaction of Pseudomonas syringae pv . phaseolicola with its host (pathogenicity) and nonhost plants (hypersensitive reaction) was previously cloned and partially characterized . In this study we defined the limits and determined the nucleotide sequence of a hrp locus (hrpS), located near the right end of the hrp cluster . The largest open reading frame (ORF302) in hrpS has a coding capacity for a 302-amino-acid polypeptide . The predicted amino acid sequence of the translation product of ORF302 (HrpS) shows significant similarity to several procaryotic regulatory proteins, including the NtrC, NifA, and DctD proteins of Rhizobium spp., the NtrC and NifA proteins of Klebsiella pneumoniae, and the TyrR protein of Escherichia coli . These proteins regulate diverse operons involved in nitrogen fixation, transport and metabolism of amino acids, and transport of C-4 dicarboxylic acids . The HrpS protein appears to be the shortest naturally occurring member of this family of proteins, corresponding for the most part to the highly conserved central domain of these proteins, which contains a putative ATP-binding site . A C-terminal segment analogous to the less-well-conserved domain, involved in DNA binding of NtrC and NifA, is also present in HrpS . These similarities suggest that HrpS is a regulatory protein . In line with this prediction is the finding that a functional hrpS gene is necessary for the activation of another hrp locus during the plant-bacterium interaction.

J Bacteriol, 1989 Sep, 171(9), 4714 - 7
An osmoregulated dipeptide in stressed Rhizobium meliloti; Smith LT et al.; One common mechanism of cellular adaptation to osmotic stress is the accumulation of organic solutes in the cytosol . We have used natural-abundance 13C nuclear magnetic resonance to identify all organic solutes that accumulate to significant levels in Rhizobium meliloti . Our studies led to the discovery of a new dipeptide, N-acetylglutaminylglutamine amide (NAGGN), which is accumulated during osmotic stress . Only rarely have peptides been shown to function in bacteria, and furthermore, this is the first example of a peptide playing a role in osmoregulation . Evidence for the biological role of NAGGN in osmotic-stress protection is presented.

J Bacteriol, 1989 Sep, 171(9), 4583 - 8
Immunogold localization of the NodC and NodA proteins of Rhizobium meliloti; Johnson D et al.; Monospecific, polyclonal antibodies to the nodC and nodA gene products of Rhizobium meliloti were used in combination with immunogold labeling and transmission electron microscopy to localize the NodC and NodA proteins in cultures of R . meliloti . Both NodC and NodA were detected in the cytoplasm and cell envelope in thin sections of free-living rhizobia treated with luteolin, a known inducer of nod gene expression; however, only NodC was detected on cell surfaces when immunolabeling was performed with intact induced cells . In view of biochemical data characterizing NodC as an outer membrane protein with a large extracellular domain, the pattern of immunolabeling on thin sections suggests that NodC is produced on free cytoplasmic ribosomes prior to assembly in the membrane . The pattern of NodA labeling on thin sections is consistent with biochemical data detecting NodA in both soluble and membrane fractions of NodA-overexpressing strains of R . meliloti.

J Bacteriol, 1989 Sep, 171(9), 4549 - 55
Genetic derepression of a developmentally regulated lipopolysaccharide antigen from Rhizobium leguminosarum 3841; Wood EA et al.; Monoclonal antibody AFRC MAC 203 recognizes a developmentally regulated lipopolysaccharide antigen in Rhizobium leguminosarum bv . viciae 3841 . Transposon-induced mutants that constitutively expressed MAC 203 antigen were isolated . These strains were morphologically normal, showed no gross abnormalities in lipopolysaccharide size distribution on sodium dodecyl sulfate-polyacrylamide gels, and induced normal nitrogen-fixing nodules . However, the mutants lacked lipopolysaccharide epitopes recognized by another rat monoclonal antibody, AFRC MAC 281, suggesting that the corresponding epitopes may be interconverted or share a common precursor . In conjugational crosses, the transposon insertion associated with both the loss of MAC 281 antigen and the constitutive expression of MAC 203 antigen showed linkage to the chromosomal rif allele . A derivative of strain 3841 with a deletion spanning the nod-fix region of the symbiotic plasmid showed no altered expression pattern for MAC 203 antigen, suggesting that the relevant genetic determinants map to genomic sites that are not associated with nifA or any known genes on the symbiotic plasmid.

J Bacteriol, 1989 Sep, 171(9), 4543 - 8
Expression of a cell surface antigen from Rhizobium leguminosarum 3841 is regulated by oxygen and pH; Kannenberg EL et al.; Rhizobium leguminosarum bv . viciae 3841 was grown in liquid suspension culture to investigate how culture conditions could affect the expression of a developmentally regulated cell surface antigen associated with lipopolysaccharide . The antigen, which is recognized by monoclonal antibody AFRC MAC 203, was expressed when cultures were grown at neutral pH under low-oxygen conditions (less than 7.5% {vol/vol} O2 in the gas phase) . Antigen was also expressed in aerobically grown cultures at pH values below 5.3 . The nature of the nitrogen and the carbon sources had no effect on antigen expression except by indirect changes on the pH of the culture medium; similarly, growth in 0.3 M NaCl did not result in antigen expression . The induction of MAC 203 antigen by low-oxygen or low-pH culture conditions is discussed in the context of tissue-specific expression within the legume root nodule.

J Bacteriol, 1989 Sep, 171(9), 4537 - 42
Developmental regulation of a Rhizobium cell surface antigen during growth of pea root nodules; VandenBosch KA et al.; A monoclonal antibody, AFRC MAC 203, was used to examine the expression of a nodule-induced cell surface antigen associated with lipopolysaccharide in Rhizobium leguminosarum bv . viciae 3841 . Silver-enhanced immunogold-labeled tissue sections revealed that, in very young tissues of pea root nodules, the nodule-induced form of lipopolysaccharide antigen was not expressed either by rhizobia in the infection thread or by bacteria recently released into the plant cell cytoplasm . In the more mature regions of the nodule, the antigen was expressed by membrane-enclosed bacteroids, including immature forms that had not yet expressed the enzyme nitrogenase and were not yet Y shaped . Immunogold labeling of thin sections revealed that the MAC 203 antigen, but not the nitrogenase, was also expressed by bacteria in infection threads situated in and between bacteroid-containing plant cells in mature nodule tissue.

J Bacteriol, 1989 Sep, 171(9), 4686 - 93
Subcellular localization of the nodD gene product in Rhizobium leguminosarum; Schlaman HR et al.; In Rhizobium strains the transcription of symbiosis plasmid-localized nod genes, except nodD, is induced by plant flavonoids and requires the nodD gene product . In order to localize NodD protein in R . leguminosarum, a NodD protein-specific antiserum was raised against a lacZ'-'nodD gene fusion product . Using these antibodies, we determined that the NodD protein is located exclusively in the cytoplasmic membrane of wild-type R . leguminosarum biovar viciae cells . This localization is independent of the presence of inducers . In a Rhizobium strain that overproduced the NodD protein, the protein was present both in the cytoplasmic membrane and the cytosol, indicating an influence of the protein abundance on its ultimate subcellular localization . It was estimated that 20 to 80 molecules of NodD protein were present per wild-type Rhizobium cell . A model which combines the localization and the DNA-binding properties of the NodD protein as well as the observed association of flavonoids with the cytoplasmic membrane is discussed.

J Bacteriol, 1989 Sep, 171(9), 5087 - 94
Regulation of glutamine synthetase II activity in Rhizobium meliloti 104A14; Shatters RG et al.; Most rhizobia contain two glutamine synthetase (GS) enzymes: GSI, encoded by glnA, and GSII, encoded by glnII . We have found that WSU414, a Rhizobium meliloti 104A14 glutamine auxotroph derived from a glnA parental strain, is an ntrA mutant . The R . meliloti glnII promoter region contains DNA sequences similar to those found in front of other genes that require ntrA for their transcription . No GSII was found in the glnA ntrA mutant, and when a translational fusion of glnII to the Escherichia coli lacZ gene was introduced into WSU414, no beta-galactosidase was expressed . These results indicate that ntrA is required for glnII expression . The ntrA mutation did not prevent the expression of GSI . In free-living culture, the level of GSII and of the glnII-lacZ fusion protein was regulated by altering transcription in response to available nitrogen . No GSII protein was detected in alfalfa, pea, or soybean nodules when anti-GSII-specific antiserum was used.

J Bacteriol, 1989 Sep, 171(9), 5079 - 86
Isolation, characterization, and complementation of Rhizobium meliloti 104A14 mutants that lack glutamine synthetase II activity; Somerville JE et al.; The glutamine synthetase (GS)-glutamate synthase pathway is the primary route used by members of the family Rhizobiaceae to assimilate ammonia . Two forms of glutamine synthetase, GSI and GSII, are found in Rhizobium and Bradyrhizobium species . These are encoded by the glnA and glnII genes, respectively . Starting with a Rhizobium meliloti glnA mutant as the parent strain, we isolated mutants unable to grow on minimal medium with ammonia as the sole nitrogen source . For two auxotrophs that lacked any detectable GS activity, R . meliloti DNA of the mutated region was cloned and partially characterized . Lack of cross-hybridization indicated that the cloned regions were not closely linked to each other or to glnA; they therefore contain two independent genes needed for GSII synthesis or activity . One of the cloned regions was identified as glnII . An R . meliloti glnII mutant and an R . meliloti glnA glnII double mutant were constructed . Both formed effective nodules on alfalfa . This is unlike the B . japonicum-soybean symbiosis, in which at least one of these GS enzymes must be present for nitrogen-fixing nodules to develop . However, the R . meliloti double mutant was not a strict glutamine auxotroph, since it could grow on media that contained glutamate and ammonia, an observation that suggests that a third GS may be active in this species.

Mol Gen Genet, 1989 Sep, 218(3), 536 - 44
Characterization and nucleotide sequence of a novel gene fixW upstream of the fixABC operon in Rhizobium leguminosarum; Hontelez JG et al.; On the Rhizobium leguminosarum PRE sym plasmid, fixABC and a novel gene fixW were identified upstream of the regulatory gene nifA . The molecular masses of FixABC, 29, 44 and 50 kDa respectively, were estimated by polyacrylamide gel electrophoresis (PAGE) and of FixW, 25 kDa, by PAGE and nucleotide sequencing . Hybridization studies using bacteroid mRNA as a probe showed that fixABC is one operon which can be transcribed independently of fixW . Nucleotide sequencing revealed that both fixW and fixA are preceded by a nif consensus promoter . The fixA promoter partly overlaps the 3'-terminal coding region of fixW, indicating that readthrough from fixW into fixA is possible . Two open reading frames, ORF71 and ORF79, precede fixW and form one operon with fixW . ORF71 contains sequences homologous to the fixA promoter and 5'-terminal coding region . One more duplication of fixA sequences was detected, also located within the sym plasmid nif/fix clusters . One duplication of fixW sequences was found . No fixW homologue could be found in other nitrogen fixing organisms except in a number of R . leguminosarum strains.

Mol Microbiol, 1989 Sep, 3(9), 1183 - 9
Cloning a genomic region required for a high-affinity iron-uptake system in Rhizobium meliloti 1021; Gill PR Jr et al.; A collection of transposon-induced mutants of Rhizobium meliloti 1021 defective in siderophore-mediated iron assimilation were obtained and classified as biosynthetic, transport or regulatory . Several of the mutations were cloned and the adjacent sequences were used to acquire complementing DNA from the wild type . A single genomic region of about 35kb complemented all of the mutants deficient in production of the siderophore.

J Bacteriol, 1989 Sep, 171(9), 4821 - 30
Rhizobium leguminosarum exopolysaccharide mutants: biochemical and genetic analyses and symbiotic behavior on three hosts; Diebold R et al.; Ten independently generated mutants of Rhizobium leguminosarum biovar phaseoli CFN42 isolated after Tn5 mutagenesis formed nonmucoid colonies on all agar media tested and lacked detectable production of the normal acidic exopolysaccharide in liquid culture . The mutants were classified into three groups . Three mutants harbored Tn5 insertions on a 3.6-kilobase-pair EcoRI fragment and were complemented to have normal exopolysaccharide production by cosmids that shared an EcoRI fragment of this size from the CFN42 genome . The Tn5 inserts of five other mutants appeared to be located on a second, slightly smaller EcoRI fragment . Attempts to complement mutants of this second group with cloned DNA were unsuccessful . The mutations of the other two mutants were located in apparently adjacent EcoRI fragments carried on two cosmids that complemented those two mutants . The latter two mutants also lacked O-antigen-containing lipopolysaccharides and induced underdeveloped nodules that lacked nitrogenase activity on bean plants . The other eight mutants had normal lipopolysaccharides and wild-type symbiotic proficiencies on bean plants . Mutants in each of these groups were mated with R . leguminosarum strains that nodulated peas (R . leguminosarum biovar viciae) or clovers (R . leguminosarum biovar trifolii) . Transfer of the Tn5 mutations resulted in exopolysaccharide-deficient R . leguminosarum biovar viciae or R . leguminosarum biovar trifolii transconjugants that were symbiotically deficient in all cases . These results support earlier suggestions that successful symbiosis with peas or clovers requires that rhizobia be capable of acidic exopolysaccharide production, whereas symbiosis with beans does not have this requirement.

Mol Plant Microbe Interact, 1989 Sep-Oct, 2(5), 224 - 32
Isolation and characterization of a Rhizobium loti gene required for effective nodulation of Lotus pedunculatus; Ward LJ et al.; A Rhizobium loti gene required for effective invasion of the host Lotus pedunculatus has been identified by transposon Tn5 mutagenesis . Cosmids that complemented a previously isolated mutation (239) at this invasion (inv) locus were identified by in planta complementation and used to construct a physical map of the gene region . The insertion site of Tn5 in PN239 was mapped to a 7.5-kb EcoRI fragment, which complemented the mutation when subcloned into pLAFR1 . Further Tn5 mutagenesis of the 7.5-kb fragment was carried out in Escherichia coli using bacteriophage lambda 467, and the mutations homogenotized into R . loti NZP2037 . Three additional Fix- mutations were isolated, and these were found to map adjacent to the position of the original mutation in strain PN239 . All the other Tn5 insertions isolated in the 7.5-kb fragment gave a Fix+ phenotype on L . pedunculatus . Electron microscopic examination of the L . pedunculatus nodules induced by the isolated Fix- mutants showed that bacteria were either blocked in release from the infection threads or were unable to undergo normal bacteroid development . The inv locus as defined by the Tn5 insertions was sequenced, and a single open-reading frame (ORF) of 576 bp, corresponding to a polypeptide of 21.3 kDa, was identified . The position and orientation of this ORF were consistent with those of the isolated Tn5 Fix- insertions.

Plant Mol Biol, 1989 Sep, 13(3), 319 - 25
Regulation of plant genes specifically induced in nitrogen-fixing nodules: role of cis-acting elements and trans-acting factors in leghemoglobin gene expression; de Bruijn FJ et al.; Transgenic alfalfa plants harboring a gene fusion between the soybean leghemoglobin (lbc3) promoter region and the chloramphenicol acetyl transferase (cat) gene were used to determine the influence of rhizobial mutants on lb gene expression in nodules . The promoter region of the Sesbania rostrata glb3 (Srglb3) leghemoglobin gene was examined for the presence of conserved motifs homologous to binding site 1 and 2 of the soybean lbc3 promoter region, found to interact with a trans-acting factor present in soybean nodule nuclear extracts (Jensen EO, Marcker KA, Schell J, de Bruijn FJ, EMBO J 7:1265-1271, 1988) . Subfragments of the S . rostrata glb3 (Srglb3) promoter region were examined for binding to trans-acting factors from nodule nuclear extracts . In addition to the binding sites previously identified (Metz BA, Welters P, Hoffmann HJ, Jensen EO, Schell J, de Bruijn FJ, Mol Gen Genet 214: 181-191), several other sites were found to interact with trans-acting factors . In most cases the same trans-acting factor(s) were shown to be involved . One fragment (202) was found to bind specifically to a different factor (protein) which was extremely heat-resistant (100 degrees C) . The appearance of this factor was shown to be developmentally regulated since the expected protein-DNA complexes were first observed around 12 days after infection, concomitant with the production of leghemoglobin proteins . Fragments of the Srglb3 5' upstream region were fused to the beta-glucuronidase reporter gene with its own CAAT and TATA box region or those of the cauliflower mosaic virus 35S and nopaline synthase (nos) promoters.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1989 Aug 25, 264(24), 14039 - 42
Isolation and characterization of the unusual lipopolysaccharide component, 2-amino-2-deoxy-2-N-(27-hydroxyoctacosanoyl)-3-O-(3-hydroxy- tetradecanoyl)-gluco-hexuronic acid, and its de-O-acylation product from the free lipid A of Rhizobium trifolii ANU843; Hollingsworth RI et al.; Two new, unusual lipid A components have been isolated and characterized from the free lipid A of Rhizobium trifolii ANU843 . 2-Amino-2-deoxy-2-N-(27-hydroxyoctacosanoyl)-3-O-(3-hydroxy- tetradecanoyl)-gluco-hexuronic acid and its de-O-acylation product were purified from the chloroform/methanol extract of a mild acid hydrolysate of the lipopolysaccharide by chromatography on C18 reverse-phase columns and layers . The compositions of the two compounds were determined by releasing the acyl components by exhaustive acid-catalyzed methanolysis and identifying them as their methyl esters by gas chromatography and gas chromatography/mass spectrometry . The sugar component was identified by converting it to the alditol acetate derivative of glucosamine in a two-step reduction and identifying it as such by gas chromatography/mass spectrometry . The linkages of the fatty acyl components to the sugar residue and the configuration of the sugar component was confirmed by 1H and 13C NMR spectroscopy . The complete structures of the two compounds were further confirmed by fast atom bombardment mass spectrometry . It is still unsure whether the de-O-acylated derivative was formed from the di-acyl compound by de-O-acylation during acid hydrolysis . These structures represent the first report of 2-amino-2-deoxy-gluco-hexuronic acid in the free lipid A of a Gram-negative bacterium and confirms our earlier contention (Hollingsworth, R.I., and Carlson, R . W . (1989) J . Biol . Chem . 264, 9000-9303) of the involvement of 27-hydroxyoctacosanoic acid in the structure of the lipopolysaccharide of Rhizobium trifolii ANU843.

J Bacteriol, 1989 Aug, 171(8), 4370 - 7
Accumulation of a nod gene inducer, the flavonoid naringenin, in the cytoplasmic membrane of Rhizobium leguminosarum biovar viciae is caused by the pH-dependent hydrophobicity of naringenin; Recourt K et al.; Most Sym plasmid-localized nodulation genes of Rhizobium leguminosarum bv . viciae are only expressed upon activation of the NodD protein by plant flavonoids, e.g., naringenin (S . A . J . Zaat, C . A . Wijffelman, H . P . Spaink, A . A . N . van Brussel, and B . J . J . Lugtenberg, J . Bacteriol, 169:198-204, 1987) . As part of a study on the mechanism of NodD protein activation, the mechanism of uptake and the intracellular fate of {3H}naringenin were studied . Naringenin was accumulated by Rhizobium cells without apparent metabolic conversion to an 80-fold-higher concentration in a process which did not require any of the other Sym plasmid-localized nod genes . Naringenin accumulation was nonsaturable, highly reversible, and not inhibited by the presence of other flavonoids or the metabolic inhibitors potassium cyanide, sodium azide, 2,4-dinitrophenol, and carbonyl cyanide m-chlorophenylhydrazone . These data indicate an accumulation mechanism without high affinity sites which does not use cellular energy . In vitro, naringenin has high affinity for the cytoplasmic membrane . This binding was pH dependent, very high at pH 5.7 and not present anymore at pH 9.7 . A similar pH dependency was found for the affinity of naringenin for the olive oil fraction of a biphasic olive oil-water system . pH-dependent changes in the UV spectrum indicate ionization of naringenin at high pH to a negatively charged form . Since it has recently been shown that the nodD gene product is located in the cytoplasmic membrane (H . R . M . Schlaman, H . P . Spaink, R . J . H . Okker, and B . J . J . Lugtenberg, J . Bacteriol., in press), our data are consistent with a model in which the un-ionized form of naringenin accumulates in the cytoplasmic membrane and activates, in a metabolically unaltered form, the NodD protein.

J Bacteriol, 1989 Aug, 171(8), 4425 - 35
Gene transfer system for Rhodopseudomonas viridis; Lang FS et al.; A gene transfer system for Rhodopseudomonas viridis was established which uses conjugation with Escherichia coli S17-I as the donor and mobilizable plasmids as vectors . Initially, plasmids of the incompatibility group P1 (pRK290 and pRK404) were used . The more effective shuttle vectors between E . coli and R . viridis, pKV1 and pKVS1, were derived from plasmid pBR322 and showed the highest conjugation frequency (10(-2} thus far demonstrated in purple bacteria . It was also demonstrated that Rhizobium meliloti can be used as a donor for conjugation with R . viridis . From a genomic cosmid library of R . viridis constructed in the vector pHC79, clones that coded for subunits H (puh operon), L, M and cytochrome c (puf operon) of the photosynthetic reaction center were isolated and characterized . For linkage of the two operons on the genome, cosmids that overlapped with the operon-carrying clones were identified . The relative positions of the two operons could not be determined, but the operons must be more than 100 kilobase pairs apart . Thus, the genomic organization of the reaction center in R . viridis is different from that of Rhodobacter capsulatus, for which a distance of about 39 kilobase pairs was determined . From a spontaneous mutant of R . viridis that is resistant to the herbicide terbutryn, the puf operon was cloned in pKVS1 and transferred by conjugation into R . viridis wild-type cells . The resulting exconjugants were resistant to the herbicide, which demonstrated that the puf operon on pKVS1 constructions was functionally expressed in R . viridis.

Plant Mol Biol, 1989 Aug, 13(2), 163 - 74
Additional nodulation genes on the Sym plasmid of Rhizobium leguminosarum biovar viciae; Canter Cremers H et al.; A Rhizobium leguminosarum biovar viciae strain lacking a 40 kb DNA region of the Sym plasmid pRL1IJ to the left (3' side) of gene nodE failed to nodulate Vicia sativa plants . Therefore this DNA region was investigated for the presence of additional nodulation genes . Complementation experiments indicated that the DNA region to the left (3' side) of nodE is functionally homologous between R . leguminosarum bv . viciae and R . leguminosarum bv . trifolii . In this DNA region, three nodulation genes were identified, nodT, nodM and nodL . TnphoA insertions in the nodT gene, about 4.5 kb to the left of nodE, lead to a delay in nodulation on Trifolium subterraneum, but not on V . sativa plants . TnphoA insertions in gene nodM have no detectable influence on nodulation . Finally, TnphoA insertions in the nodL gene affected nodulation so that only rarely nodules were induced on the inoculated plants . The nucleotide sequence of this gene is presented . On the basis of the sequence a membrane integrated protein is predicted with a molecular weight of 20.1 kDa . Microscopical analysis of the infection process by nodL mutants indicate a role for nodL in maintaining the stability of the infection thread . Experiments using transcriptional lacZ fusions suggest that nodL belongs to the same transcriptional unit as nodF,E . Very low expression of nodL seems to be sufficient for biological activity.

J Gen Microbiol, 1989 Aug, 135 ( Pt 8), 2329 - 34
Transposon mutagenesis and complementation of the fructokinase gene in Rhizobium leguminosarum biovar trifolii; McLaughlin RE et al.; Transposon Tn5 was used to generate a fructokinase mutation in Rhizobium leguminosarum biovar trifolii BAL . The section of the genome containing Tn5 was cloned into the EcoRI site of the vector pHC79 and isolated by direct selection on medium containing kanamycin and tetracycline . Total EcoRI digestion was used to obtain a single fragment containing Tn5 and flanking DNA sequences . The flanking DNA was used as a probe to isolate an intact fructokinase gene from a pLAFR1 cosmid clone bank of the parental strain . A cosmid showing homology to the probe was tri-parentally conjugated into the fructokinase-negative strain, complementing the mutation . The complemented mutant exhibited the wild-type phenotype, with an increase in fructokinase production presumably due to multiple copies of the gene.

J Bacteriol, 1989 Aug, 171(8), 4154 - 61
Implication of nifA in regulation of genes located on a Rhizobium meliloti cryptic plasmid that affect nodulation efficiency; Sanjuan J et al.; We examined the contribution of a cryptic plasmid, pRmeGR4b, to the nodulation of Medicago sativa by strain GR4 of Rhizobium meliloti . A 905-base-pair PstI DNA fragment in pRmeGR4b was found to hybridize DNA of the R . meliloti fixA promoter region as a probe . Sequence analysis of the PstI fragment showed a 206-base-pair region displaying high homology with the DNA upstream of the RNA start points of the P1 and P2 symbiotic promoters . Putative nif promoter consensus sequences were conserved in this DNA segment . Expression of DNA downstream of the nif promoterlike sequence, monitored by beta-galactosidase activity of different lacZ fusions, was demonstrated to depend on a functional nifA gene, both in microaerobically free-living cells and in nodules . Individual transposon Tn3-HoHo1 insertions in this DNA region caused a reduced nodulation competitiveness . This new symbiotic region, occupying approximately 5 kilobases of pRmeGR4b DNA, was called nfe (nodule formation efficiency).

Plant Mol Biol, 1989 Aug, 13(2), 175 - 88
Analysis of the major inducers of the Rhizobium nodA promoter from Vicia sativa root exudate and their activity with different nodD genes; Zaat SA et al.; Root exudate of Vicia sativa contains 7 inducers for the nodA promoter of Rhizobium leguminosarum biovar viciae . Six of these inducers are flavanones . One inducer was identified as 3,5,7,3'-tetrahydroxy-4'-methoxyflavanone, and a second inducer most likely is 7,3'-dihydroxy-4'-methoxyflavanone . The inducing activity of these compounds and the other inducers depends on the nodD gene present in the test strains, which originated either from R . leguminosarum biovars viciae or trifolii, or from R . meliloti . Three inducers are 'common', three others almost exclusively induce the nodA promoter in the presence of the R . leguminosarum biovar viciae nodD gene, and the last one is active with the noD genes of either R . leguminosarum biovar viciae or that of R . meliloti . Testing of a large number of flavonoids revealed two classes of structural features required for inducing ability: (i) features required for induction in general, and (ii), features restricting the inducing ability to (a) specific nodD gene(s) . These features are discussed in relation to current models of the process of nodD-mediated transcription activation of the inducible nod genes.

Mol Gen Genet, 1989 Aug, 218(2), 315 - 22
Differential regulation of soybean chalcone synthase genes in plant defence, symbiosis and upon environmental stimuli; Wingender R et al.; Four independent recombinant lambda clones hybridizing to parsley chalcone synthase (CHS) cDNA were isolated from a soybean (Glycine max) genomic library . Restriction fragment length polymorphism (RFLP) analysis indicated that the CHS gene family comprises six members . The CHS genes were found to be clustered with three genes on a 10 kb segment and pairs on others . DNA sequences of the 5'-, the coding-, and the 3' untranslated regions were determined for three different genes . A consensus alignment of the 5' regions revealed extensive homology between them for up to 150 bp upstream of the TATA box . Developmental regulation of CHS was observed in uninfected and in rhizobium-infected roots . Regulation at the level of transcription by different stimuli was investigated in the root, stem and cotyledons of soybean seedlings . Our results suggest a co-operative induction of CHS genes by wounding and elicitor treatment of cotyledons . The most rapid transcript accumulation, however, was observed in roots and stems . The induction of CHS genes by light was found to be UV dependent . A possible involvement of different members of the CHS gene family in response to elicitor versus UV treatment was analysed by the use of gene specific probes, and unexpectedly revealed that only CHS 1 transcription was induced by either elicitor or UV treatment of seedlings.

J Bacteriol, 1989 Jul, 171(7), 3961 - 7
Lipopolysaccharide mutants of Rhizobium meliloti are not defective in symbiosis; Clover RH et al.; Mutants of Rhizobium meliloti selected primarily for bacteriophage resistance fall into 13 groups . Mutants in the four best-characterized groups (class A, lpsB, lpsC, and class D), which map to the rhizobial chromosome, appear to affect lipopolysaccharide (LPS) as judged by the reactivity with monoclonal antibodies and behavior on sodium dodecyl sulfate-polyacrylamide gels of extracted LPS . Mutations in all 13 groups, in an otherwise wild-type genetic background, are Fix+ on alfalfa . This suggests that LPS does not play a major role in symbiosis . Mutations in lpsB, however, are Fix- in one particular genetic background, evidently because of the cumulative effect of several independent background mutations . In addition, an auxotrophic mutation evidently equivalent to Escherichia coli carAB is Fix- on alfalfa.

Appl Environ Microbiol, 1989 Jul, 55(7), 1730 - 4
Influence of soil variables on in situ plasmid transfer from Escherichia coli to Rhizobium fredii; Richaume A et al.; A model system was established to determine whether intergeneric plasmid transfer occurs in soil and how various soil variables affect the rate of plasmid transfer . The donor bacterium, Escherichia coli HB101 carrying plasmid pBLK1-2 (pRK2073::Tn5), and the recipient bacterium, Rhizobium fredii USDA 201, were inoculated into a sterile Adelphia fine-sandy-loam soil . Transconjugants were enumerated by direct plating on antibiotic-amended HM {N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; 2-(N-morpholino) ethanesulfonic acid} salts medium . Randomly chosen transconjugants were verified by serological typing and Southern hybridization with a Tn5 gene probe . The maximum transfer frequency was observed after 5 days of incubation (1.8 x 10(-4) per recipient) . The influences of clay (0 to 50% addition), organic matter (0 to 15% addition), soil pH (4.3 to 7.25), soil moisture (2 to 40%), and soil incubation temperature (5 to 40 degrees C) on plasmid transfer were examined . Maximum transfer frequencies were noted at a clay addition of 15%, an organic matter addition of 5%, a soil pH of 7.25, a soil moisture content of 8%, and a soil incubation temperature of 28 degrees C . These results indicate that intergeneric plasmid transfer may occur in soil and that soil variables may significantly affect the rate of transfer.

J Bacteriol, 1989 Jul, 171(7), 3926 - 32
Cloning and sequencing of the gltX gene, encoding the glutamyl-tRNA synthetase of Rhizobium meliloti A2; Laberge S et al.; The gltX gene, coding for the glutamyl-tRNA synthetase of Rhizobium meliloti A2, was cloned by using as probe a synthetic oligonucleotide corresponding to the amino acid sequence of a segment of the glutamyl-tRNA synthetase . The codons chosen for this 42-mer were those most frequently used in a set of R . meliloti genes . DNA sequence analysis revealed an open reading frame of 484 codons, encoding a polypeptide of Mr 54,166 containing the amino acid sequences of an NH2-terminal and various internal fragments of the enzyme . Compared with the amino acid sequence of the glutamyl-tRNA synthetase of Escherichia coli, the N-terminal third of the R . meliloti enzyme was strongly conserved (52% identity); the second third was moderately conserved (38% identity) and included a few highly conserved segments, whereas no significant similarity was found in the C-terminal third . These results suggest that the C-terminal part of the protein is probably not involved in the recognition of substrates, a feature shared with other aminoacyl-tRNA synthetases.

Plant Mol Biol, 1989 Jul, 13(1), 1 - 12
Production of root hair deformation factors by Rhizobium meliloti nodulation genes in Escherichia coli: HsnD (NodH) is involved in the plant host-specific modification of the NodABC factor; Banfalvi Z et al.; The role of the hsnD (nodH) gene in the determination of the host-specific nodulation ability of Rhizobium meliloti was studied by expressing the common nodulation genes (nodABC) with or without the hsnD gene in Escherichia coli and testing for biological activity on various leguminous plants . In this way, four categories of plants were established . Upon infection with E . coli carrying the nodABC construct, root hair deformation (Had) was detected on clovers while the hsnD gene was additionally needed for the elicitation of the same response on alfalfa and sweet clover . A weak root hair deformation was seen on siratro by inoculation with E . coli harbouring the nodABC genes and was highly increased when hsnD was also introduced . Cowpea and Desmodium did not respond to any of the E . coli strains constructed . Exudates or cytosolic fractions of the respective E . coli derivatives elicited the same root hair deformation as the intact bacteria . These data indicate that not only the nodABC gene products but also the hsnD product are involved in the synthesis of Had factors . Subclones expressing only the nodA, nodB, or nodC genes or the same genes in pairs (nodAB, nodBC, nodAC) did not provide a compound with activity comparable to the NodABC factor, suggesting that all three genes are required for the production of the Had factor which is active on clover . Coinoculation of alfalfa plants with two strains of E . coli, one carrying the nodABC genes and the other expressing only hsnD, or combining exudates or cytosolic fractions from these strains did not result in root hair deformation on alfalfa . These data indicate that the HsnD protein itself or its product is not an additional alfalfa-specific extracellular signal but more likely is enzymatically involved in the modification of the basic compound determined by the nodABC genes.

Mol Microbiol, 1989 Jul, 3(7), 943 - 55
The nifA gene product from Rhizobium leguminosarum biovar trifolii lacks the N-terminal domain found in other NifA proteins; Iismaa SE et al.; The nifA gene has been identified between the fixX and nifB genes in the clover microsymbiont Rhizobium leguminosarum biovar trifolii (R.I . bv . trifolii) strain ANU843 . Expression of the nifA gene is induced in the symbiotic state and site-directed mutagenesis experiments indicate that nifA expression is essential for symbiotic nitrogen fixation . Interestingly, the predicted R.I . bv . trifolii NifA protein lacks an N-terminal domain that is present in the homologous proteins from R.I . bv . viciae, Rhizobium meliloti, Bradyrhizobium japonicum, Klebsiella pneumoniae and all other documented NifA proteins . This indicates that this N-terminal domain is not essential for NifA function in R.I . bv . trifolii.

Mol Microbiol, 1989 Jul, 3(7), 879 - 89
Characterization of Rhizobium phaseoli Sym plasmid regions involved in nodule morphogenesis and host-range specificity; Cevallos MA et al.; Two nodulation regions from the symbiotic plasmid (pSym) of Rhizobium phaseoli CE-3 were identified . The two regions were contained in overlapping cosmids pSM927 and pSM991 . These cosmids, in a R . phaseoli pSym-cured strain background, induced ineffective nodules on Phaseolus vulgaris roots . Transconjugants of Rhizobium meliloti harbouring pSM991 induced nodule-like structures on bean roots, suggesting that this cosmid contains host-range determinants . Analysis of deletions and insertional mutations in the sequences of pSM991 indicated that the genes responsible for the induction and development of nodules in P . vulgaris are organized in two regions 20 kb apart . One region, located in a 6.8 kb EcoRI fragment, includes the common nodABC genes . The other region, located in a 3.5 kb EcoRI fragment, contains information required for host-range determination.

FEMS Microbiol Lett, 1989 Jul 1, 51(1), 15 - 19
Comparative study of the symbiotic plasmid DNA in free living bacteria and bacteroids of Rhizobium leguminosarum; Vierny C et al.; The symbiotic plasmid (pSym) DNA present in bacteroids of strain RCR1001 of Rhizobium leguminosarum biovar viceae has been compared qualitatively and quantitatively with that present in free living bacteria by hybridization experiments with appropriate probes . A decrease in the relative amount of pSym DNA was observed in bacteroids as compared to bacteria . No rearrangements of the symbiotically expressed pSym borne genes were detected in bacteroids.

J Bacteriol, 1989 Jul, 171(7), 4045 - 53
Symbiotic properties of rhizobia containing a flavonoid-independent hybrid nodD product; Spaink HP et al.; A hybrid nodD gene consisting of 75% of the nodD1 gene of Rhizobium meliloti at the 5' end and 27% of the nodD gene of Rhizobium trifolii at the 3' end activates the six tested inducible nod promoters of Rhizobium leguminosarum, R . trifolii, or R . meliloti to maximal levels, even in the absence of flavonoids . In strains containing such a constitutive activating nodD gene, transcription of nod genes started at the same site as in flavonoid-induced strains containing a wild-type nodD gene . In contrast to heterologous wild-type nodD products, the constitutive activating nodD gene does not cause a limitation of the host range . Furthermore, R . leguminosarum, R . trifolii, and R . meliloti strains containing the constitutive activating nodD gene induce (pseudo) nodules on tropical leguminous plants . Comparison of the symbiotic properties of rhizobia containing the constitutive nodD hybrid gene with those of rhizobia containing various wild-type nodD genes indicates that the activation of the nodD product by flavonoids is of crucial importance during the process of infection thread formation and, surprisingly, also during nitrogen fixation.

Mol Plant Microbe Interact, 1989 Jul-Aug, 2(4), 181 - 94
Nucleotide sequence and protein products of two new nodulation genes of Rhizobium meliloti, nodP and nodQ; Schwedock J et al.; Previous studies had suggested the existence of nodulation (nod) genes downstream of nodG in Rhizobium meliloti strain 1021 . We have established the DNA sequence and analyzed the translation products of the genes located in this position . Computer analysis of the DNA sequence revealed a number of overlapping putative open-reading frames (ORFs), so we constructed several clones that contained either full-length or truncated ORFs . The protein products of these clones were expressed in both R . meliloti and Escherichia coli in vitro transcription-translation systems . These assays unambiguously defined the expressed ORFs, which we named nodP and nodQ . In addition, we found homology to these genes, via Southern hybridizations, elsewhere in the genome of R . meliloti strain 1021, and in other species of Rhizobium . The nodP gene also displayed homology to E . coli . A computer search revealed significant homology between NodQ and the GDP binding domain of elongation factor Tu (EF-Tu).

J Bacteriol, 1989 Jul, 171(7), 3989 - 95
Evidence for divalent cation (Ca2+)-stabilized oligomeric proteins and covalently bound protein-peptidoglycan complexes in the outer membrane of Rhizobium leguminosarum; de Maagd RA et al.; Two unusual characteristics of some outer membrane proteins of Rhizobium leguminosarum are described . First, most of the major outer membrane proteins could only be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after lysozyme treatment of the isolated cell envelopes, suggesting a very strong, possibly covalent, interaction of these proteins with the peptidoglycan . These peptidoglycan-associated outer membrane proteins belonged to two distinct groups of immunologically related proteins, groups II and III, as defined by typing with monoclonal antibodies . As members of both groups of proteins could be radioactively labeled by growing cells in the presence of N-{3H}acetylglucosamine, we propose that variation in the apparent molecular weight of the antigens within each group is caused by varying numbers of peptidoglycan subunit residues on only two or three different outer membrane proteins . Second, group III outer membrane proteins, with masses of 35 to 46 kilodaltons, formed oligomers stabilized by divalent cations which resisted complete denaturation in 2% sodium dodecyl sulfate at 100 degrees C . Reconstitution experiments showed that of the divalent cations tested, Ca2+ and, to a lesser extent, Mn2+ and Sr2+ were the best stabilizers.

J Biol Chem, 1989 Jun 5, 264(16), 9300 - 3
27-Hydroxyoctacosanoic acid is a major structural fatty acyl component of the lipopolysaccharide of Rhizobium trifolii ANU 843; Hollingsworth RI et al.; A new hydroxylated, very long-chain fatty acid has been isolated and characterized from the lipopolysaccharide (LPS) of Rhizobium trifolii ANU 843 . The lipid A of the organism was degraded by mild alkali and borohydride and the products methylated, peracetylated, and fractionated on a C18 reverse-phase column . The major lipid fraction was reduced with lithium triethylborohydride, methylated, peracetylated, and subjected to thin layer chromatography . The methylated peracetylated acid and the reduced diacetylated diol (1,27-dihydroxyoctacosane diacetate) were isolated and characterized by mass spectrometry and 1H NMR spectroscopy using homonuclear decoupling . The identity and linkage of the new fatty acid in the lipopolysaccharide was confirmed by 1H NMR spectroscopy of purified lipid A fractions and similar NMR studies of lipid A after acylation by phenylisocyanate . In the native LPS, the 27-hydroxy C-28 fatty acid is acylated at the 27-hydroxy position by other 3-hydroxy fatty acids . About 50% of the total fatty acid content of the LPS of R . trifolii ANU 843 is 27-hydroxyoctacosanoic acid . This oxyacyloxy structure involving 27-hydroxyoctacosanoic appears to be the major structural feature of the lipid A of this organism.

J Biol Chem, 1989 Jun 5, 264(16), 9294 - 9
A new core tetrasaccharide component from the lipopolysaccharide of Rhizobium trifolii ANU 843; Hollingsworth RI et al.; A second core oligosaccharide fragment has been isolated and characterized from the lipopolysaccharide (LPS) of Rhizobium trifolii ANU 843 . The oligosaccharide is a tetrasaccharide composed of galactose, galacturonic acid, mannose, and 3-deoxy-D-manno-2-octulosonic acid . The mannose residue is alpha-linked to the 4-position of 3-deoxy-D-manno-2-octulosonic acid and the galacturonic acid residue is alpha-linked to the 6-position of mannose . The galactose residue, which is acetylated at the 4-position, is attached to the 4-position of mannose by an alpha-linkage . All of the aldoses are in the pyranose form . The composition of the tetrasaccharide was determined by gas-liquid chromatography of the alditol acetate derivatives of the component monosaccharides . The configuration of anomeric linkages was determined by 1H NMR spectroscopy . Fast atom bombardment-mass spectrometry (FAB-MS) was performed on acetylated, per(trideutero)acetylated and underivatized tetrasaccharide giving sequence information in addition to information on the residue which was acetylated . Similar studies were performed on the oligosaccharide after reduction with sodium cyanoborohydride and peracetylation with labeled and unlabeled acetic anhydride as before . Further linkage and sequence analysis was obtained from methylation analysis, and from electron impact mass spectrometry of the per(trideutero)acetylated oligosaccharide and from collision-induced dissociation fast atom bombardment tandem mass spectrometry using linked scans at constant B/E on the cyanoborohydride-reduced, per (trideutero)acetylated oligosaccharide . The exact location of the acetyl group was deduced from 1H NMR double resonance experiments in conjunction with mass spectrometric data.

Microbiologia, 1989 Jun, 5(1), 13 - 23
{Utilization of nitrate by bacteroids and cytosol of nodules formed by Rhizobium leguminosarum}; Fernandez-Lopez M et al.; Nitrite production by nodules and roots of pea plants (Pisum sativum L., cultivar Alaska) inoculated with Rhizobium leguminosarum strain 3855 has been studied . Nitrate reductase (NR) activity and nitrite reductase (NiR) activity of the bacteroidal and cytosolic fractions of the nodules were also determined, as well as the nitrite content of the nodules cytosol . Nitrite production by nodules and roots from plants treated with 5 mM KNO3 was higher than that of nodules and roots from plants not treated with nitrate, and regardless of the nitrate treatment, nitrite production increased with the incubation period . The presence of nitrate, propanol or both compounds in the incubation mixtures significantly increased the nitrite production by nodules and roots . Nitrite reductase activity was detected in fresh by isolated bacteroids of R . leguminosarum strain 3855, although the presence of nitrate reductase activity could not be detected both in bacteroids of nodules isolated from plants treated or not with 5 mM KNO3 . After isolation, when bacteroids were incubated in a mixture with nitrate, nitrate reductase activity developed after incubation for 12 h . Consequently, there was an increase in nitrite reductase activity, which resulted in the disappearance of the nitrite previously accumulated in the incubation medium . Nitrate utilization by bacteroids was not detected until 5 h from the beginning of the incubation period . Since the presence of chloramphenicol or rifampicin in the incubation medium prevented the development of the nitrate reductase activity, such activity was induced in bacteroids . Nitrite content and nitrate reductase and nitrite reductase activities of the cytosol from nodules of pea plants treated or not with 5 mM KNO3 varied with the buffer used for nodules homogenization . However, no nitrite was found when nodules were homogenized with ethanol, what indicates that nitrite accumulation in the cytosol occurs during the homogenization process of the nodules.

J Bacteriol, 1989 Jun, 171(6), 3354 - 65
The central domain of Rhizobium meliloti NifA is sufficient to activate transcription from the R . meliloti nifH promoter; Huala E et al.; The Rhizobium meliloti nifA product (NifA) shares extensive homology in its central region and at its C-terminal end with Rhizobium leguminosarum DctD and with NtrC from several species . All three proteins are transcriptional activators of NtrA (RpoN)-RNA polymerase-dependent promoters . Several large deletions of R . meliloti NifA were constructed to investigate the role of the conserved and divergent domains of NifA in transcriptional activity and posttranscriptional regulation by oxygen . The ability of NifA expressed from the Escherichia coli lacZ promoter to activate the R . meliloti nifH promoter in E . coli and R . meliloti was tested under a range of defined atmospheric oxygen partial pressures . Deletion of the divergent N-terminal domain of NifA had little effect on NifA activity and no effect on oxygen sensitivity . Deletion of the conserved C-terminal helix-turn-helix motif of NifA did not eliminate NifA-dependent activation of the nifH promoter, although it did decrease NifA activity about twofold in E . coli and 10-fold in R . meliloti . A NifA carrying both the N-terminal and C-terminal deletions and consisting of only the central highly conserved domain and 50 divergent amino acids retained the ability to activate transcription from the nifH promoter . The transcriptional activity of the conserved central domain is consistent with the prediction that the core domain is the part of NifA which interacts with the transcriptional machinery to stimulate transcription.

J Bacteriol, 1989 Jun, 171(6), 3471 - 8
Rhizobium meliloti anthranilate synthase gene: cloning, sequence, and expression in Escherichia coli; Bae YM et al.; We determined the DNA sequence of the Rhizobium meliloti gene encoding anthranilate synthase, the first enzyme of the tryptophan pathway . Sequences similar to those seen for the two subunits of the enzyme as found in all other procaryotic species studied are present in a single open reading frame of 729 codons . This apparent gene fusion joins the C terminus of the large subunit (TrpE) to the N terminus of the small subunit (TrpG) through a short connecting segment . We designate the fused gene trpE(G) . The gene is flanked by a typical rho-independent terminator at the 3' end and a complex regulatory region at the 5' end resembling those of operons under transcriptional attenuation control . The location of the promoter was determined by S1 nuclease protection, using Rhizobium mRNA . Although this promoter was inactive in Escherichia coli, mutations eliciting activity were easily obtained . One of these was a C----T change at position -9 in the -10 region . The +1 position of the mRNA is the first base of the initiation codon of the leader peptide, implying that unlike trpE(G), which has a normal Shine-Dalgarno sequence, the leader peptide gene lacks a ribosome-binding site.

Mol Microbiol, 1989 Jun, 3(6), 813 - 23
Analysis of C4-dicarboxylate transport genes in Rhizobium meliloti; Yarosh OK et al.; A 5.1 kbp DNA fragment was isolated which complemented C4-dicarboxylate transport mutants (dct) of Rhizobium meliloti . Characterization of this fragment by subcloning, transposon mutagenesis, and complementation analysis revealed three loci, designated dctA, dctB, and dctD . TnphoA-generated alkaline phosphatase fusions to dctA suggested that this gene encodes the structural transport protein and allowed the determination of its direction of transcription . Analysis of the fusions in various mutant backgrounds demonstrated that dctB, dctD, and ntrA products are required for dctA expression . The dctA fusion was constitutively expressed in a dctA mutant background, but was not expressed in dctA dctB or dctA dctD double mutants . This suggests that the constitutive expression in a dctA mutant background is mediated through dctB and dctD . Three independent second-site Dct+ revertant mutations in ntrA mutant strains mapped to the dct locus . Succinate transport in these revertant strains was constitutive, whereas in the wild type, succinate transport was inducible . These results are consistent with the direct requirement of the ntrA gene product for dctA expression . Alfalfa plants inoculated with the dctB and dctD mutants showed reduced nitrogen-fixing activity . Nodules induced by dctA mutants failed to fix nitrogen . These symbiotic phenotypes are consistent with previous suggestions that dctA expression in bacteroids can occur independently of dctB and dctD.

Mol Microbiol, 1989 Jun, 3(6), 745 - 55
The Rhizobium meliloti host range nodQ gene encodes a protein which shares homology with translation elongation and initiation factors; Cervantes E et al.; The Rhizobium meliloti nod region IIb is involved in host-range determination: (i) the presence of region IIb is necessary for transfer of alfalfa root hair curling ability to Rhizobium leguminosarum biovar trifolii; (ii) a mutation in region IIb extends the R . meliloti infection host range to Vicia sativa nigra; (iii) dominance of R . meliloti nod genes over R . leguminosarum biovar viciae nod genes is abolished by mutations in region IIb . The nucleotide sequence of this region has been determined . Genes corresponding to the two open reading frames identified are designated nodP and nodQ . The predicted amino acid sequence of the NodQ protein shows homology with translation initiation and elongation factors . The consensus sequence involved in the GTP-binding domain is conserved.

J Bacteriol, 1989 Jun, 171(6), 3324 - 30
Identification of Bradyrhizobium nod genes involved in host-specific nodulation; Deshmane N et al.; Three loci important for soybean nodulation by Bradyrhizobium japonicum were delimited by Tn5 mutagenesis on a 5.3-kilobase EcoRI fragment adjacent to the nodABC genes . Results of hybridization studies suggested that this region is conserved in Bradyrhizobium species but absent in all Rhizobium species . lacZ translational fusions of two of the loci contained in this region were found to be inducible by host-produced flavonoid chemicals via a mechanism requiring a functional nodD gene product . A mutation in one of the loci was found to result in an alteration of the host range of B . japonicum . This mutation appears to block nodulation at the step at which plant root cortical cell division is induced.

J Bacteriol, 1989 Jun, 171(6), 3218 - 27
Ntr-like promoters and upstream regulatory sequence ftr are required for transcription of a developmentally regulated Caulobacter crescentus flagellar gene; Mullin DA et al.; The flbG (hook operon or transcription unit II) and flaN (transcription unit I) operons of Caulobacter crescentus have a -12, -24 nucleotide sequence motif that is very similar to those of the Nif and Ntr promoters of enteric bacteria and Rhizobium spp . and a conserved ftr (flagellar gene transcription regulation) sequence, previously designated II-1 (D . A . Mullin, S . A . Minnich, L.-S . Chen, and A . Newton, J . Mol . Biol . 195:939-943, 1987) at approximately -100 . We have used site-directed mutagenesis to examine the role of these sequences in the transcriptional regulation of these periodically expressed flagellar genes . Mutations in the flbG promoter that removed the conserved GC at -12, -13, the GG at -24, -25, or an AC base pair at -18, -19 in the nonconserved sequence between the -12, -24 elements completely eliminated detectable transcription . Mutations at other positions resulted in either a slight decrease (position 26), no change (position 15), or an elevated level (position -16 or -19) of the flbG transcript . By contrast, most of these flbG promoter mutations resulted in greatly elevated levels of transcription from the opposing flaN operon . Similar experiments were used to confirm the location of the flaN promoter to a -12, -24 Nif and Ntr sequence motif . Deletion of all or part of the ftr element or point mutations in the sequence drastically reduced the level of flbG transcript and resulted in increased levels of the flaN transcript . Thus, the conserved sequences at -12 and -24 in flbG and flaN are required for transcription of these genes in vivo, and the ftr element is required for transcription of flbG . This analysis also suggested that the ftr sequence and sequences in the flbG promoter are required for the negative autoregulation of the flbG and flaN operons . We speculate that the flbG and flaN promoters and the ftr element interact in some way to mediate the negative control of these divergent transcription units.

Gene, 1989 May 15, 78(1), 111 - 20
Direct selection for curing and deletion of Rhizobium plasmids using transposons carrying the Bacillus subtilis sacB gene; Hynes MF et al.; We have constructed derivatives of the transposon Tn5 carrying the mob site (oriT) of plasmid RP4, and an nptI-sacB-sacR cassette {Ried and Collmer, Gene 57 (1987) 239-246} . The mob site, in conjunction with the antibiotic-resistance markers carried on the transposons, allows identification of transposon inserts in cryptic plasmids by mobilisation to other strains . The sacB-sacR genes allow direct selection for the loss or curing of plasmids, because only strains which no longer contain an active sacB gene are able to grow on media containing sucrose . We have tested these transposons in four strains of Rhizobium leguminosarum and two strains of Rhizobium meliloti, and have been able to demonstrate curing of several large cryptic plasmids, and generation of large deletions in many other plasmids . This method has enabled us to show that the R . leguminosarum plasmids pRL12JI and pR1eVF39f carry auxotrophic markers, and that the plasmid pR1eVF39c carries genes which affect colony morphology.

Genetics, 1989 May, 122(1), 7 - 18
A family of activator genes regulates expression of Rhizobium meliloti nodulation genes; Mulligan JT et al.; Nodulation (nod) gene expression in Rhizobium meliloti requires plant inducers and the activating protein product of the nodD gene . We have examined three genes in R . meliloti which have nodD activity and sequence homology . These three nodD genes are designated nodD1, nodD2 and nodD3, and have distinctive properties . The nodD1 gene product activates expression of the nodABC operon, as measured by a nodC-lacZ fusion or by transcript analysis, in the presence of crude seed or plant wash or the inducer, luteolin . The nodD3 gene product can cause a high basal (uninduced) level of nodC-lacZ expression and nodABC transcripts which is relatively unaffected by inducers . The effect of nodD3 is dependent on the presence of another gene, syrM (symbiotic regulator) . By primer extension analysis we determined that the transcription start site is the same for nodD1 plus luteolin or nodD3-syrM mediated expression of nodA and nodH mRNAs . syrM also enhances the expression of another symbiotically important trait, production of extracellular polysaccharide . This regulatory effect of syrM requires locus syrA, which is linked to nodD3 and syrM . The syrM-syrA mediated increase in polysaccharide production requires at least some of the previously identified exo genes and may be a parallel regulatory event to the syrM-nodD3 control of nod promoters.

Proc Natl Acad Sci U S A, 1989 May, 86(9), 3055 - 9
A second exopolysaccharide of Rhizobium meliloti strain SU47 that can function in root nodule invasion; Zhan HJ et al.; Rhizobium meliloti strain SU47 produces the calcofluor-binding exopolysaccharide, succinoglycan, that is required for alfalfa root nodule invasion . Strains derived from R . meliloti SU47 secreted an acidic exopolysaccharide, EPSb, that replaced succinoglycan in nodule invasion . EPSb, which has not formerly been identified among the Rhizobiaceae, consisted of the repeating unit 4,6-O-(1-carboxyethylidene)-alpha-D-Galp1----3(X-O-Ac)-beta-D-G lcp1----3 . EPSb synthesis occurred either in strains containing a mutation in a locus designated mucR or in strains with a recombinant cosmid pMuc . mucR mapped slightly counterclockwise from pyr49 on the chromosome, while pMuc contained genes mapping to the megaplasmid pRmeSU47b . In exoA, -F, and -H mutants, which are deficient in normal succinoglycan secretion and nodule invasion, a transposon Tn5 insertion in mucR or the presence of pMuc resulted in EPSb secretion and a restoration of nodule invasion on Medicago sativa and Melilotus alba . Mutants in exoB and exoC were incapable of succinoglycan and EPSb secretion as well as nodule invasion . A mutant that secreted succinoglycan but was incapable of EPSb secretion invaded nodules normally.

Mol Endocrinol, 1989 May, 3(5), 881 - 4
Human placental 17 beta-hydroxysteroid dehydrogenase is homologous to NodG protein of Rhizobium meliloti; Baker ME; The amino acid sequence of human placental 17 beta-hydroxysteroid dehydrogenase (17 beta-OH-steroid dehydrogenase) was found to be similar to that of the NodG protein of Rhizobium meliloti . The computer-based comparison score is 11.5 SD higher than that obtained with 2500 comparisons of randomized sequences of these proteins . The probability of getting such a score by chance is 6 x 10(-31) . 17 beta-OH-steroid dehydrogenase is also similar to Klebsiella aerogenes ribitol dehydrogenase and Escherichia coli glucitol-6-phosphate dehydrogenase . We propose that the steroid recognition site on 17 beta-OH-steroid dehydrogenase evolved from an ancestral recognition site for polyols such as ribitol and glucitol-6-phosphate.

Mol Plant Microbe Interact, 1989 May-Jun, 2(3), 97 - 106
Extension of host range of Rhizobium leguminosarum bv . trifolii caused by point mutations in nodD that result in alterations in regulatory function and recognition of inducer molecules; McIver J et al.; The positive activation of several nodulation genes in strain ANU843 of Rhizobium leguminosarum biovar trifolii is mediated by the product of the nodD gene and by the interaction of NodD with plant-secreted inducer and anti-inducer compounds . We have mutagenized the nodD gene of strain ANU843 with nitrosoguanidine and have found that the ability of the mutated nodD products to interact with inducer and anti-inducer compounds is affected by the amino acid sequence in at least two key regions, including a novel area between amino acids 77 and 123 . Several novel classes of mutants were recognized by phenotypic and molecular analysis of the mutant nodD genes . Classes 1 and 4 mutants were able to induce nodA expression independently of the addition of inducer and anti-inducer compounds and were unable to mediate autoregulation of the nodD gene . Classes 2 and 3 mutants retained several properties of the wild-type nodD, including the ability to interact with inducer and anti-inducer compounds and the capacity to autoregulate nodD expression . In addition, class 2 mutants showed an inducer-independent ability to mediate nodA expression to 10-fold higher levels over control strains . The class 3 mutant showed reactivity to compounds that had little or no inducing ability with the wild-type nodD . An alteration in NodD function was demonstrated with classes 2 and 3 mutants, which showed greatly enhanced ability to complement a Tn5-induced mutation in the nodD1 gene of strain NGR234 and to restore nodulation ability on the tropical legume siratro . Mutants of nodD possessing inducer-independent ability to activate nod gene expression (classes 1, 2, and 4) were capable of extending the host range of R . l . bv . trifolii to the nonlegume Parasponia . DNA sequence analysis showed that single base changes were responsible for the altered phenotypic properties of five of six mutants examined . Four of the six mutations affected amino acid residues in a putative receiver domain in the N-terminal end of the nodD protein.

Proc Natl Acad Sci U S A, 1989 May, 86(10), 3484 - 8
OxyR, a positive regulator of hydrogen peroxide-inducible genes in Escherichia coli and Salmonella typhimurium, is homologous to a family of bacterial regulatory proteins; Christman MF et al.; The oxyR gene is required for the induction of a regulon of hydrogen peroxide-inducible genes in Escherichia coli and Salmonella typhimurium . The E . coli oxyR gene has been cloned and sequenced, revealing an open reading frame (305 amino acids) that encodes a 34.4-kDa protein, which is produced in maxicells carrying the oxyR clone . The OxyR protein shows homology to a family of positive regulatory proteins including LysR in E . coli and NodD in Rhizobium . Like them, oxyR appears to be negatively autoregulated: an oxyR::lacZ gene fusion produced 5-fold higher levels of beta-galactosidase activity in oxyR null mutants compared to oxyR+ controls, and extracts from an OxyR-overproducing strain were able to protect regions (-27 to +21) of the oxyR promoter from DNase I digestion . DNA sequence analysis of the oxyR2 mutation, which causes overexpression of oxyR-regulated proteins in the absence of oxidative stress, showed that the oxyR2 phenotype is due to a missense mutation (C.G to T.A transition) that changes alanine to valine at amino acid position 234 of OxyR.

Gene, 1989 Apr 15, 77(1), 141 - 53
Structural and functional analysis of nitrogenase genes from the broad-host-range Rhizobium strain ANU240; Badenoch-Jones J et al.; The genes encoding the structural components of nitrogenase, nifH, nifD and nifK, from the fast-growing, broad-host-range Rhizobium strain ANU240 have been identified and characterized . They are duplicated and linked in an operon nifHDK in both copies . Sequence analysis of the nifH gene from each copy, together with partial sequence analysis of the nifD and nifK genes, and restriction endonuclease analysis suggested that the duplication is precise . Comparison of the Fe-protein sequence from strain ANU240 with that from other nitrogen-fixing organisms revealed that, despite its broad host range and certain physiological properties characteristic of Bradyrhizobium strains, ANU240 is more closely related to the narrow-host-range Rhizobium strains than to the broad-host-range Bradyrhizobium strains . The promoter regions of both copies of the nif genes contain the consensus sequence characteristic of nif promoters, and functional analysis of the two promoters suggested that both nif operons are transcribed in nodules.

J Biol Chem, 1989 Apr 5, 264(10), 5710 - 4
The effect of interspecies transfer of Rhizobium host-specific nodulation genes on acidic polysaccharide structure and in situ binding by host lectin; Philip-Hollingsworth S et al.; Host specificity in the Rhizobium-legume symbiosis is controlled in the bacterium by host specific nodulation (hsn) genes residing on its symbiotic plasmid . We have examined the structure of the major acidic heteropolysaccharide produced by recombinant hybrid strains of Rhizobium leguminosarum carrying cloned R . trifolii hsn genes with those produced by the parent donor and recipient strains . Alteration of the nod gene composition of R . leguminosarum strain 300 by introduction of an 8-kilobase set of hsn genes (nodFERL and nodMN) from R . trifolii strain ANU843, resulted in a hybrid strain which conferred efficient white clover infection and nodulation, production of the R . trifolii-type acidic polysaccharide, and an increased proportion of bacterial cells which bound to the white clover lectin, trifoliin A, in the external root environment . 1H NMR studies indicated that the structure of the polysaccharide from the hybrid recombinant differed from that of the R . leguminosarum strain 300 recipient in site and stoichiometry of acetate and stoichiometry of 3-hydroxybutyrate substituents . In contrast, the polysaccharide from a different hybrid recombinant strain containing only R . trifolii nodFERL genes had the acetylation pattern of the R . leguminosarum recipient but was substituted with 3-hydroxybutyrate at a level between that made by R . trifolii and R . leguminosarum . This latter recombinant strain displays sparse infection and nodulation of white clover roots . Immunofluorescence studies indicated that the R . leguminosarum recombinant strain containing the full complement of R . trifolii hsn genes (nodFERL and nodMN) gained the ability to interact with the excreted lectin, trifoliin A, in the white clover root environment, whereas the recombinant strain containing R . trifolii nodFERL only, was significantly attenuated in this cell-lectin interaction . These results indicate that the acylation pattern of the acidic polysaccharide synthesized by these hybrid recombinants of R . leguminosarum is influenced by the introduced hsn genes of R . trifolii and suggest that the acidic polysaccharide of R . trifolii and the interaction of these bacteria with the host lectin may contribute to host specificity in the white clover-R . trifolii symbiosis.

Indian J Biochem Biophys, 1989 Apr, 26(2), 120 - 2
Enzymes of carbohydrate metabolism in fast-growing Rhizobium grown on hexoses or succinate; Mandal NC et al.; Enzymatic evidence supports that succinate mediates repression of hexose-catabolising enzymes in fast-growing Rhizobium sp . (Cicer arietinum) . Enzymes of the Embden-Myerhof-Parnas, Entner-Doudoroff and pentose phosphate pathways were found present in hexose-grown cells but not in succinate-grown cells . These however could be induced by the presence of hexoses.

Mol Gen Genet, 1989 Apr, 216(2-3), 293 - 302
The Rhizobium meliloti fdxN gene encoding a ferredoxin-like protein is necessary for nitrogen fixation and is cotranscribed with nifA and nifB; Klipp W et al.; Sequencing of the Rhizobium meliloti DNA region downstream of nifA revealed the existence of nifB, fdxN and ORF3 . The molecular weight of the fdxN protein (Mr 6830) and the distribution of cysteine residues in its deduced amino acid sequence is typical for low molecular weight bacterial ferredoxins . Interposon insertion and plasmid integration mutagenesis demonstrated that FdxN is essential for nitrogen fixation in R . meliloti, whereas the predicted translation product of ORF3 (Mr 8708) is not necessary for this process . In contrast, ferredoxin-like proteins, which are encoded by nifB-associated genes, are not required for nitrogen fixation in all other organisms analysed so far . Plasmid integration mutagenesis additionally revealed that nifA, nifB and fdxN form one transcriptional unit . This result was confirmed by complementation analysis of polar interposon insertion mutants of nifA, nifB and fdxN and by complementation of a non-polar nifA deletion mutant . A DNA sequence resembling a typical nif consensus promoter, which is preceded by two putative NifA-binding sites, is located in front of nifB . This nifB promoter can be activated in Escherichia coli by the nifA gene product of Klebsiella pneumoniae to the same level as that of the R . meliloti nifH promoter . In contrast, R . meliloti NifA stimulates the nifH promoter more efficiently than the nifB promoter . This low-level activation of the nifB promoter may be the reason why transcription of nifB and fdxN is initiated primarily at a promoter in front of nifA.

J Bacteriol, 1989 Apr, 171(4), 1932 - 41
Identification of a gene linked to Rhizobium meliloti ntrA whose product is homologous to a family to ATP-binding proteins; Albright LM et al.; The ntrA gene of Rhizobium meliloti has recently been identified and shown to be required for a diverse set of metabolic functions (C . W . Ronson, B . T . Nixon, L . M . Albright, and F . M . Ausubel, J . Bacteriol . 169:2424-2431, 1987) . As a result of sequencing the ntrA gene and its flanking regions from R . meliloti, we identified an open reading frame directly upstream of ntrA, ORF1, whose predicted product is homologous to a superfamily of ATP-binding proteins involved in transport, cell division, nodulation, and DNA repair . The homology of ORF1 to this superfamily and its proximity to ntrA led us to investigate its role in symbiosis by mutagenesis and expression studies . We were unable to isolate an insertion mutation in ORF1, suggesting that ORF1 may code for an essential function . We identified the start of transcription for the ntrA gene in vegetative cells and bacteroids and showed that ORF1 and ntrA are transcriptionally unlinked . ORF1 appears to be in an operon with one or more upstream genes.

EMBO J, 1989 Apr, 8(4), 1279 - 86
fixK, a gene homologous with fnr and crp from Escherichia coli, regulates nitrogen fixation genes both positively and negatively in Rhizobium meliloti; Batut J et al.; Nitrogen fixation genes are shown to undergo a complex positive and negative regulation in Rhizobium meliloti . Activation of fixN by fixLJ is shown to require a third regulatory gene, fixK . As fixK is activated by fixLJ, we propose a cascade model for fixN regulation such that fixLJ activates fixN via fixK . In addition fixK negatively regulates expression of the nif-specific activator nifA as well as its own expression by autoregulation . Thus nifA and fixK are subject to a mixed regulation, positive (by fixLJ) and negative (by fixK) . The sequence of fixK shows homology with the Escherichia coli regulators fnr and crp, which makes fixK the third characterized member of this family of prokaryotic regulators.

Plant Cell, 1989 Apr, 1(4), 391 - 401
Two glutamine synthetase genes from Phaseolus vulgaris L . display contrasting developmental and spatial patterns of expression in transgenic Lotus corniculatus plants; Forde BG et al.; The gln-gamma gene, which specifies the gamma subunit of glutamine synthetase in Phaseolus vulgaris L., has been isolated and the regulatory properties of its promoter region analyzed in transgenic Lotus corniculatus plants . A 2-kilobase fragment from the 5'-flanking region of gln-gamma conferred a strongly nodule-enhanced pattern of expression on the beta-glucuronidase reporter gene . Parallel studies on the promoter of another glutamine synthetase gene (gln-beta) showed that a 1.7-kilobase fragment directed 20-fold to 140-fold higher levels of beta-glucuronidase expression in roots than in shoots . Histochemical localization of beta-glucuronidase activity in nodules of the transgenic plants indicated that the chimeric gln-gamma gene was expressed specifically in the rhizobially infected cells; expression of the gln-beta construct was detected in both cortical and infected regions of young nodules, and became restricted to the vascular tissue as the nodule matured . We conclude that gln-beta and gln-gamma genes are differentially expressed both temporally and spatially in plant development and that the cis-acting regulatory elements responsible for conferring these contrasting expression patterns are located within a 2-kilobase region upstream of their coding sequences.

J Bacteriol, 1989 Mar, 171(3), 1467 - 75
Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti; Pleier E et al.; The genomic region that codes for the flagellin subunits of the complex flagellar filaments of Rhizobium meliloti was cloned and sequenced . Two structural genes, flaA and flaB, that encode 395- and 396-amino-acid polypeptides, respectively, were identified . These exhibit 87% sequence identity . The amino acid sequences of tryptic peptides suggest that both of these subunit proteins are represented in the flagellar filaments . The N-terminal methionine was absent from the mature flagellin subunits . Their derived primary structures show almost no relationship to flagellins from Escherichia coli, Salmonella typhimurium, or Bacillus subtilis but exhibit up to 60% similarity to the N- and C-terminal portions of flagellin from Caulobacter crescentus . It is suggested that the complex flagellar filaments of R . meliloti are unique in being assembled from heterodimers of two related flagellin subunits . The tandemly arranged flagellin genes were shown to be transcribed separately from unusual promoter sequences.

J Bacteriol, 1989 Mar, 171(3), 1736 - 8
Rhizobium meliloti regulatory gene fixJ activates transcription of R . meliloti nifA and fixK genes in Escherichia coli; Hertig C et al.; When present in Escherichia coli on the multicopy expression vector pUC19, a Rhizobium meliloti regulatory gene, fixJ, belonging to a two-component regulatory system, activated the expression of two R . meliloti symbiotic genes, nifA and fixK . Primer extension by reverse transcription showed that FixJ stimulates nifA expression in E . coli by activating pnifA.

Indian J Exp Biol, 1989 Mar, 27(3), 288 - 9
Construction of a gene bank of Rhizobium leguminosarum Rld 164; Stanfield S et al.; The total genomic DNA of R . leguminosarum Rld164 (trp, sms, azi) was cloned in the EcoR1 site of the wide host and conjugally transferable cosmid vector pLAFR1 . The average insert size in the gene clones of the bank was found to be 21.3 Kb . The strain R . leguminosarum Rld7 (leu-1) was employed as recepient to conjugally transfer and thus isolate the complementary leu+ allele carrying clones from the gene bank.

J Bacteriol, 1989 Mar, 171(3), 1673 - 82
Rhizobium meliloti 1021 has three differentially regulated loci involved in glutamine biosynthesis, none of which is essential for symbiotic nitrogen fixation; de Bruijn FJ et al.; We have cloned and characterized three distinct Rhizobium meliloti loci involved in glutamine biosynthesis (glnA, glnII, and glnT) . The glnA locus shares DNA homology with the glnA gene of Klebsiella pneumoniae, encodes a 55,000-dalton monomer subunit of the heat-stable glutamine synthetase (GS) protein (GSI), and complemented an Escherichia coli glnA mutation . The glnII locus shares DNA homology with the glnII gene of Bradyrhizobium japonicum and encodes a 36,000-dalton monomer subunit of the heat-labile GS protein (GSII) . The glnT locus shares no DNA homology with either the glnA or glnII gene and complemented a glnA E . coli strain . The glnT locus codes for an operon encoding polypeptides of 57,000, 48,000, 35,000, 29,000, and 28,000 daltons . glnA and glnII insertion mutants were glutamine prototrophs, lacked the respective GS form (GSI or GSII), grew normally on different nitrogen sources (Asm+), and induced normal, nitrogen-fixing nodules on Medicago sativa plants (Nod+ Fix+) . A glnA glnII double mutant was a glutamine auxotroph (Gln-), lacked both GSI and GSII forms, but nevertheless induced normal Fix+ nodules . glnT insertion mutants were prototrophs, contained both GSI and GSII forms, grew normally on different N sources, and induced normal Fix+ nodules . glnII and glnT, but not glnA, expression in R . meliloti was regulated by the nitrogen-regulatory genes ntrA and ntrC and was repressed by rich N sources such as ammonium and glutamine.

Plasmid, 1989 Mar, 21(2), 142 - 6
Plasmid localization and mapping of two Azospirillum brasilense loci that affect exopolysaccharide synthesis; Michiels K et al.; Two Azospirillum brasilense loci that correct Rhizobium meliloti exoB and exoC mutants for exopolysaccharide (EPS) synthesis have been identified previously (K . W . Michiels, J . Vanderleyden, A . P . Van Gool, E . R . Signer, J . Bacteriol., 1988b) . A . brasilense exo mutants produce EPS of lower molecular weight than the wild type strain . Here, we show by hybridization that these exo loci are located on a 90-MDa plasmid in A . brasilense Sp7 . In four other Azospirillum strains but not in A . lipoferum SpBr17, the loci are likewise located on a plasmid of approximately the same size . Transposon Tn5 insertions in these loci were isolated and mapped on the cloned DNA by restriction analysis . Hybridization of restriction digests of purified 90-MDa plasmid DNA with probes containing the exo loci confirmed their plasmid location . This is the first report on plasmid localization of genes in Azospirillum.

Plasmid, 1989 Mar, 21(2), 120 - 8
ISR1, a transposable DNA sequence resident in Rhizobium class IV strains, shows structural characteristics of classical insertion elements; Priefer UB et al.; ISR1 is a small transposable element, identified in Rhizobium class IV strains by its high frequent mutagenic insertion into plasmid RP4 . Hybridization studies showed that ISR1 is present in, multiple copies in Rhizobium class IV strains . Nucleotide sequence analysis revealed that ISR1 has a length of 1260 bp and is characterized by perfect inverted repeats of 13 nucleotides followed by a stretch of 28/29 nucleotides with imperfect homology . The insertion under study generated a target site duplication of 4 bp . ISR1 carries a large open reading frame, encoding a putative polypeptide of 278 amino acids (ORFA*), and three smaller ones in antiparallel direction (ORFs A1, A2, A3) . Two of them are completely covered by the large open reading frame . No significant homology to 17 other known insertion sequence elements could be detected, either at nucleotide or at amino acid levels.

Plant Cell, 1989 Mar, 1(3), 373 - 9
Nodule-specific kinases phosphorylating nuclear factors in isolated nuclei; Suzuki H et al.; In vitro phosphorylation of total nuclear proteins from soybean (Glycine max L) nodules formed by Bradyrhizobium japonicum 61A76 showed several differences in comparison with those from uninfected roots or embryonic-axes nuclei . Three types of protein phosphorylations were observed in nodule nuclei: Ca(2+)- and calmodulin-independent, Ca(2+)- and calmodulin-dependent, and Ca(2+)-dependent but calmodulin-independent . In addition, Ca(2+)-dependent dephosphorylation of some nuclear proteins was observed in nodule nuclei . The first and second types of phosphorylations were also present in root nuclei, but the trifluoperazine-insensitive and Ca(2+)-dependent phosphorylation (indicating calmodulin independence) occurs only in nodules . The latter appears to phosphorylate a nodule-specific protein of 65 kilodaltons and this protein was purified from other nuclear phosphorylated proteins . In addition, some nuclear proteins from uninfected tissue were found to be phosphorylated or dephosphorylated by kinases or phosphatases that originated from the nodule nuclei . These data suggest that some activities of nuclear factors in nodules may be regulated by specific phosphorylation or dephosphorylation during symbiotic interactions with rhizobia.

Cell, 1989 Feb 24, 56(4), 661 - 72
A novel exopolysaccharide can function in place of the calcofluor-binding exopolysaccharide in nodulation of alfalfa by Rhizobium meliloti; Glazebrook J et al.; We have found that R . meliloti strain Rm1021, which is known to synthesize a Calcofluor-binding exopolysaccharide (EPS I), also has a cryptic capacity to synthesize a second exopolysaccharide (EPS II) . Structural analysis of EPS II has shown that it differs in many respects from EPS I . Genetic analysis indicates that EPS II synthesis requires the products of at least seven loci on the second symbiotic megaplasmid of R . meliloti, and is induced by a mutation, expR101, which causes increased transcription of these genes . Synthesis of EPS II suppresses the symbiotic defects of EPS I-deficient strains on Medicago sativa (alfalfa), but not on four other plants that are normally hosts for Rm1021 . These observations suggest that structural features of bacterial exopolysaccharides are involved in the determination of host range . The implications of these results for models of exopolysaccharide function, such as serving as signals to the plant or shielding the bacteria from plant defense responses, are discussed.

J Bacteriol, 1989 Feb, 171(2), 1136 - 42
Immunological characterization of Rhizobium leguminosarum outer membrane antigens by use of polyclonal and monoclonal antibodies; de Maagd R et al.; Surface antigens of Rhizobium leguminosarum biovar viciae strain 248 were characterized by using polyclonal and monoclonal antibodies . With Western immunoblotting as the criterion, an antiserum raised against living whole cells recognized mainly flagellar antigens and the O-antigen-containing part of the lipopolysaccharide (LPS) . Immunization of mice with a peptidoglycan-outer membrane complex yielded eight monoclonal antibodies, of which three reacted with LPS and five reacted with various sets of outer membrane protein antigens . The observation that individual monoclonal antibodies react with sets of related proteins is discussed . Studies of the influence of calcium deficiency and LPS alterations on surface antigenicity showed that in normally grown wild-type cells, the O-antigenic side chain of LPS blocks binding of an antibody to a deeper-lying antigen . This antigen is accessible to antibodies in cells grown under calcium limitation as well as in O-antigen-lacking mutant cells . Two of the antigen groups which can be distinguished in cell envelopes of free-living bacteria were depleted in cell envelopes of isolated bacteroids, indicating that the monoclonal antibodies could be useful tools for studying the differentiation process from free-living bacteria to bacteroids.

J Bacteriol, 1989 Feb, 171(2), 1151 - 7
Localization and symbiotic function of a region on the Rhizobium leguminosarum Sym plasmid pRL1JI responsible for a secreted, flavonoid-inducible 50-kilodalton protein; de Maagd RA et al.; A previously described (R . A . de Maagd, C . A . Wijffelman, E . Pees, and B . J . J . Lugtenberg, J . Bacteriol . 170:4424-4427, 1988) Sym plasmid-dependent, naringenin-inducible 50-kilodalton protein of Rhizobium leguminosarum biovar viciae is further characterized in this paper . The protein was overproduced by constructing a strain containing multiple copies of the R . meliloti nodD gene, which facilitated its purification . An antiserum was used to screen Tn5 insertion mutants located in the pRL1JI region found to be responsible for the production of the 50-kilodalton protein . These inserts define a new nod locus left of the nod genes identified previously . Mutations in this region affect the nodulation ability in a way which is dependent on the bacterial background as well as on the host plant . The mutants nodulate normally in a strain RBL1532 (R . leguminosarum biovar viciae strain 248, cured of its Sym plasmid) background on all three tested host plant species . In contrast, in a strain RBL5045 (R . leguminosarum biovar trifolii strain RCR5, cured of its Sym plasmid) background, nodulation on Vicia sativa is severely impaired, whereas nodulation on Vicia hirsuta and Trifolium subterraneum is apparently unaltered.

J Bacteriol, 1989 Feb, 171(2), 1143 - 50
Isolation and characterization of mutants of Rhizobium leguminosarum bv . viciae 248 with altered lipopolysaccharides: possible role of surface charge or hydrophobicity in bacterial release from the infection thread; de Maagd RA et al.; Effects of alterations in lipopolysaccharide (LPS) structure of Rhizobium leguminosarum bv . viciae on effective symbiosis and on a number of cell surface characteristics were studied . Tn5 mutants with altered LPSs were screened for their inability to bind monoclonal antibody 3, one of three monoclonal antibodies to the tentative O-antigenic part of the wild-type LPS of strain 248 . Ten class I LPS mutants completely lacked the O-antigen-containing LPS species . The class II LPS mutant had a severely diminished amount of an antigenically altered O-antigen-containing LPS . The class III LPS mutant had normal amounts of an altered, O-antigen-containing LPS . Class I and II mutants, but not the class III mutant, showed abnormal nodule development (i.e., blocked in the stage of bacterial release from the infection thread) resulting in nodules in which very few, at the most, plant cells contained bacteroids and which were unable to fix nitrogen . Class I and II mutants were nonmotile and were more sensitive to hydrophobic compounds than the parent strain . The most striking difference between the symbiotically defective class I and II LPS mutants on one hand and the wild-type strain and the class III mutant on the other hand was that the class I and II mutants have a more hydrophobic cell surface and a higher electrophoretic mobility . A role for an O-antigen-containing LPS in bacterial release from the infection thread, through its effects on general physicochemical cell surface characteristics, is proposed.

J Biol Chem, 1989 Jan 25, 264(3), 1461 - 6
Host-range related structural features of the acidic extracellular polysaccharides of Rhizobium trifolii and Rhizobium leguminosarum; Philip-Hollingsworth S et al.; Proton nuclear magnetic resonance (1H NMR) and fast atom bombardment mass spectrometric analyses were performed on enzymatically derived oligosaccharides from the acidic excreted polysaccharides (EPS) from representative bacterial strains of the pea-nodulating symbiont, Rhizobium leguminosarum (128C53, 128C63, and 300) and the clover-nodulating symbiont, Rhizobium trifolii (NA-30, ANU843, 0403, TA-1, LPR5035, USDA20.102, and 4S) . The results revealed structural similarities and differences between EPS of these two species . Octasaccharide units containing galactose, glucuronic acid, alpha-L-threo-hex-4-enopyranosyluronic acid, and glucose in a molar ratio of 1:1:1:5 were obtained from the EPS of the three R . leguminosarum strains and had the same primary glycosyl sequence and location of pyruvate, acetate, and 3-hydroxybutyrate substituents . About 80% of the galactose residues were acylated with 3-hydroxybutyrate, and there were two acetyl groups per repeating unit distributed between the 2 glucose residues of the main chain-derived sequence of the octasaccharides . In contrast, the R . trifolii strains had varied EPS structures, each of which differed from the common R . leguminosarum EPS structure . The EPS from one group of R . trifolii strains (0403 and LPR5035) most closely resembled the R . leguminosarum EPS but differed in that a lower number of galactose and glucose residues were substituted by 3-hydroxybutyryl and acetyl groups, respectively . The EPS from a second group of R . trifolii strains (ANU843, TA-1, and NA-30) was even more different than the R . leguminosarum EPS . These R . trifolii octasaccharides bore a single acetyl group on O-3 of the glucuronic acid residue . In addition, the level of acylation by 3-hydroxybutyryl groups was 50% of that present in the R . leguminosarum EPS . The remaining two strains of R . trifolii (USDA20.102 and 4S) had very different patterns of acylation to each other and to all of the other strains . The EPS from strain USDA20.102 practically lacked 3-hydroxybutyryl groups and had a unique degree and pattern of acetylation . The oligomers from the EPS of R . trifolii strain 4S completely lacked 3-hydroxybutyryl groups and galactose . The latter EPS contained only one O-1-carboxyethylidene group and had a different degree and pattern of acetylation . Interestingly, these two latter strains differ from the other R . trifolii strains in nodulation rates on rare clover species in the Trifolium cross-inoculation group . Thus, we define several groups of R . trifolii based upon their EPS structures and establish their similarities and distinct differences with the EPS of R . leguminosarum.(ABSTRACT TRUNCATED AT 400 WORDS)

J Bacteriol, 1989 Jan, 171(1), 573 - 6
Protoporphyrinogen oxidation, a step in heme synthesis in soybean root nodules and free-living rhizobia; Jacobs NJ et al.; Extracts of the crude bacteroid fraction of symbiotically grown Bradyrhizobium japonicum were much more active in oxidizing protoporphyrinogen to protoporphyrin than were extracts of cells grown under free-living conditions, especially when assayed in atmospheres containing only traces of oxygen . This correlates with the higher heme content of the microaerophilic nodules . Furthermore, the high level of oxidative activity in the crude bacteroid fraction was associated with an uncharacterized membrane fraction, probably of plant origin, that was separable from the bacteroids by Percoll gradient centrifugation.

J Bacteriol, 1989 Jan, 171(1), 569 - 72
Roles of flagella, lipopolysaccharide, and a Ca2+-dependent cell surface protein in attachment of Rhizobium leguminosarum biovar viciae to pea root hair tips; Smit G et al.; The relationship between Ca2+-dependent cell surface components of Rhizobium leguminosarum biovar viciae, motility, and ability to attach to pea root hair tips was investigated . In contrast to flagella and lipopolysaccharide, a small protein located on the cell surface was identified as the Ca2+-dependent adhesin.

J Bacteriol, 1989 Jan, 171(1), 531 - 7
Characterization of three choline transport activities in Rhizobium meliloti: modulation by choline and osmotic stress; Pocard JA et al.; Choline has both a nutritional and osmoregulatory role in Rhizobium meliloti (T . Bernard, J . A . Pocard, B . Perroud, and D . Le Rudulier, Arch . Microbiol . 143:359-364, 1986) . In view of this fact, choline transport was studied in R . meliloti 102F34 to determine how the rate of choline uptake is modulated . The effects of the cultural conditions on the kinetics of transport are presented . A high-affinity activity and a low-affinity activity were found in cells grown in minimal medium . The addition of 0.3 M NaCl or other osmolytes to the medium resulted in a marked decrease in the high-affinity activity, whereas the low-affinity activity remained fairly constant . Furthermore, results from osmotic upshock and downshock experiments indicate that the response of the cell to high osmolarity is rapid; hence, the mechanism of regulation by salt likely does not involve gene induction . A second high-affinity transport activity was induced by choline itself . Like the constitutive low-affinity transport activity, this activity was not greatly altered when the cells were grown in media of elevated osmotic strength . We conclude that although all three kinetically distinct transport systems are efficient at low osmolarity, only the induced high- and low-affinity activities are important for osmoregulation . The characteristics of the three transport activities from R . meliloti are compared with those from other bacterial species that use choline for growth and/or osmoregulation.

Mol Microbiol, 1989 Jan, 3(1), 87 - 93
Transcription of rhiA, a gene on a Rhizobium leguminosarum bv . viciae Sym plasmid, requires rhiR and is repressed by flavanoids that induce nod genes; Economou A et al.; Strains of Rhizobium leguminosarum biovar viciae specifically make an abundant protein (Rhi) in free-living culture but not in bacteroids . Genes needed for Rhi synthesis are on a Sym plasmid and here we show that one of these genes, rhiA, is the structural gene that specifies this polypeptide . Transcription of rhiA requires a regulatory gene, rhiR, located close to rhiA and to nod genes involved in nodulation . Mutations in rhiA or rhiR do not appear to affect symbiotic nitrogen fixation . Transcription of rhiA is repressed in cells grown in the presence of the flavanone hesperetin or the flavone apigenin, both of which are potent inducers of transcription of nod genes . This was deduced from the use of rhiA-lacZ fusions; however, when the Rhi polypeptide was detected in SDS gels, there was no apparent difference in the intensity of its staining in extracts obtained from cells grown with or without these flavanoid nod gene inducer molecules . However, a mutation in a nodulation gene, nolR, also closely linked to the nod and rhi genes, caused a severe depression in the amount of Rhi (as seen on gels) that was made in cells grown in the presence of inducer flavanoids.

J Bacteriol, 1989 Jan, 171(1), 8 - 15
Rhizobium leguminosarum CFN42 genetic regions encoding lipopolysaccharide structures essential for complete nodule development on bean plants; Cava JR et al.; Eight symbiotic mutants defective in lipopolysaccharide (LPS) synthesis were isolated from Rhizobium leguminosarum biovar phaseoli CFN42 . These eight strains elicited small white nodules lacking infected cells when inoculated onto bean plants . The mutants had undetectable or greatly diminished amounts of the complete LPS (LPS I), whereas amounts of an LPS lacking the O antigen (LPS II) greatly increased . Apparent LPS bands that migrated between LPS I and LPS II on sodium dodecyl sulfate-polyacrylamide gels were detected in extracts of some of the mutants . The mutant strains were complemented to wild-type LPS I content and antigenicity by DNA from a cosmid library of the wild-type genome . Most of the mutations were clustered in two genetic regions; one mutation was located in a third region . Strains complemented by DNA from two of these regions produced healthy nitrogen-fixing nodules . Strains complemented to wild-type LPS content by the other genetic region induced nodules that exhibited little or no nitrogenase activity, although nodule development was obviously enhanced by the presence of this DNA . The results support the idea that complete LPS structures, in normal amounts, are necessary for infection thread development in bean plants.

J Bacteriol, 1989 Jan, 171(1), 465 - 72
Isolation of a Rhizobium phaseoli cytochrome mutant with enhanced respiration and symbiotic nitrogen fixation; Soberon M et al.; Cultured cells of a Rhizobium phaseoli wild-type strain (CE2) possess b-type and c-type cytochromes and two terminal oxidases: cytochromes o and aa3 . Cytochrome aa3 was partially expressed when CE2 cells were grown on minimal medium, during symbiosis, and in well-aerated liquid cultures in a complex medium (PY2) . Two cytochrome mutants of R . phaseoli were obtained and characterized . A Tn5-mob-induced mutant, CFN4201, expressed diminished amounts of b-type and c-type cytochromes, showed an enhanced expression of cytochrome oxidases, and had reduced levels of N,N,N',N'-tetramethyl-p-phenylenediamine, succinate, and NADH oxidase activities . Nodules formed by this strain had no N2 fixation activity . The other mutant, CFN4205, which was isolated by nitrosoguanidine mutagenesis, had reduced levels of cytochrome o and higher succinate oxidase activity but similar NADH and N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activities when compared with the wild-type strain . Strain CFN4205 expressed a fourfold-higher cytochrome aa3 content when cultured on minimal and complex media and had twofold-higher cytochrome aa3 levels during symbiosis when compared with the wild-type strain . Nodules formed by strain CFN4205 fixed 33% more N2 than did nodules formed by the wild-type strain, as judged by the total nitrogen content found in plants nodulated by these strains . Finally, low-temperature photodissociation spectra of whole cells from strains CE2 and CFN4205 reveal cytochromes o and aa3 . Both cytochromes react with O2 at -180 degrees C to give a light-insensitive compound . These experiments identify cytochromes o and aa3 as functional terminal oxidases in R . phaseoli.

J Bacteriol, 1989 Jan, 171(1), 383 - 91
Escherichia coli sigma 54 RNA polymerase recognizes Caulobacter crescentus flbG and flaN flagellar gene promoters in vitro; Ninfa AJ et al.; A set of the periodically regulated flagellar (fla) genes of Caulobacter crescentus contain conserved promoter sequence elements at -24 and -12 that are very similar to the sequence of the nitrogen assimilation (Ntr) and nitrogen fixation (Nif) promoters of enteric bacteria and Rhizobium spp . Transcription from Ntr and Nif promoters requires RNA polymerase containing sigma 54 instead of the usual sigma 70 and, in the case of the Ntr promoters, is activated by the transcription factors NRI and NRII . We have now demonstrated that the C . crescentus flbG and flaN promoters, which contain the Ntr/Nif type of consensus sequence, are utilized by purified Escherichia coli sigma 54 RNA polymerase (E sigma 54) in the presence of NRI and NRII but not by the purified sigma 70 RNA polymerase (E sigma 70) of E . coli . Oligonucleotide-generated flbG promoter deletions that removed the highly conserved GG dinucleotide at -24 or the GC dinucleotide at -12 or altered the spacing between the -24 and -12 sequence elements prevented utilization of the flbG promoter by the E . coli E sigma 54 . Transversions of T to G at positions -26 and -15 also inactivated flbG promoter function in the E . coli cell-free transcription system, while a transition of G to A at position -16 in the nonconserved spacer region had no effect . The C . crescentus flaO and flbF promoters, which do not contain the Ntr/Nif-type promoter consensus sequence, were not utilized by either purified E sigma 54 or E sigma 70 from E . coli . Our results help to define the features of the Ntr/Nif-type consensus sequence required for promoter utilization by purified E . coli E sigma 54 and support the idea that C . crescentus may contain a specialized polymerase with similar promoter specificity required for expression of a set of fla genes.

Plant Cell, 1989 Jan, 1(1), 65 - 72
A non-nodulating alfalfa mutant displays neither root hair curling nor early cell division in response to Rhizobium meliloti; Dudley ME et al.; The early events in the alfalfa-Rhizobium meliloti symbiosis include deformation of epidermal root hairs and the approximately concurrent stimulation of cell dedifferentiation and cell division in the root inner cortex . These early steps have been studied previously by analysis of R . meliloti mutants . Bacterial strains mutated in nodABC, for example, fail to stimulate either root hair curling or cell division events in the plant host, whereas exopolysaccharide (exo) mutants of R . meliloti stimulate host cell division but the resulting nodules are uninfected . As a further approach to understanding early symbiotic interactions, we have investigated the phenotype of a non-nodulating alfalfa mutant, MnNC-1008 (NN) (referred to as MN-1008) . Nodulating and non-nodulating plants were inoculated with wild-type R . meliloti and scored for root hair curling and cell divisions . MN-1008 was found to be defective in both responses . Mutant plants inoculated with Exo- bacteria also showed no cell division response . Therefore, the genetic function mutated in MN-1008 is required for both root hair curling and cell division, as is true for the R . meliloti nodABC genes . These observations support the model that the distinct cellular processes of root hair curling and cell division are triggered by related mechanisms or components, or are causally linked.

Chin J Biotechnol, 1989, 5(3), 133 - 9
Expression of Bradyrhizobium japonicum nodulation gene in Rhizobium fredii nod mutants; Zhou LM et al.; The B . japonicum USDA 110 pLAFR1 gene library was transferred to Tn5-induced Rhizobium fredii USDA 191-4 Nod- mutants with helper plasmid pRK2013 . SmR TcR transconjugants occurred with a frequency of 5 x 10(-4) . The transconjugants were purified and used to inoculate germinated soybean seeds . Seven nodules were obtained in the nodulation experiment . The fast-growing Nod+ SmR TcR Rhizobium fredii strain was isolated from all nodules . Each isolate had acquired a new plasmid with a molecular mass of approximately 51 kb . This recombinant plasmid was transferred to E . coli HB101 by helper pRK2013 at a frequency of 1 x 10(-4) . In addition to the vector fragment, all the recombinant plasmids gave 23 kb and 6.5 kb fragments on EcoRI digestion . A DNA-DNA hybridization test with a 32P-labelled nod gene probe (prepared from pRmSL26) confirmed that the 20 kb EcoRI-BamHI DNA fragment of the plasmids exhibited sequence homology with an R . meliloti nod gene probe.

Acta Biochim Pol, 1989, 36(3-4), 257 - 62
Anaerobic processes in yellow lupin (Lupinus luteus L.) root nodules; Mazurowa H et al.; The lupin root nodule homogenate was separated by centrifugation in the Percoll density gradient into the Rhizobium bacteroid fraction and plant subcellular components . High activities of alcohol dehydrogenase and lactate dehydrogenase in the soluble fraction of host plant, and high capability of the isolated bacteroids to oxidize ethanol, malate, lactate and acetaldehyde evidence functional interrelationship between the plant and bacteroids.

Nucleic Acids Res, 1988 Dec 23, 16(24), 11469 - 88
The DNA-binding domain of the transcriptional activator protein NifA resides in its carboxy terminus, recognises the upstream activator sequences of nif promoters and can be separated from the positive control function of NifA; Morett E et al.; The positive control protein NifA activates transcription of nitrogen fixation promoters in Klebsiella pneumoniae . NifA is believed to bind to specific sites, the upstream activator sequences (UAS's), of the nif promoters which it activates . We have now shown by mutation of the carboxy terminus of NifA that this is the DNA-binding domain and that the DNA-binding and positive activator functions of NifA can be separated . Mutational analysis of the nifH UAS and in vivo methylation protection analysis of the interaction of NifA with the nifH promoter demonstrates that the UAS is recognised by the carboxy terminus of NifA . The UAS's of K . pneumoniae nif promoters are also required for activation by the Rhizobium meliloti NifA indicating that this activator also possesses DNA-binding activity.

J Bacteriol, 1988 Dec, 170(12), 5925 - 7
High-frequency transformation of Rhizobium meliloti; Courtois J et al.; Transformation of R factor RP4 and its derivative pRK290 from Escherichia coli to Rhizobium meliloti is reported . The efficiency of transformation was in the range of 10(-5) per viable cell . In addition, chromosomal DNA prepared from one R . meliloti strain resistant to streptomycin was transferred to the isoleucine-valine-requiring mutant susceptible to streptomycin.

J Bacteriol, 1988 Dec, 170(12), 5489 - 99
Rhizobium meliloti host range nodH gene determines production of an alfalfa-specific extracellular signal; Faucher C et al.; The Rhizobium meliloti nodH gene is involved in determining host range specificity . By comparison with the wild-type strain, NodH mutants exhibit a change in host specificity . That is, although NodH mutants lose the ability to elicit root hair curling (Hac-), infection threads (Inf-), and nodule meristem formation (Nod-) on the homologous host alfalfa, they gain the ability to be Hac+ Inf+ Nod+ on a nonhomologous host such as common vetch . Using root hair deformation (Had) bioassays on alfalfa and vetch, we have demonstrated that sterile supernatant solutions of R . meliloti cultures, in which the nod genes had been induced by the plant flavone luteolin, contained symbiotic extracellular signals . The wild-type strain produced at least one Had signal active on alfalfa (HadA) . The NodH- mutants did not produce this signal but produced at least one factor active on vetch (HadV) . Mutants altered in the common nodABC genes produced neither of the Had factors . This result suggests that the nodABC operon determines the production of a common symbiotic factor which is modified by the NodH product into an alfalfa-specific signal . An absolute correlation was observed between the specificity of the symbiotic behavior of rhizobial cells and the Had specificity of their sterile filtrates . This indicates that the R . meliloti nodH gene determines host range by helping to mediate the production of a specific extracellular signal.

Mol Gen Genet, 1988 Dec, 215(1), 134 - 8
Identification of the Klebsiella pneumoniae glnB gene: nucleotide sequence of wild-type and mutant alleles; Holtel A et al.; The glnB gene of Klebsiella pneumoniae, which encodes the nitrogen regulation protein PII, has been cloned and sequenced . The gene encodes a 12429 dalton polypeptide and is highly homologous to the Escherichia coli glnB gene . The sequences of a glnB mutation which causes glutamine auxotrophy and of a Tn5 induced Gln+ suppressor of this mutation were also determined . The glutamine auxotrophy was deduced to be the result of a modification of the uridylylation site of PII, and the suppression was shown to be caused by Tn5 insertion in glnB . The 3' end of an open reading frame of unknown function was identified upstream of glnB and may be part of an operon containing glnB . Potential homologues of glnB encoding polypeptides extremely similar in sequence to PII were identified upstream of published sequences of the glutamine synthetase structural gene (glnA) in Rhizobium leguminosarum, Bradyrhizobium japonicum and Azospirillum brasilense.

Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 9133 - 7
Synthesis of an opine-like compound, a rhizopine, in alfalfa nodules is symbiotically regulated; Murphy PJ et al.; We show that the promoter of the mos locus, which encodes genes required for the synthesis of a nodule-specific, opine-like compound, a rhizopine, in alfalfa nodules is regulated by the symbiotic nitrogen-fixation regulatory gene nifA . The 5'-regulatory region and amino-terminal end of the first open reading frame of the mos locus are highly homologous to the 5'-regulatory region and amino-terminal portion of the Rhizobium meliloti nifH gene . The coordinate regulation of mos and nif genes suggests that the mos locus plays a symbiotic role . We propose that the rhizopine enhances the survival of the bacterial partner in the symbiosis.

J Bacteriol, 1988 Dec, 170(12), 5718 - 27
Interference between Rhizobium meliloti and Rhizobium trifolii nodulation genes: genetic basis of R . meliloti dominance; Debelle F et al.; Transfer of an IncP plasmid carrying the Rhizobium meliloti nodFE, nodG, and nodH genes to Rhizobium trifolii enabled R . trifolii to nodulate alfalfa (Medicago sativa), the normal host of R . meliloti . Using transposon Tn5-linked mutations and in vitro-constructed deletions of the R . meliloti nodFE, nodG, and nodH genes, we showed that R . meliloti nodH was required for R . trifolii to elicit both root hair curling and nodule initiation on alfalfa and that nodH, nodFE, and nodG were required for R . trifolii to elicit infection threads in alfalfa root hairs . Interestingly, the transfer of the R . meliloti nodFE, nodG, and nodH genes to R . trifolii prevented R . trifolii from infecting and nodulating its normal host, white clover (Trifolium repens) . Experiments with the mutated R . meliloti nodH, nodF, nodE, and nodG genes demonstrated that nodH, nodF, nodE, and possibly nodG have an additive effect in blocking infection and nodulation of clover.

Gene, 1988 Nov 15, 71(1), 57 - 64
Cloning and characterization of a novel beta-galactosidase-coding gene from Rhizobium meliloti; Fanning S et al.; The Rhizobium meliloti (Rm) lacZ gene provides a convenient model to investigate patterns of gene regulation in these agronomically important bacteria . A gene encoding beta-galactosidase (beta Gal) activity was cloned from R . meliloti by complementing a lactose-negative Escherichia coli mutant . A series of Sau3A subclones was generated in pBR322, and the coding region for the beta Gal-coding gene was localized to a 2.4-kb core fragment . In E . coli 'maxicells', these lacZ subclones produced a 79-kDa polypeptide, irrespective of the fragment size demonstrating that the translation initiation signal(s) are located on the 2.4-kb fragment . Transposon Tn5 mutagenesis and BAL 31 deletion analysis showed that the expression of the Rm lacZ gene in E . coli was dependent on the tetracycline-resistance promoter of pBR322 . The cloned sequence was required for beta Gal synthesis in Rhizobium since mutants generated by reverse genetics lack this enzyme and were specifically defective in lactose catabolism.

Mol Gen Genet, 1988 Nov, 214(3), 496 - 502
Characterization of the fixABC region of Azorhizobium caulinodans ORS571 and identification of a new nitrogen fixation gene; Kaminski PA et al.; The fast growing strain, Azorhizobium caulinodans ORS571, isolated from stem nodules of the tropical legume Sesbania rostrata, can grow in the free-living state at the expense of molecular nitrogen . Five point mutants impaired in nitrogen fixation in the free-living state have been complemented by a plasmid containing the cloned fix-ABC region of strain ORS571 . Genetic analysis of the mutants showed that one was impaired in fixC, one in fixA and the three others in a new gene, located upstream from fixA and designated nifO . Site-directed Tn5 mutagenesis was performed to obtain Tn5 insertions in fixB and fixC . The four genes are required for nitrogen fixation both in the free-living state and under symbiotic conditions . The nucleotide sequence of nifO was established . The gene is transcribed independently of fixA and does not correspond to fixX, recently identified in Rhizobium meliloti and R . leguminosarum . Biochemical analysis of the five point mutants showed that they synthesized normal amounts of nitrogenase components . It is unlikely that fixA, fixC and nifO are involved in electron transport to nitrogenase . FixC could be required for the formation of a functional nitrogenase component 2.

Appl Environ Microbiol, 1988 Nov, 54(11), 2825 - 32
Genetic diversity and relationships among isolates of Rhizobium leguminosarum biovar phaseoli; Pinero D et al.; Fifty-one isolates of Rhizobium leguminosarum biovar phaseoli from various geographic and ecological sources, largely in Mexico, were characterized by the electrophoretic mobilities of 15 metabolic enzymes, and 46 distinctive multilocus genotypes (electrophoretic types {ETs}) were distinguished on the basis of allele profiles at the enzyme loci . Mean genetic diversity per enzyme locus among the 46 ETs was 0.691, the highest value yet recorded for any species of bacterium . The occurrence of strong nonrandom associations of alleles over loci suggested a basically clonal population structure, reflecting infrequent recombination of chromosomal genes . Multilocus genotypic diversity was unusually high, with the most strongly differentiated pairs of ETs having distinctive alleles at all 15 loci and major clusters of ETs diverging at genetic distances as large as 0.89 . This great diversity in the chromosomal genome raises the possibility that R . leguminosarum biovar phaseoli is a polyphyletic assemblage of strains . As other workers have suggested, the inclusion of all strains capable of nodulating beans in a single biovar or species is genetically unrealistic and taxonomically misleading . A biologically meaningful classification of Rhizobium spp . should be based on assessment of variation in the chromosomal genome rather than on phenotypic characters, especially those mediated for the most part or wholly by plasmid-borne genes, such as host relationships.

Mol Microbiol, 1988 Nov, 2(6), 743 - 8
Comparison of the chemotactic behaviour of Rhizobium leguminosarum with and without the nodulation plasmid; Armitage JP et al.; The chemotactic behaviour of a strain of Rhizobium leguminosarum biovar viciae was investigated . The flavanoids apigenin and naringenin, inducers of transcription of the nodulation (nod) genes, were both potent attractants but hesperitin, another flavone nod gene inducer, was not . The response of strains containing the Sym plasmid pRL1Jl to apigenin and naringenin was significantly greater than the response of a strain cured of the plasmid, although both strains gave a positive response . Addition of the flavanol kaempferol, an antagonist of nod gene induction, had no detectable effect on the chemotactic response to naringenin or apigenin, but was itself found to be an attractant . The attractant response to a variety of amino acids and sugars was not affected by the presence of the Sym plasmid . Homoserine, the most abundant nitrogenous compound in legume exudates, was also found to be an attractant . However, although the Sym plasmid is required for the biovar to metabolize homoserine as a carbon source, it was not required for the chemotactic response . A group of membrane proteins showed increased methylation in response to stimulation with serine . There was no measurable change in methylation after stimulation with apigenin.

J Bacteriol, 1988 Nov, 170(11), 5401 - 4
Isolation and characterization of Azospirillum brasilense loci that correct Rhizobium meliloti exoB and exoC mutations; Michiels KW et al.; The occurrence in Azospirillum brasilense of genes that code for exopolysaccharide (EPS) synthesis was investigated through complementation studies of Rhizobium meliloti Exo- mutants . These mutants are deficient in the synthesis of the major acidic EPS of Rhizobium species and form empty, non-nitrogen-fixing root nodules on alfalfa (J . A . Leigh, E . R . Signer, and G . C . Walker, Proc . Natl . Acad . Sci . USA 82:6231-6235, 1985) . We demonstrated that the exoC mutation of R . meliloti could be corrected for EPS production by several cosmid clones of a clone bank of A . brasilense ATCC 29145 . However, the EPS produced differed in structure from the wild-type R . meliloti EPS, and the symbiotic deficiency of the exoC mutation was not reversed by any of these cosmid clones . The exoB mutation could be corrected not only for EPS production but also for the ability to form nitrogen-fixing nodules on alfalfa by one particular cosmid clone of A . brasilense . Tn5 insertions in the cloned DNA were isolated and used to construct Azospirillum mutants with mutations in the corresponding loci by marker exchange . It was found that these mutants failed to produce the wild-type high-molecular-weight EPS, but instead produced EPSs of lower molecular weight.

Plasmid, 1988 Nov, 20(3), 249 - 58
Construction of transposons carrying the transfer functions of RP4; Johnson DA; The transfer genes and origin of transfer of the wide host range plasmid RP4 have been cloned into the transposons Tn1 and Tn5 . The newly constructed transposons can be used to mutagenize bacterial plasmids or the chromosome in species such as Escherichia coli or Rhizobium . It is then possible to mobilize the plasmid or chromosome using the transfer functions provided in cis by the transposon . These constructs may aid chromosome mapping in many gram-negative species by allowing the wider use of the RP4 conjugal transfer system combined with the potential ability to select the site of insertion and thus the site of the origin of transfer.

Gene, 1988 Oct 15, 70(1), 139 - 51
Cloning and characterization of the 5-aminolevulinate synthase gene(s) from Rhodobacter sphaeroides; Tai TN et al.; The 5-aminolevulinate synthase gene (hemA) from Rhizobium meliloti was used to probe a genomic lambda bank derived from Rhodobacter sphaeroides DNA . Two phage clones were found to bear homology to the Rhizobium probe . Southern hybridization analysis of the two lambda phage clones, which we designated lambda Hem 10 and lambda Hem 12, showed that the homology to the Rhizobium hemA gene was localized to a 3.1-kb SalI fragment derived from lambda Hem 10 and a 7.0-kb SalI fragment derived from lambda Hem 12 . Each of the SalI fragments was subsequently cloned into the multiple cloning site of pUC19 in both orientations relative to the lac promoter . Restriction analysis confirmed that each SalI fragment was unique . It was also shown from Southern hybridization analysis that the regions of homology within each of the R . sphaeroides restriction fragments and the Rhizobium probe were different . Further, we have tentatively concluded that each R . sphaeroides hemA gene shows a relatively low degree of homology to the other . Data obtained from in vitro transcription-translation studies in a homologous R . sphaeroides cell-free system, and complementation of hemA mutations of both Escherichia coli and R . sphaeroides by either of the putative hemA clones suggested the presence of a gene encoding 5-aminolevulinate synthase on each DNA sequence . The fact that 5-aminolevulinate synthase activity could be demonstrated in mutant strains complemented in trans with either cloned DNA fragment further supported this conclusion.

J Bacteriol, 1988 Oct, 170(10), 4569 - 75
Phosphoglycerol substituents present on the cyclic beta-1,2-glucans of Rhizobium meliloti 1021 are derived from phosphatidylglycerol; Miller KJ et al.; The synthesis of periplasmic cyclic beta-1,2-glucans is a property unique to species of the family Rhizobiaceae . For this reason, it is generally believed that these molecules may play an important role in the plant infection process . In the present study, we determined that the cyclic beta-1,2-glucans produced by Rhizobium meliloti 1021 were predominantly anionic in character and contained both phosphoglycerol and succinic acid substituents . In addition, we demonstrated that phosphatidylglycerol was the source of the phosphoglycerol substituents present on these oligosaccharides and that greater than 60% of the total phospholipid turnover in this organism involved this substitution reaction.

J Bacteriol, 1988 Sep, 170(9), 4424 - 7
Detection and subcellular localization of two Sym plasmid-dependent proteins of Rhizobium leguminosarum biovar viciae; de Maagd RA et al.; The previously described Sym plasmid-dependent 24-kilodalton rhi protein of Rhizobium leguminosarum biovar viciae was localized in the cytosol fraction . Another Sym plasmid-dependent protein of 50 kilodaltons is secreted into the growth medium, and its expression is dependent on both the nodD gene and a nod gene inducer.

J Bacteriol, 1988 Sep, 170(9), 3793 - 802
Induction of nitrogen-fixing nodules on clover requires only 32 kilobase pairs of DNA from the Rhizobium trifolii symbiosis plasmid; Innes RW et al.; Overlapping subclones from the Rhizobium trifolii symbiosis plasmid pRt843a were generated by using in vivo and in vitro methods . Subclones were assayed for symbiotic phenotype by introducing them into a derivative of R . trifolii ANU843 cured of its symbiosis plasmid and testing the transconjugant strains for the ability to induce nitrogen-fixing nodules on clover . One subclone spanning 32 kilobase pairs (kb) of DNA from pRt843a was found to restore nitrogen fixation ability . This subclone included all known nodulation genes of R . trifolii ANU843 and the nitrogenase structural genes nifHDK . In addition, regions homologous to fixABC, nifA, nifB, nifE, and nifN genes of other nitrogen-fixing bacteria were identified in this 32-kb subclone by DNA-DNA hybridization . Transposon mutagenesis of this subclone confirmed that regions containing these nif and fix genes were required for induction of nitrogen-fixing nodules on clover . In addition, a region located 5 kb downstream of the nifK gene was found to be required for induction of nitrogen-fixing nodules . No homology to known nif and fix genes could be detected in this latter region.

J Cell Biochem, 1988 Sep, 38(1), 35 - 49
Respiration supported nitrogenase activity of isolated Rhizobium meliloti bacteroids; Miller RW et al.; Bacteroids having a high level of respiration-supported nitrogenase activity were isolated from nitrogen-fixing alfalfa root nodules . Gentle maceration under anaerobic conditions in the presence of sodium succinate and a fatty acid scavenging agent were employed in this method . A large proportion of isolated bacteroids retained a triple membrane structure as shown by transmission electron microscopy . Dicarboxylic acids of the TCA cycle (malate, fumarate, succinate), but not glutamate or aspartate, supported sufficient respiratory activity to supply the nitrogenase system with ATP and reducing equivalents and to protect the nitrogenase system from inactivation by 4% oxygen over a period of 20-30 min . Sugars did not support nitrogenase activity in intact bacteroids . The properties of the isolated bacteroids were ascribed to minimal damage to the cytoplasmic membrane and peribacteroidal membrane during isolation . With succinate as substrate and oxygen as terminal electron acceptor, initial nitrogenase activity was determined at 4% oxygen in the gas phase of the assay system employed . At this oxygen concentration, the sustained rate of acetylene reduction by respiring bacteroids was linear up to 30 min . Bacteroid activity declined rapidly with time of exposure to oxygen above 4% in the gas phase . The optimum temperature range for this activity was 10-20 degrees C . Nitrogenase activity was measurable at incubation temperatures below 10 degrees C under 4% oxygen . Functionally intact bacteroids had little nitrogenase activity under anaerobic conditions in the presence of an external source of ATP and reductant . Treatment of the bacteroids with chlorpromazine eliminated respiration-supported activity and rendered the bacteroid cell membrane permeable to external ATP . Bacteroids treated with chlorpromazine had high acetylene reducing activity with external ATP and dithionite in the absence of oxygen.

J Bacteriol, 1988 Sep, 170(9), 4406 - 10
Bacterial-type ferredoxin genes in the nitrogen fixation regions of the cyanobacterium Anabaena sp . strain PCC 7120 and Rhizobium meliloti; Mulligan ME et al.; The nucleotide sequence of a region located downstream of the nifB gene, both in the cyanobacterium Anabaena sp . strain PCC 7120 and in Rhizobium meliloti, has been determined . This region contains a gene (fdxN) whose predicted polypeptide product strongly resembles typical bacterial ferredoxins . Cyanobacteria have not previously been shown to contain bacterial-type ferredoxins . The presence of this gene suggests that nitrogen-fixing cyanobacteria have at least four distinct ferredoxins.

J Bacteriol, 1988 Sep, 170(9), 4257 - 65
Symbiotic loci of Rhizobium meliloti identified by random TnphoA mutagenesis; Long S et al.; We have developed a system for using TnphoA (TnphoA is Tn5 IS50L::phoA), which generates fusions to alkaline phosphatase (C . Manoil and J . Beckwith, Proc . Natl . Acad . Sci . USA 82:8129-8133, 1985), in Rhizobium meliloti . Active fusions expressing alkaline phosphatase can arise only when this transposon inserts in genes encoding secreted or membrane-spanning proteins . By confining our screening to 1,250 TnphoA-generated mutants of R . meliloti that expressed alkaline phosphatase, we efficiently identified 25 symbiotically defective mutants, all of which formed ineffective (Fix-) nodules on alfalfa . Thirteen of the mutants were unable to synthesize an acidic exopolysaccharide (exo::TnphoA) that is required for nodule invasion . Twelve of the mutations created blocked at later stages of nodule development (fix::TnphoA) and were assigned to nine symbiotic loci . One of these appeared to be a previously undescribed locus located on the pRmeSU47a megaplasmid and to encode a membrane protein . Two others were located on the pRmeSU47b megaplasmid: one was a new locus which was induced by luteolin and encoded a membrane protein, and the other was dctA, the structural gene for dicarboxylic acid transport . The remaining six loci were located on the R . meliloti chromosome . One of these was inducible by luteolin and encoded a membrane protein which determined lipopolysaccharide structure . Three additional chromosomal loci also appeared to encode membrane proteins necessary for symbiosis . The remaining two chromosomal loci encoded periplasmic proteins required for symbiosis.

J Bacteriol, 1988 Sep, 170(9), 4249 - 56
Rhizobium meliloti mutants that overproduce the R . meliloti acidic calcofluor-binding exopolysaccharide; Doherty D et al.; The acidic Calcofluor-binding exopolysaccharide of Rhizobium meliloti Rm1021 plays one or more critical roles in nodule invasion and possibly in nodule development . Two loci, exoR and exoS, that affect the regulation of synthesis of this exopolysaccharide were identified by screening for derivatives of strain Rm1021 that formed mucoid colonies that fluoresced extremely brightly under UV light when grown on medium containing Calcofluor . The exopolysaccharide produced in large quantities by the exoR95::Tn5 and exoS96::Tn5 strains was indistinguishable from that produced by the parental strain Rm1021, and its synthesis required the function of at least the exoA, exoB, and exoF genes . Both the exoR and exoS loci were located on the chromosome, and the exo96::Tn5 mutation was 84% linked to the trp-33 mutation by phi M12 transduction . Synthesis of the Calcofluor-binding exopolysaccharide by strain Rm1021 was greatly stimulated by starvation for ammonia . In contrast, the exoR95::Tn5 mutant produced high levels of exopolysaccharide regardless of the presence or absence of ammonia in the medium . The exoS96::Tn5 mutant produced elevated amounts of exopolysaccharide in the presence of ammonia, but higher amounts were observed after starvation for ammonia . The presence of either mutation increased the level of expression of exoF::TnphoA and exoP::TnphoA fusions (TnphoA is Tn5 IS50L::phoA) . Analyses of results obtained when alfalfa seedlings were inoculated with the exoR95::Tn5 strain indicated that the mutant strain could not invade nodules . However, pseudorevertants that retained the original exoR95::Tn5 mutation but acquired unlinked suppressors so that they produced an approximately normal amount of exopolysaccharide were able to invade nodules and fix nitrogens . The exoS95::Tn5 strain formed Fix+ nodules, although some minor variability was observed.

J Bacteriol, 1988 Sep, 170(9), 4239 - 48
Genetic analysis of a cluster of genes required for synthesis of the calcofluor-binding exopolysaccharide of Rhizobium meliloti; Long S et al.; Rhizobium meliloti produces an acidic, Calcofluor-binding exopolysaccharide which plays a role in nodulation of alfalfa plants by this bacterium . We constructed and mapped 102 transposon insertions in a 48-kilobase (kb) region previously shown to contain several exo genes . Mutations affecting production of the Calcofluor-binding exopolysaccharide were clustered in a 22-kb region and fell into 12 complementation groups . Strains carrying mutations in seven of the complementation groups (exoA, exoB, exoF, exoL, exoM, exoP, and exoQ) produced no Calcofluor-binding exopolysaccharide and induced non-nitrogen-fixing nodules on alfalfa . Mutants in an eighth complementation group, exoH (Leigh et al., Cell 51:579-587, 1987), produce an altered exopolysaccharide and also induce the formation of non-nitrogen-fixing nodules . Mutants in the remaining four complementation groups produced less Calcofluor-binding material than the wild type . Mutants carrying mutations in two of these complementation groups (exoK and exoN) formed apparently normal, nitrogen-fixing nodules, while mutants in the other two groups (exoG and exoJ) formed normal nodules less efficiently than the wild type.

Cell, 1988 Aug 26, 54(5), 671 - 83
Cascade regulation of nif gene expression in Rhizobium meliloti; David M et al.; We report the discovery of two genes from Rhizobium meliloti, fixL and fixJ, which are positive regulators of symbiotic expression of diverse nitrogen fixation (nif and fix) genes . nif gene regulation is shown to consist of a cascade: the fixLJ genes activate nifA, which in turn activates nifHDK and fixABCX . Like nifA, fixN can be induced in free-living microaerobic cultures of R . meliloti, indicating a major physiological role for oxygen in nif and fix gene regulation . Microaerobic expression of fixN and nifA depends on fixL and fixJ . The FixL and FixJ proteins belong to a family of two-component regulatory systems widely spread among prokaryotes and responsive to the cell environment . We propose that FixL, which has features of a transmembrane protein, senses an environmental signal and transduces it to FixJ, a transcriptional activator of nif and fix genes.

J Bacteriol, 1988 Aug, 170(8), 3327 - 32
Characterization of polysaccharides of Rhizobium meliloti exo mutants that form ineffective nodules; Leigh JA et al.; Mutants of Rhizobium meliloti SU47 with defects in the production of the Calcofluor-binding expolysaccharide succinoglycan failed to gain entry into alfalfa root nodules . In order to define better the polysaccharide phenotypes of these exo mutants, we analyzed the periplasmic oligosaccharide cyclic (1-2)-beta-D-glucan and lipopolysaccharide (LPS) in representative mutants . The exoC mutant lacked the glucan and had abnormal LPS which appeared to lack a substantial portion of the O side chain . The exoB mutant had a spectrum of LPS species which differed from those of both the wild-type parental strain and the exoC mutant . The presence of the glucan and normal LPS in the exoA, exoD, exoF, and exoH mutants eliminated defects in these carbohydrates as explanations for the nodule entry defects of these mutants . We also assayed for high- and low-molecular-weight succinoglycans . All of the exo mutants except exoD and exoH completely lacked both forms . For the Calcofluor-dim exoD mutant, the distribution of high- and low-molecular-weight forms depended on the growth medium . The haloless exoH mutant produced high-molecular-weight and only a trace of low-molecular-weight succinoglycan; the succinyl modification was missing, as was expected from the results of previous studies . The implications of these observations with regard to nodule entry are discussed.

J Bacteriol, 1988 Aug, 170(8), 3396 - 403
Mutants of Rhizobium meliloti defective in succinate metabolism; Finan TM et al.; We characterized mutants of Rhizobium meliloti SU47 that were unable to grow on succinate as the carbon source . The mutants fell into five groups based on complementation of the succinate mutations by individual recombinant plasmids isolated from a R . meliloti clone bank . Enzyme analysis showed that mutants in the following groups lacked the indicated common enzyme activities: group II, enolase (Eno); group III, phosphoenolpyruvate carboxykinase (Pck); group IV, glyceraldehyde-3-phosphate dehydrogenase (Gap), and 3-phosphoglycerate kinase (Pgk) . Mutants in groups I and V lacked C4-dicarboxylate transport (Dct-) activity . Wild-type cells grown on succinate as the carbon source had high Pck activity, whereas no Pck activity was detected in cells that were grown on glucose as the carbon source . It was found that in free-living cells, Pck is required for the synthesis of phosphoenolpyruvate during gluconeogenesis . In addition, the enzymes of the lower half of the Embden-Meyerhoff-Parnas pathway were absolutely required for gluconeogenesis . Eno, Gap, Pck, and one of the Dct loci (ntrA) mapped to different regions of the chromosome; the other Dct locus was tightly linked to a previously mapped thi locus, which was located on the megaplasmid pRmeSU47b.

Eur J Biochem, 1988 Jul 15, 175(1), 141 - 9
Amino acid sequences of hemoglobins I and II from root nodules of the non-leguminous Parasponia rigida-rhizobium symbiosis, and a correction of the sequence of hemoglobin I from Parasponia andersonii; Kortt AA et al.; The amino acid sequence of hemoglobins I (pI 6.15 as oxyhemoglobin) and II (pI 5.64 as oxyhemoglobin) from the nitrogen-fixing root nodules of Parasponia rigida have been determined by protein sequencing . The sequence of hemoglobin I (pI 6.16, as oxyhemoglobin) from Parasponia andersonii was re-examined and the corrected primary structure, now in agreement with that predicted from the DNA sequence, is reported . The three Parasponia hemoglobins contain 161 amino acid residues (Mr approximately equal to 18,700 including the heme) with a single cysteine residue and five methionine residues . The N-terminal serine is blocked by an acetyl group . The primary structure of the Parasponia hemoglobins is highly conserved . Hemoglobins I from the two species of Parasponia are identical; both show microheterogeneity at position 30 (Asp/Glu substitution) and hemoglobin I from P . rigida shows microheterogeneity at position 150 (Ala/Val) while hemoglobin I from P . andersonii has only an Ala at 150 . P . rigida hemoglobin II shows no microheterogeneity at these positions, having Asp and Val residues respectively, and it contains a single amino acid change of a Gln for an Arg at position 85, which accounts for the 0.5 unit difference in isoelectric point observed between hemoglobins I and II . The sequence data are consistent with allelic heterogeneity at a single locus rather than different genes.

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