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, in