Gene, 1995 Aug 30, 162(1), 21 - 7 Characterization of the cmcH genes of Nocardia lactamdurans and Streptomyces clavuligerus encoding a functional 3'-hydroxymethylcephem O-carbamoyltransferase for cephamycin biosynthesis; Coque JJ et al.; Sequencing of ORF10 (gene cmcH) of the Nocardia lactamdurans cephamycin gene cluster proved that it encodes a protein with a deduced molecular mass of 57,149 Da . This protein showed significant similarity to the putative O-carbamoyltransferases (O-Cases) encoded by the nodU genes of Rhizobium fredii and Bradyrhizobium japonicum, involved in the synthesis of nodulation factors . The carbamoyl-phosphate (CP)-binding amino-acid sequence of human OTCase is conserved in the cmcH product . A similar cmcH (80% identify in a 160-nt fragment) in the cephamycin (CmC) cluster of cmc genes of Streptomyces clavuligerus was partially sequenced . The cmcH gene is closely linked to and in the same orientation as cefF in both organisms . Both cmcH were subcloned in pIJ702 and expressed in Streptomyces lividans . Extracts of transformants could carbamoylate decarbamoylcefuroxime . A similar cmcH was found by Southern hybridization in Streptomyces cattleya, but not in Streptomyces griseus or Streptomyces lipmanii which produce non-carbamoylated CmC. Gene, 1995 Aug 8, 161(1), 63 - 7 Sequences of nifX, nifW, nifZ, nifB and two ORF in the Frankia nitrogen fixation gene cluster; Harriott OT et al.; The actinomycete Frankia alni fixes N2 in root nodules of several non-leguminous plants . It is one of the few known N2-fixing members of the high-GC Gram+ lineage of prokaryotes . Thus, we have undertaken a study of its nitrogen fixation gene (nif) organization to compare with that of the more extensively characterized proteobacteria . A cosmid (pFN1) containing the nif region of Fa CpI1 was isolated from a cosmid library using the nifHDK genes of Fa CpI1 as a probe . A 4.5-kb BamHI fragment that mapped downstream from the previously characterized nifHDK genes was cloned and sequenced . Based on nt and aa sequence similarities to nif from other N2-fixing bacteria, eight ORF were identified and designated nifX, orf3, orf1, nifW, nifZ, nifB, orf2 and nifU . A region that hybridized to Rhizobium meliloti and Klebsiella pneumoniae nifA did not appear to contain a nifA-like gene . We have revised the map of the Fa nif region to reflect current information. Gene, 1995 Aug 8, 161(1), 33 - 8 Identification and activity of two insertion sequence elements in Rhodococcus sp . strain IGTS8; Denome SA et al.; Two putative insertion sequence (IS) elements, IS1166 and IS1295, were identified on a plasmid present in Rhodococcus sp . IGTS8 . Four copies of IS1166 were present in strain IGTS8: one copy on each of two separate plasmids and two copies on a third plasmid or in the chromosome . Of eight rhodococci tested, only R . zopfii contained a copy of an IS1166-like element . Two mutants of strain IGTS8 were isolated in which an additional copy of IS1166 was present, suggesting that at least one copy of this element may be able to transpose . IS1166 and IS1295 are new members of a family of IS elements which includes IS6120 from Mycobacterium smegmatis, IST2 from Thiobacillus ferrooxidans, IS256 from Staphylococcus aureus and ISRm3 from Rhizobium meliloti . The 24-25-bp inverted repeats of these six elements are highly similar and, with the exception of IS1295, their transposases exhibit moderate identities . In particular, the predicted amino-acid sequence of the transposase from IS1166 is 86% identical to that from the known transposable element IS6120. Gene, 1995 Aug 8, 161(1), 27 - 31 Rhizobium leguminosarum NodT is related to a family of outer-membrane transport proteins that includes TolC, PrtF, CyaE and AprF; Rivilla R et al.; Cells containing a protein fusion consisting of the Rhizobium leguminosarum bv . viciae nodulation protein, NodT, fused to PhoA, produced alkaline phosphatase activity, indicating that the N terminus of NodT could translocate PhoA across the inner membrane . Cellular fractionation suggested that the NodT::PhoA fusion is targetted to the outer membrane . NodT resembles a family of bacterial outer membrane proteins including TolC, PrtF, CyaE and AprF, which are involved in secretion . By analogy, NodT (together with the inner membrane putative transport proteins NodI and NodJ) is proposed to be involved in the secretion of nodulation factors. Mol Microbiol, 1995 Aug, 17(4), 687 - 99 In Rhizobium meliloti, the operon associated with the nod box n5 comprises nodL, noeA and noeB, three host-range genes specifically required for the nodulation of particular Medicago species; Ardourel M et al.; In Rhizobium meliloti, the genes required for nodulation of legume hosts are under the control of DNA regulatory sequences called nod boxes . In this paper, we have characterized three host-specific nodulation genes, which form a flavonoid-inducible operon down-stream of the nod box n5 . The first gene of this operon is identical to the nodL gene identified by Baev and Kondorosi (1992) in R . meliloti strain AK631 . The product of the second gene, NoeA, presents some homology with a methyltransferase . nodL mutants synthesize Nod factors lacking the O-acetate substituent . In contrast, in strains carrying a mutation in either noeA or noeB, no modification in Nod-factor structure or production could be detected . On particular hosts, such as Medicago littoralis, mutants of the n5 operon showed a very weak nodule-forming ability, associated with a drastic decrease in the number of infection threads, while nodulation of Medicago truncatula or Melilotus alba was not affected . Thus, nodL noeA and noeB are host-specific nodulation genes . By using a gain-of-function approach, we showed that the presence of nodL, and hence of O-acetylated Nod factors, is a major prerequisite for confering the ability to nodulate alfalfa upon the heterologous bacterium Rhizobium tropici. Arch Microbiol, 1995 Aug, 164(2), 142 - 51 A pAO1-encoded molybdopterin cofactor gene (moaA) of Arthrobacter nicotinovorans: characterization and site-directed mutagenesis of the encoded protein; Menendez C et al.; A gene homologous to moaA, the gene responsible for the expression of a protein involved in an early step in the synthesis of the molybdopterin cofactor of Escherichia coli, was found to be located 2.7-kb upstream of the nicotine dehydrogenase (ndh) operon on the catabolic plasmid pAO1 of Arthrobacter nicotinovorans . The MoaA protein, containing 354 amino acids, migrated on an SDS-polyacrylamide gel with an apparent molecular weight of 40,000, in good agreement with the predicted molecular weight of 38,880 . The pAO1-encoded moaA gene from A . nicotinovorans was expressed in E . coli as an active protein that functionally complemented moaA mutants . Its deduced amino acid sequence shows 43% identity to the E . coli MoaA, 44% to the NarAB gene product from Bacillus subtilis, and 42% to the gene product of two contiguous ORFs from Methanobacterium formicicum . N-terminal sequences, including the motif CxxxCxYC, are conserved among the MoaA and NarAB proteins . This motif is also present in proteins involved in PQQ cofactor synthesis in almost all the NifB proteins reported so far and in the fixZ gene product from Rhizobium leguminosarum . Mutagenesis of any of these three conserved cysteine residues to serine abolished the biological activity of MoaA, while substitution of the tyrosine by either serine, phenylalanine, or alanine did not alter the capacity of the protein to complement the moaA mutation in E . coli . A second Cys-rich domain with the motif FCxxC(13x)C is found close to the C-terminus of MoaA and NarAB proteins . These two Cys-rich sequences may be involved in the coordination of a metal ions . The pAO1 copy of moaA may not be unique in the A . nicotinovorans genome since the molybdopterin cofactor oxidation products were detected in cell extracts from a plasmidless strain. Plant Physiol, 1995 Aug, 108(4), 1587 - 95 Structural requirements of synthetic and natural product lipo-chitin oligosaccharides for induction of nodule primordia on Glycine soja; Stokkermans TJ et al.; Rhizobia synthesize a class of lipo-chitin oligosaccharides that induce root hair deformation and induce the initiation of nodule structures on legume roots . These lipo-chitin oligosaccharides are tetra- and penta-lipo-oligosaccharides of N-acetylglucosamine with an acyl substitution on the nonreducing end and are commonly known as Nod factors . In this study, we demonstrate that synthetic analogs of natural product Nod factors have the same biological activities . To determine structure-activity relationships, a collection of synthetic and natural product lipo-chitin oligosaccharides was assayed on Glycine soja . All biologically active lipo-chitin oligosaccharides induced both root hair deformation and nodule initiations on G . soja . The most active lipo-chitin oligosaccharides deformed root hairs at 10(-15) M and induced nodules at 1 ng of lipo-chitin oligosaccharide per spot inoculation . Plant responses demonstrate an interdependence of backbone length and the presence of substitutions on the reducing end . Lipo-chitin oligosaccharides containing four N-acetylglucosamine residues were active only without a reducing end modification, whereas lipo-chitin oligosaccharides containing five N-acetylglucosamine residues were active only with reducing end modification . The plant thus recognizes lipo-chitin oligosaccharides without reducing end substitutions despite the importance of these modifications for host range. Eur J Biochem, 1995 Aug 1, 231(3), 742 - 6 A Rhizobium meliloti ferredoxin (FdxN) purified from Escherichia coli donates electrons to Rhodobacter capsulatus nitrogenase; Riedel KU et al.; The fdxN gene from Rhizobium meliloti encoding a bacterial-type ferredoxin (FdxN) was expressed in Escherichia coli under the control of the lac promoter . The fdxN gene product was purified under anaerobic conditions by ion-exchange chromatography and gel-filtration steps using an antiserum raised against an FdxN-LacZ fusion protein as a detection system . The purified ferredoxin was shown to be identical to the predicted R . meliloti FdxN protein in its amino acid composition and N-terminal amino acid sequence . Chemical determination of the iron content revealed 8.6 +/- 0.6 mol Fe/mol FdxN . The ultraviolet/visible absorption spectrum of the FdxN protein in the oxidized form exhibited maxima at 284 nm and 378 nm, with an A378/A284 ratio of 0.7 . EPR spectroscopy revealed a rhombic signal when FdxN was partially reduced, and a broad signal indicative of spin-spin interaction when fully reduced, suggesting the presence of two Fe-S cluster/ferredoxin polypeptide . Our data suggest that FdxN contains two {4Fe-4S} clusters . Purified FdxN was able to mediate electron transport between illuminated chloroplasts and Rhodobacter capsulatus nitrogenase in vitro. Proc Natl Acad Sci U S A, 1995 Aug 1, 92(16), 7352 - 6 Lipid A biosynthesis in Rhizobium leguminosarum: role of a 2-keto-3-deoxyoctulosonate-activated 4' phosphatase; Price NP et al.; Lipid A from several strains of the N2-fixing bacterium Rhizobium leguminosarum displays significant structural differences from Escherichia coli lipid A, one of which is the complete absence of phosphate groups . However, the first seven enzymes of E . coli lipid A biosynthesis, leading from UDP-GlcNAc to the phosphorylated intermediate, 2-keto-3-deoxyoctulosonate (Kdo2)-lipid IVA, are present in R . leguminosarum . We now describe a membrane-bound phosphatase in R . leguminosarum extracts that removes the 4' phosphate of Kdo2-lipid IVA . The 4' phosphatase is selective for substrates containing the Kdo domain . It is present in extracts of R . leguminosarum biovars phaseoli, viciae, and trifolii but is not detectable in E . coli and Rhizobium meliloti . A nodulation-defective strain (24AR) of R . leguminosarum biovar trifolii, known to contain a 4' phosphatase residue on its lipid A, also lacks measurable 4' phosphatase activity . The Kdo-dependent 4' phosphatase appears to be a key reaction in a pathway for generating phosphate-deficient lipid A. J Bacteriol, 1995 Aug, 177(15), 4289 - 96 Suppression of the Fix- phenotype of Rhizobium meliloti exoB mutants by lpsZ is correlated to a modified expression of the K polysaccharide; Reuhs BL et al.; The rhizobial production of extracellular polysaccharide (EPS) is generally required for the symbiotic infection of host plants that form nodules with an apical meristem (indeterminate nodules) . One exception is Rhizobium meliloti AK631, an exoB mutant of Rm41, which is deficient in EPS production yet infects and fixes nitrogen (i.e., is Fix+) on alfalfa, an indeterminate nodule-forming plant . A mutation of lpsZ in AK631 results in a Fix- strain with altered phage sensitivity, suggesting that a cell surface factor may substitute for EPS in the alfalfa-AK631 symbiosis . Biochemical analyses of the cell-associated polysaccharides of AK631 and Rm5830 (AK631 lpsZ) demonstrated that the lpsZ mutation affected the expression of a surface polysaccharide that is analogous to the group II K polysaccharides of Escherichia coli; the polysaccharide contains 3-deoxy-D-manno-2-octulosonic acid or a derivative thereof in each repeating unit . Rm5830 produced a polysaccharide with altered chromatographic and electrophoretic properties, indicating a difference in the molecular weight range . Similar results were obtained in a study of Rm1021, a wild-type isolate that lacks the lpsZ gene: the introduction of lpsZ into Rm1021 exoB (Rm6903) both suppresses the Fix- phenotype and results in a modified expression of the K polysaccharide . Chromatography and electrophoresis analysis showed that the polysaccharide extracted from Rm6903 lpsZ+ differed from that of Rm6903 in molecular weight range . Importantly, the effect of LpsZ is not structurally specific, as the introduction lpsZ+ into Rhizobium fredii USDA257 also resulted in a molecular weight range change in the structurally distinct K polysaccharide produced by that strain . This evidence suggests that LpsZ has a general effect on the size-specific expression of rhizobial K polysaccharides. Can J Microbiol, 1995 Aug, 41(8), 674 - 84 The ntrBC genes of Azospirillum brasilense are part of a nifR3-like-ntrB-ntrC operon and are negatively regulated; Machado HB et al.; A cosmid able to complement the Nif- and nitrate-dependent growth phenotypes of the Azospirillum brasilense mutant FP9 was isolated from a genomic library of the wild-type strain FP2 . A 6-kb DNA region was sequenced and showed two open reading frames (ORFs) identified as the ntrB and ntrC genes . An ORF1 located upstream from the ntrB gene and coding for a 36-kDa polypeptide showed similarity to the nifR3 gene of Rhodobacter capsulatus and the ORF1 of Rhizobium leguminosarum, both located upstream from the ntrB gene in a complex operon . Two other unidentified ORFs (ORF5 and partial ORF4) coding for hydrophobic polypeptides were also observed . delta ORF1-ntrBC, ORF1, ntrB, and ntrC mutants obtained by recombination of suicide plasmids containing an insertion of a promoterless lacZ kanamycin cassette showed decreased nitrogenase activities and were unable to grow on nitrate as the sole N source . These phenotypes were restored by complementation with plasmids containing the ntrC gene . Analysis of lacZ transcriptional fusions suggested that the ORF1-ntrBC operon in Azospirillum brasilense is expressed from a promoter located upstream from the ORF1 and that it is negatively regulated by the ntrC gene product. Wei Sheng Wu Xue Bao, 1995 Aug, 35(4), 242 - 9 {Cloning and sequencing of ntrBC genes from Azospirillum brasilense}; Yan D et al.; A gene library of Azospirillum brasilense Yu62 was constructed in EMBL3 . The library was screened with PCR amplified fragment as a special probe . Ten positive plaques (EA1-EA10) were selected . Detection results showed they contained two different types of clones, representing as EA4 and EA9 respectively . Southern hybridization of EA4 displayed that target gene was located in a 2.9kb EcoRI fragment . Sequence of this fragment had allowed the position and identification of ntrC gene, which encoding a protein of 53469, consisted of 480 amino acids . In the upstream of ntrC, a complete ntrB coding region was also found, which encoding a protein of 43487, consisted of 400 amino acids . Homologous analysis of the deduced amino acid sequences of ntrC and ntrB from different bacteria demonstrated that A . brasilense was closer to Rhizobia than to other free-living diazotrophs. FEBS Lett, 1995 Jul 24, 368(3), 536 - 40 Rhizobium tropici nodulation factor sulfation is limited by the quantity of activated form of sulfate; Poupot R et al.; Rhizobium tropici is a broad host-range symbiont of Phaseolus vulgaris . This bacterium produces a mixture of sulfated and non-sulfated N-methylated pentameric nodulation (Nod) factors . To understand the genetic bases of the partial sulfation of R . tropici Nod factors, which might be involved in the broad host-range of this species, we introduced in R . tropici CFN299 the recombinant plasmid pGMI515 carrying a set of nodulation (nod) genes of R . meliloti, including those involved in the sulfation of R . meliloti Nod factors . The CFN299 (pGMI515) transconjugant produced only sulfated Nod factors, but approximately half of them were no more N-methylated . Mutations in R . meliloti nodH gene did not decrease the Nod factor sulfation whereas inactivation of the nodPQ genes restored the production of a mixture of sulfated and non-sulfated molecules . These results suggest that the limiting step in R . tropici Nod factor sulfation is the production of activated sulfate donors . Mutations in the R . meliloti nodFEG and nodH genes did not change the N-methylation pattern, whereas mutations in nodPQ increased the degree of N-methylation, suggesting a metabolic link between sulfation and methylation of R . tropici Nod factors. Mol Plant Microbe Interact, 1995 Jul-Aug, 8(4), 584 - 92 The enhancement of ammonium assimilation in Rhizobium etli prevents nodulation of Phaseolus vulgaris; Mendoza A et al.; The modification of the ammonium assimilation pathway of Rhizobium etli (GS-GOGAT) by adding an additional ammonium assimilation enzyme, GDH, strongly affects its symbiotic interaction with beans . The plasmid pAM1a, based in the stable vector pTR101 (M . Weinstein, R . C . Roberts, and D . R . Helsinki, J . Bacteriol . 174,7486-7489, 1992), containing the Escherichia coli gdhA gene flanked by two transcription-translation terminators was constructed . The expression of GDH in both, the wild type (CFN42/pAM1a) and a ntrC- mutant (CFN2012/pAM1a) R . etli strains, gave a similar metabolic effect, i.e., high GDH and reduced GOGAT activities, and an increased synthesis and excretion of several amino acids . The total inhibition of bean nodulation was observed when the minimum optimal inoculum of R . etli CFN42/pAM1a was used; however, an effective symbiosis occurred with the CFN2012/pAM1a mutant strain . While a total inhibition of the induction of the nodA gene by bean root exudate or by naringenin was observed in the CFN42/pAM1a strain, at 10 mM ammonium, the CFN2012/pAM1a showed an optimal nodA gene induction . A correlation between nodA gene induction, Nod factor production, and nodulation was observed . We conclude that in R . etli, there is a down-regulation of nod gene expression and nodulation when a high internal nitrogen content is built up by the presence of a functional GDH and that NtrC is involved in such regulation . An instability of the plasmid harboring the gdhA gene was observed during symbiosis, indicating a strong selection against cells containing this plasmid. Mol Plant Microbe Interact, 1995 Jul-Aug, 8(4), 549 - 59 Structural and functional conservation of the rhizopine catabolism (moc) locus is limited to selected Rhizobium meliloti strains and unrelated to their geographical origin; Rossbach S et al.; Rhizopine (L-3-O-methyl-scyllo-inosamine; 3-O-MSI) synthesis (mos) and catabolism (moc) genes were originally isolated from Rhizobium meliloti strain L5-30 (Murphy et al., Proc . Natl . Acad . Sci . U.S.A., 84:493, 1987) . These genes have been postulated to give a competitive advantage to this strain in the rhizosphere, since the ability to utilize the unusual nutritional mediator rhizopine as nitrogen and carbon source appears to be correlated with the ability of Moc+ bacteria to efficiently infect alfalfa plants . This study examines the distribution of rhizopine catabolism (moc) genes among different soil bacteria . By using oligonucleotide primers homologous to the moc genes and the polymerase chain reaction (PCR), moc genes were shown to be absent from a random collection of 100 different soil isolates . However, screening 50 different electrophoretic type strains of a worldwide R . meliloti collection (Eardly et al., Appl . Environ . Microbiol . 56:187, 1990) revealed the presence of moc genes in three additional strains, S33, 102F51, and 74B3 . These three strains were found to be able to synthesize rhizopine in planta (Mos+) and to catabolize it (Moc+) . To determine the relatedness of the Mos+/Moc+ strains to each other and to other R . meliloti strains, we used the rep-PCR method to generate genomic fingerprints, and to create a phylogenetic tree with the help of an optical imaging system and data analysis program (AMBIS) . Because of the apparent infrequent occurrence of moc genes among soil bacteria, we suggest that the use of moc genes as a selectable marker trait for tracking genetically manipulated organisms is feasible. Mol Plant Microbe Interact, 1995 Jul-Aug, 8(4), 492 - 8 Characterization of a Rhizobium meliloti proline dehydrogenase mutant altered in nodulation efficiency and competitiveness on alfalfa roots; Jimenez-Zurdo JI et al.; Rhizobium meliloti strain GRM8 is able to transform ornithine into proline by means of an ornithine cyclodeaminase and, therefore, has the ability to use either of these amino acids as its sole carbon and nitrogen source . By Tn5 insertion mutagenesis we obtained a GRM8 mutant derivative strain (LM1) unable to catabolize either ornithine or proline . DNA hybridization studies showed that the LM1 mutant carries a single Tn5 insertion within a chromosomally located gene that, as deduced from a partial nucleotide sequence, encodes a proline dehydrogenase (ProDH) . Enzymatic assays confirmed the lack of ProDH activity in cell extracts of strain LM1 and revealed that production of this enzyme is inducible in the parental strain by proline and ornithine . Ultrastructural nodule microscopy analysis, acetylene reduction assays, and dry-weight determinations of nodulated alfalfa plants showed no obvious defect in the nitrogen fixation process of the ProDH- mutant LM1 . However, nodulation tests and competition assays demonstrated that in R . meliloti ProDH is required for nodulation efficiency and competitiveness on alfalfa roots. Respir Physiol, 1995 Jul, 101(1), 1 - 10 Hypoxia-inducible gene expression; Fandrey J; When oxygen is lacking the cellular production of some hormones, cytokines and glycolytic enzymes can be dramatically increased by a hypoxia-induced increase in the expression of the respective genes that encode for these proteins . The most progress in understanding how the transcription of genes is increased under hypoxic conditions has been made by studying the hypoxia-inducible expression of the erythropoietin gene . Elucidating the oxygen sensitive enhancer elements of the erythropoietin gene has prompted studies on other oxygen-regulated genes . The transcription-regulating proteins that are induced with hypoxia bind to closely related regulatory DNA sequences that control the expression of the genes for erythropoietin, the vascular endothelial growth factor and a number of glycolytic enzymes . It became evident that the hypoxia-inducible enhancer may be part of a widespread oxygen-sensing mechanism acting in a wide variety of mammalian cells . Comparison with the oxygen sensor system in the bacterium Rhizobium meliloti revealed some similarities with the putative oxygen sensor in mammalian cells . This sensor is thought to respond to hypoxia by inducing the signalling cascade that results in binding of the transcription factors to their respective enhancer elements to induce transcription of the respective gene. Plant J, 1995 Jul, 8(1), 111 - 9 Early nodulin gene expression during Nod factor-induced processes in Vicia sativa; Vijn I et al.; Rhizobium leguminosarum bv . viciae-secreted Nod factors are able to induce root hair deformation, the formation of nodule primordia and the expression of early nodulin genes in Vicia sativa (vetch) . To obtain more insight into the mode of action of Nod factors the expression of early nodulin genes was followed during Nod factor-induced root hair deformation and nodule primordium formation . The results of these studies suggested that the expression of VsENOD5 and VsENOD12 is not required for root hair deformation . In the Nod factor-induced primordia both VsENOD12 and VsENOD40 are expressed in a spatially controlled manner similar to that found in Rhizobium-induced nodule primordia . In contrast, VsENOD5 expression has never been observed in Nod factor-induced primordia, showing that the induction of VsENOD5 and VsENOD12 expression are not coupled . VsENOD5 expression is induced in the root epidermis by Nod factors and in Rhizobium-induced nodule primordia only in cells infected by the bacteria, suggesting that the Nod factor does not reach the inner cortical cells. Appl Environ Microbiol, 1995 Jul, 61(7), 2775 - 9 Phylogenetic relationships and host range of Rhizobium spp . that nodulate Phaseolus vulgaris L; Hernandez-Lucas I et al.; We determined the nucleotide sequences of 16S rRNA gene segments from five Rhizobium strains that have been isolated from tropical legume species . All share the capacity to nodulate Phaseolus vulgaris L., the common bean . Phylogenetic analysis confirmed that these strains are of two different chromosomal lineages . We defined the host ranges of two strains of Rhizobium etli and three strains of R . tropici, comparing them with those of the two most divergently related new strains . Twenty-two of the 43 tested legume species were nodulated by three or more of these strains . All seven strains have broad host ranges that include woody species such as Albizia lebbeck, Gliricidia maculata, and Leucaena leucocephala. J Bacteriol, 1995 Jul, 177(14), 3979 - 84 Oxygen control of the Bradyrhizobium japonicum hemA gene; Page KM et al.; The hemA gene of Bradyrhizobium japonicum, which encodes the first enzyme in the heme biosynthetic pathway, is regulated by oxygen . Up to ninefold induction of beta-galactosidase activity is seen when cultures of B . japonicum containing either a plasmid-encoded or a chromosomally integrated hemA-lacZ fusion are shifted to restricted aeration . The oxygen effect is mediated via the FixLJ two-component regulatory system, which regulates the expression of a number of genes involved in the nitrogen fixation process in response to low-oxygen conductions; oxygen induction is lost when the hemA-lacZ fusion is expressed in strains of B . japonicum carrying mutations in fixL or fixJ . The B . japonicum hemA promoter region contains a sequence identical to the Escherichia coli Fnr binding site (positions -46 to -33 relative to the hemA transcription start site) . Fnr is a regulatory protein necessary for the oxygen-regulated expression of anaerobic respiratory genes . Activity of a hemA-lacZ fusion construct in which the Fnr box-like sequence was replaced with a BglII site is not induced in B . japonicum cultures grown under restricted aeration . The fnr homolog fixK is FixLJ dependent . Collectively, these data suggest a role for the rhizobial Fnr-like protein, FixK, in the regulation of hemA . Furthermore, the coregulation of hemA with symbiotically important genes via FixLJ is consistent with the idea that hemA is required in the nodule as well as under free-living conditions. Microbiology, 1995 Jul, 141 ( Pt 7), 1691 - 705 beta-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other gram-negative bacteria; Wilson KJ et al.; A series of transposons are described which contain the gusA gene, encoding beta-glucuronidase (GUS), expressed from a variety of promoters, both regulated and constitutive . The regulated promoters include the tac promoter which can be induced by IPTG, and nifH promoters which are symbiotically activated in legume nodules . One transposon contains gusA with a strong Shine-Dalgarno translation initiation context, but no promoter, and thus acts as a promoter-probe transposon . In addition, a gus operon deletion strain of Escherichia coli, and a transposon designed for use in chromosomal mapping using PFGE, are described . The GUS transposons are constructed in a mini-Tn5 system which can be transferred to Gram-negative bacteria by conjugation, and will form stable genomic insertions . Due to the absence of GUS activity in plants and many bacteria of economic importance, these transposons constitute powerful new tools for studying the ecology and population biology of bacteria in the environment and in association with plants, as well as for studies of the fundamental molecular basis of such interactions . The variety of assays available for GUS enable both quantitative assays and spatial localization of marked bacteria to be carried out. Microbiology, 1995 Jul, 141 ( Pt 7), 1683 - 90 Rhizobium meliloti lacking mosA synthesizes the rhizopine scyllo-inosamine in place of 3-O-methyl-scyllo-inosamine; Rao JP et al.; The Rhizobium meliloti Rm220-3 mos locus producing the rhizopine scyllo-inosamine (SI) in nodules is shown to consist of three ORFs (ORF1, mosB and mosC) arranged in an operon structure . This differs from the R . meliloti L5-30 mos locus, which produces 3-O-methyl-scyllo-inosamine (3-O-MSI), by the complete absence of mosA . The deletion covers a region of 1235 nt and includes the entire mosA gene as well as the sequence both upstream and downstream . As a result, Rm220-3 mos ORF1 shares a 5' region common with L5-30 ORF1 but includes an additional 10 bp insertion and a section in the L5-30 mosA and mosB intercistronic region . Antibodies against L5-30 Mos proteins detected MosB and MosC proteins in Rm220-3-induced nodules but no translation product for either ORF1 or mosA was detected . A construct prepared from the L5-30 mos locus which has a truncated mosA gene produces SI rather than 3-O-MSI, confirming this gene is involved in a methylation step in the production of 3-O-MSI . Further, changes made to this ORF result in reduced levels of the rhizopine. Mol Microbiol, 1995 Jul, 17(2), 387 - 97 NodS is an S-adenosyl-L-methionine-dependent methyltransferase that methylates chitooligosaccharides deacetylated at the non-reducing end; Geelen D et al.; In response to phenolic compounds exuded by the host plant, symbiotic Rhizobium bacteria produce signal molecules (Nod factors), consisting of lipochitooligosaccharides with strain-specific substitutions . In Azorhizobium caulinodans strain ORS571 these modifications are an O-arabinosyl group, an O-carbamoyl group, and an N-methyl group . Several lines of evidence indicate that the nodS gene located in the nodABCSUIJ operon is implicated in the methylation of Nod factors . Previously we have shown that NodS is an S-adenosyl-L-methionine (SAM)-binding protein, essential for the L-{3H-methyl}-methionine labelling of ORS571 Nod factors in vivo . Here, we present an in vitro assay showing that NodS from either A . caulinodans or Rhizobium species NGR234 methylates end-deacetylated chitooligosaccharides, using {3H-methyl}-SAM as a methyl donor . The enzymatic and SAM-binding activity were correlated with the nodS gene and localized within the soluble protein fraction . The A . caulinodans nodS gene was expressed in Escherichia coli and a glutathione-S-transferase-NodS fusion protein purified . This protein bound SAM and could methylate end-deacetylated chitooligosaccharides, but could not fully methylate acetylated chitooligosaccharides or unmethylated lipo-chitooligosaccharides . These data implicate that the methylation step in the biosynthesis pathway of ORS571 Nod factors occurs after deacetylation and prior to acylation of the chitooligosaccharides. Mol Microbiol, 1995 Jul, 17(2), 357 - 66 Identification of a chemotaxis operon with two cheY genes in Rhodobacter sphaeroides; Ward MJ et al.; A large chemotaxis operon was identified in Rhodobacter sphaeroides WS8-N using a probe based on the 3' terminal portion of the Rhizobium meliloti cheA gene . Two genes homologous to the enteric cheY were identified in an operon also containing cheA, cheW, and cheR homologues . The deduced protein sequences of che gene products were aligned with those from Escherichia coli and shown to be highly conserved . A mutant with an interrupted copy of cheA showed normal patterns of swimming, unlike the equivalent mutants in E . coli which are smooth swimming . Tethered cheA mutant cells showed normal responses to changes in organic acids, but increased, inverted responses to sugars . The unusual behaviour of the cheA mutant and the identification of two homologues of cheY suggests that R . sphaeroides has at least two pathways controlling motor activity . To identify functional similarity between the newly identified R . sphaeroides Che pathway and the methyl-accepting chemotaxis protein (MCP)-dependent pathway in enteric bacteria, the R . sphaeroides cheW gene was expressed in a cheW mutant strain of E . coli and found to complement, causing a partial return to a swarming phenotype . In addition, expression of the R . sphaeroides gene in wild-type E . coli resulted in the same increased tumbling and reduced swarming as seen when the native gene is overexpressed in E . coli . The identification of che homologues in R . sphaeroides and complementation by cheW suggests the presence of MCPs in an organism previously considered to use only MCP-independent sensing . The MCP-dependent pathway, appears conserved.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1995 Jun 26, 367(2), 180 - 2 Behavior of Rhizobium meliloti in oxygen gradients; Zhulin IB et al.; Rhizobium meliloti cells responded to an abrupt change in oxygen concentration by changing the cell speed (chemokinesis), but they did not alter the frequency at which swimming cells stopped briefly (aerotaxis) . Changes in cell speed upon stimulation with oxygen coincided with changes in membrane potential . The cells did not form an aerotactic band in a spatial gradient of oxygen as do the cells of other bacterial species . The fixL and fixJ genes which encode a heme-containing protein kinase that senses oxygen and a response regulator, respectively, were not involved in the behavior of R . meliloti in oxygen gradients. Nature, 1995 Jun 15, 375(6532), 577 - 81 Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation; Mukhopadhyay D et al.; Angiogenesis, the formation of new microvasculature by capillary sprouting, is crucial for tumour development . Hypoxic regions of solid tumours produce the powerful and directly acting angiogenic protein VEGF/VPF (vascular endothelial growth factor/vascular permeability factor) . We now investigate the signal transduction pathway involved in hypoxic induction of VEGF expression . Hypoxia is known to induce a tyrosine kinase cascade that results in the activation of nitrogen-fixation genes in Rhizobium meliloti, and activation of tyrosine kinases is critical in signalling triggered by growth factors and ultraviolet light . We show here that genistein, an inhibitor of protein tyrosine kinase, blocks VEGF induction . Hypoxia increases the kinase activity of pp60c-src and its phosphorylation on tyrosine 416 but does not activate Fyn or Yes . Expression of either a dominant-negative mutant form of c-Src or of Raf-1 markedly reduces VEGF induction . VEGF induction by hypoxia in c-src(-) cells is impaired, although there is a compensatory activation of Fyn . Our results provide an insight into hypoxia-triggered intracellular signalling, define VEGF as a new downstream target for c-SRC, and suggest a role for c-SRc in promoting angiogenesis. J Biol Chem, 1995 Jun 9, 270(23), 13961 - 7 Characterization of chitin synthase 2 of Saccharomyces cerevisiae . Implication of two highly conserved domains as possible catalytic sites; Nagahashi S et al.; Chitin synthase 2 of Saccharomyces cerevisiae was characterized by means of site-directed mutagenesis and subsequent expression of the mutant enzymes in yeast cells . Chitin synthase 2 shares a region whose sequence is highly conserved in all chitin synthases . Substitutions of conserved amino acids in this region with alanine (alanine scanning) identified two domains in which any conserved amino acid could not be replaced by alanine to retain enzyme activity . These two domains contained unique sequences, Glu561-Asp562-Arg563 and Gln601-Arg602-Arg603-Arg604-Trp605, that were conserved in all types of chitin synthases . Glu561 or arginine at 563, 602, and 603 could be substituted by glutamic acid and lysine, respectively, without significant loss of enzyme activity . However, even conservative substitutions of Asp562 with glutamic acid, Gln601 with asparagine, Arg604 with lysine, or Trp605 with tyrosine drastically decreased the activity, but did not affect apparent Km values for the substrate significantly . In addition to these amino acids, Asp441 was also found in all chitin synthase . The mutant harboring a glutamic acid substitution for Asp441 severely lost activity, but it showed a similar apparent Km value for the substrate . Amounts of the mutant enzymes in total membranes were more or less the same as found in the wild type . Furthermore, Asp441, Asp562, Gln601, Arg604, and Trp605 are completely conserved in other proteins possessing N-acetylglucosaminyltransferase activity such as NodC proteins of Rhizobium bacterias . These results suggest that Asp441, Asp562, Gln601, Arg604, and Trp605 are located in the active pocket and that they function as the catalytic residues of the enzyme. Proc Natl Acad Sci U S A, 1995 Jun 6, 92(12), 5273 - 7 Symbiotic induction of a MADS-box gene during development of alfalfa root nodules; Heard J et al.; In response to infection by Rhizobium, highly differentiated organs called nodules form on legume roots . Within these organs, the symbiotic association between the host plant and bacteria is established . A putative plant transcription factor, NMH7, has been identified in alfalfa root nodules . nmh7 contains a MADS-box DNA-binding region and shows homology to flower homeotic genes . This gene is a member of a multigene family in alfalfa and was identified on the basis of nucleic acid homology to plant regulatory protein genes (MADS-box-containing genes) from Antirrhinum and Arabidopsis . RNA analysis and in situ hybridization showed that expression of this class of regulatory genes is limited to the infected cells of alfalfa root nodules and is likely to be involved in the signal transduction pathway initiated by the bacterial symbiont, Rhizobium meliloti . The expression of nmh7 in a root-derived organ is unusual for this class of regulatory genes. Mol Microbiol, 1995 Jun, 16(6), 1123 - 36 A central domain of Rhizobium NodE protein mediates host specificity by determining the hydrophobicity of fatty acyl moieties of nodulation factors; Bloemberg GV et al.; Previously, we have shown that the nodE gene is a major determinant of the difference in host range between Rhizobium leguminosarum biovars viciae and trifolii . A new genetic test system for stringent functional analysis of nodE genes was constructed . By testing chimeric nodE genes constructed by the exchange of polymerase chain reaction (PCR)-generated restriction cassettes, we show that a central domain, containing only 44 non-conserved amino acid residues, determines the host specificity of the NodE protein (401 amino acid residues) . Mass spectrometric analysis of the lipo-chitin oligosaccharides (LCOs) produced by the new test strain containing the biovar viciae nodE gene shows that molecules containing a polyunsaturated C18:4 (trans-2, trans-4, trans-6, cis-11-octadecatetraenoic) fatty acyl moiety are produced, as is the case for wild-type R . leguminosarum bv . viciae . The LCOs determined by the biovar trifolii nodE gene, which was overproduced in our test strain, carry C18:2 and C18:3 fatty acyl chains containing two or three conjugated trans double bonds, respectively . Therefore, the main difference between the nodE-determined LCOs of biovar viciae and trifolii in this system is the presence or absence of one cis double bond, resulting in the very different hydrophobicity of the LCOs . Using a newly developed spot application assay, we show that the C18:2- and C18:3-containing LCOs are able to induce the formation of nodule primordia on roots of Trifolium pratense . On the basis of these and other recent results, we propose that the host range of nodulation of the R . leguminosarum biovars viciae and trifolii is determined by the degree of hydrophobicity of the polyunsaturated fatty acyl moieties of their LCOs, which is mediated by the host-specific central domain of the NodE protein. J Bacteriol, 1995 Jun, 177(11), 3133 - 42 Characterization, nucleotide sequence, and conserved genomic locations of insertion sequence ISRm5 in Rhizobium meliloti; Laberge S et al.; A target for ISRm3 transposition in Rhizobium meliloti IZ450 is another insertion sequence element, named ISRm5 . ISRm5 is 1,340 bp in length and possesses terminal inverted repeats of unequal lengths (27 and 28 bp) and contain five mismatches . An open reading frame that spans 89% of the length of one DNA strand encodes a putative transposase with significant similarity to the putative transposases of 11 insertion sequence elements from diverse bacterial species, including ISRm3 from R . meliloti . Multiple copies and variants of ISRm5 occur in the R . meliloti genome, often in close association with ISRm3 . Five ISRm5 copies in two strains were studied, and each was found to be located between 8-bp direct repeats . At two of these loci, which were shown to be highly conserved in R . meliloti, the copies of ISRm5 were found to be associated with pairs of short inverted repeats resembling transcription terminators . This structural arrangement not only may provide a conserved niche for ISRm5 but also may be a preferred target for transposition. J Bacteriol, 1995 Jun, 177(11), 3058 - 66 Fermentative and aerobic metabolism in Rhizobium etli; Encarnacion S et al.; Strains of Rhizobium etli, Rhizobium meliloti, and Rhizobium tropici decreased their capacity to grow after successive subcultures in minimal medium, with a pattern characteristic for each species . During the growth of R . etli CE 3 in minimal medium (MM), a fermentation-like response was apparent: the O2 content was reduced and, simultaneously, organic acids and amino acids were excreted and poly-beta-hydroxybutyrate (PHB) was accumulated . Some of the organic acids excreted into the medium were tricarboxylic acid (TCA) cycle intermediates, and, concomitantly, the activities of several TCA cycle and auxiliary enzymes decreased substantially or became undetectable . Optimal and sustained growth and a low PHB content were found in R . etli CE 3 when it was grown in MM inoculated at a low cell density with O2 maintained at 20% or with the addition of supplements that have an effect on the supply of substrates for the TCA cycle . In the presence of supplements such as biotin or thiamine, no amino acids were excreted and the organic acids already excreted into the medium were later reutilized . Levels of enzyme activities in cells from supplemented cultures indicated that carbon flux through the TCA cycle was maintained, which did not happen in MM . It is proposed that the fermentative state in Rhizobium species is triggered by a cell density signal that results in the regulation of some of the enzymes responsible for the flux of carbon through the TCA cycle and that this in turn determines how much carbon is available for the synthesis and accumulation of PHB . The fermentative state of free-living Rhizobium species may be closely related to the metabolism that these bacteria express during symbiosis. Mol Gen Genet, 1995 May 20, 247(4), 423 - 9 Isolation and characterisation of genes for sulphate activation and reduction in Aspergillus nidulans: implications for evolution of an allosteric control region by gene duplication; Borges-Walmsley MI et al.; A region of the Aspergillus nidulans genome carrying the sA and sC genes, encoding PAPS reductase and ATP sulphurylase, respectively, was isolated by transformation of an sA mutant with a cosmid library . The genes were subcloned and their functions confirmed by retransformation and complementation of A . nidulans strains carrying sA and sC mutations . The physical distance of 2 kb between the genes corresponds to a genetic distance of 1 cM . While the deduced amino acid sequence of the sA gene product shows homology with the equivalent MET16 gene product of Saccharomyces cerevisiae, the sC gene product resembles the equivalent MET3 yeast gene product at the N-terminal end, but differs markedly from it at the C-terminal end, showing homology to the APS kinases of several microorganisms . It is proposed that this C-terminal region does not encode a functional APS kinase, but is responsible for allosteric regulation by PAPS of the sulphate assimilation pathway in A . nidulans, and that the ATP sulphurylase encoding-gene (sC) of filamentous ascomycetes may have evolved from a bifunctional gene similar to the nodQ gene of Rhizobium meliloti. J Biol Chem, 1995 May 19, 270(20), 11783 - 8 Lipopolysaccharide core structures in Rhizobium etli and mutants deficient in O-antigen; Carlson RW et al.; Lipopolysaccharide (LPS) is a major component of the bacterial outer membrane, and for Rhizobium spp . has been shown to play a critical role in the establishment of an effective nitrogen-fixing symbiosis with a legume host . Many genes required for O-chain polysaccharide synthesis are in the lps alpha region of the CE3 genome; this region may also carry lps genes required for core oligosaccharide synthesis . The LPSs from several strains mutated in the alpha region were isolated, and their mild acid released oligosaccharides, purified by high performance anion-exchange chromatography, were characterized by electrospray- and fast atom bombardment-mass spectrometry, NMR, and methylation analysis . The LPSs from several mutants contained truncated O-chains, and the core region consisted of a (3-deoxy-D-manno-2-octulosomic acid) (Kdo)-(2-->6)-alpha-Galp-(1-->6)-{alpha-GalpA-(1-->4)}-alpha-Ma np-(1-->5)- Kdop (3-deoxy-D-manno-2-octulosomic acid) (Kdo)pentasaccharide and a alpha-GalpA-(1-->4)-{alpha-GalpA-(1-->5)}-Kdop trisaccharide . The pentasaccharide was altered in two mutants in that it was missing either the terminal Kdo or the GalA residue . These results indicate that the lps alpha region, in addition to having the genes for O-chain synthesis, contains genes required for the transfer of these 2 residues to the core region . Also, the results show that an LPS with a complete core but lacking an O-chain polysaccharide is not sufficient for an effective symbiosis. FEMS Microbiol Lett, 1995 May 15, 128(3), 255 - 63 Construction and characterization of a Rhizobium leguminosarum biovar viciae strain designed to assess horizontal gene transfer in the environment; Selbitschka W et al.; An integration vector was developed which inserts cloned DNA in a non-essential site of the Rhizobium leguminosarum biovar viciae chromosome . The expression of integrated genes is under the control of the constitutive neomycin phosphotransferase II (nptII) promoter of transposon Tn5 . The design of the vector ensures that loss of vector sequences can be detected, enabling selection of progeny containing only the requisite DNA . The newly constructed vector was employed to insert the Escherichia coli gusA gene conferring GUS activity into R . leguminosarum bv . viciae strain LRS39401 which is cured of its symbiotic plasmid (pSym) . One GUS-positive transconjugant, strain CT0370, was shown to have lost all vector sequences . Conjugal transfer of pSym2004 (a Tn5-tagged derivative of symbiotic plasmid pRL1JI, which specifies pea nodulation and symbiotic nitrogen fixation) to CT0370, restored the GUS-positive strain's symbiotic proficiency . Strain CT0370 is presently being used in a field release experiment in order to assess the extent of pSym transfer in a natural R . leguminosarum bv . viciae population under environmental conditions. FEMS Microbiol Lett, 1995 May 15, 128(3), 241 - 5 NtrBC-dependent expression from the Rhizobium meliloti dctA promoter in Escherichia coli; Allaway D et al.; Effects of the two-component sensor-regulator pairs DctBD and NtrBC upon the expression of a dctA::phoA fusion from Rhizobium meliloti were determined under excess and limiting nitrogen concentrations in Escherichia coli . Results indicated that NtrBC affected transcription from the dctA promoter on a number of regulatory levels and under different physiological conditions in the heterologous host . However, NtrBC-dependent cross-talk was not observed in free-living R . meliloti under the conditions tested . Comparisons of the predicted amino acid sequences of DctD and NtrC from various sources indicated a specific region of the NtrC from rhizobia, which may have diverged from a consensus NtrC/DctD sequence to minimise interference between the two component systems, NtrBC and DctBD. J Bacteriol, 1995 May, 177(10), 2892 - 900 The dnaA gene of Rhizobium meliloti lies within an unusual gene arrangement; Margolin W et al.; Rhizobium meliloti exists either as a free-living soil organism or as a differentiated endosymbiont bacteroid form within the nodules of its host plant, alfalfa (Medicago sativa), where it fixes atmospheric N2 . Differentiation is accompanied by major changes in DNA replication and cell division . In addition, R . meliloti harbors three unique large circular chromosome-like elements whose replication coordination may be complex . As part of a study of DNA replication control in R . meliloti, we isolated a dnaA homolog . The deduced open reading frame predicts a protein of 57 kDa that is 36% identical to the DnaA protein of Escherichia coli, and the predicted protein was confirmed by immunoblot analysis . In a comparison with the other known DnaA proteins, this protein showed the highest similarity to that of Caulobacter crescentus and was divergent in some domains that are highly conserved in other unrelated species . The dnaA genes of a diverse group of bacteria are adjacent to a common set of genes . Surprisingly, analysis of the DNA sequence flanking dnaA revealed none of these genes, except for an rpsT homolog, also found upstream of dnaA in C . crescentus . Instead, upstream of rpsT lie homologs of fpg, encoding a DNA glycosylase, and fadB1, encoding an enoyl-coenzyme A hydratase with a strikingly high (53 to 55%) level of predicted amino acid identity to two mammalian mitochondrial homologs . Downstream of dnaA, there are two open reading frames that are probably expressed but are not highly similar to any genes in the databases . These results show that R . meliloti dnaA is located within a novel gene arrangement. FEMS Microbiol Lett, 1995 May 1, 128(2), 177 - 84 Cloning of the second adenylate cyclase gene (cya2) from Rhizobium meliloti F34: sequence similarity to eukaryotic cyclases; Archdeacon J et al.; A second adenylate cyclase (cya2) gene was isolated from a Rhizobium meliloti F34 gene bank . Complemented E . coli delta cya mutants were capable of utilizing a number of, but not all, carbon sources known to be regulated by cAMP . DNA hybridization studies showed cya2 to be unique to R . meliloti strains . The cya2 nucleotide sequence was determined and found to encode a protein of 363 amino acids . Residues were identified within the C-terminal domain which are conserved in both eukaryotic adenylate and guanylate cyclases, including a putative ATP binding site . Similar residues were also found in the prokaryotic R . meliloti Cya1 protein . A R . meliloti cya1/cya2 double mutant was constructed and characterized; however, cAMP production was still observed in this strain indicating the presence of a third cya gene. Mol Plant Microbe Interact, 1995 May-Jun, 8(3), 468 - 72 Identification and characterization of a Rhizobium leguminosarum bv . phaseoli gene that is important for nodulation competitiveness and shows structural homology to a Rhizobium fredii host-inducible gene; Michiels J et al.; DNA sequence analysis of a 1.4-kb SalI-HindIII segment located approximately 2 kb upstream of the Rhizobium leguminosarum bv . phaseoli syrM gene revealed the presence of an open reading frame (ORF3) encoding a putative 295-amino acid polypeptide with a molecular mass of 33,401 Da . ORF3 is homologous to a R . fredii host-inducible gene . The proteins encoded by R . l . bv . phaseoli ORF3 and by the R . fredii host-inducible gene share 37% sequence identity . In contrast to the R . fredii host-inducible gene, expression of ORF3 is not induced in the presence of Phaseolus vulgaris root exudates or by specific flavonoids, able to induce nodulation genes in R . l . bv . phaseoli . A R . l . bv . phaseoli ORF3 mutant was constructed by site-directed deletion/replacement mutagenesis . This mutant strain is not affected in symbiotic nitrogen fixation but exhibits a delay in nodulation on Phaseolus vulgaris . Moreover, this mutant was shown to be defective in competition for nodulation. Appl Environ Microbiol, 1995 May, 61(5), 1822 - 7 Identification of Rhizobium spp . in peat-based inoculants by DNA hybridization and PCR and its application in inoculant quality control; Tas E et al.; Procedures based on DNA hybridization and PCR were developed for quality control of Rhizobium inoculants . Inoculants for pea and goat's rue were produced by Elomestari Ltd., Juva, Finland, in sterile dry fine peat by the standard procedure used by the company . The inoculants contained Rhizobium galegae HAMBI 1174 and HAMBI 1207 and an R . leguminosarum biovar vicia strain, 16HSA, either solely or in combinations of two or three strains . DNA was isolated from 1-g samples of each peat inoculant and analyzed by nonradioactive DNA-DNA hybridization and by PCR . The hybridization probes were total DNAs from pure cultures of R . galegae HAMBI 1207 and R . leguminosarum biovar viciae 16HSA and a 264-bp strain-specific fragment from the genome of R . galegae HAMBI 1174 . The total DNA probes distinguished inoculants containing R . galegae or R . leguminosarum, and the strain-specific probe distinguished inoculants containing R . galegae HAMBI 1174 . The hybridization results for R . galegae were verified in a PCR experiment by amplifying an R . galegae species-specific fragment and an R . galegae HAMBI 1174 strain-specific fragment in the same reaction . When suitable probes and primers are available, the methods described here offer promising alternatives for the quality control of peat-based inoculants. Plasmid, 1995 May, 33(3), 226 - 31 Construction and properties of cloning vectors based on a 7.2-kb Rhizobium meliloti cryptic plasmid; Froissard D et al.; Cloning vectors (pFD1001, pFD1192, pFD1194, and pFD1212) were constructed by extension of the host range of a 7.2-kb Rhizobium meliloti cryptic plasmid (pRM1132f) with the ColE1-based plasmids, pBR322, pACYC177, pACYC 184, pSUP301, or pHC179; mobilization was facilitated by introduction of the oriT region from pRK2, a broad-host-range plasmid . The vector plasmids transferred readily into a wide range of gram-negative bacteria and had relatively low copy number in R . meliloti; two constructs, pFD1001 and pFD1212, were completely stable in R . meliloti isolated from nodules of alfalfa (Medicago sativa) . A representative of the vector constructs (pFD1001) could be maintained in R . meliloti in the presence of the broad-host-range shuttle plasmid pRK290 . These two vector plasmids could be introduced into R . meliloti, either simultaneously or singly when pRK290 was the resident plasmid; however, entry of pRK290 was blocked when pFD1001 was the resident plasmid . The cloning vectors constructed in this study should prove to be useful for the genetic manipulation of Rhizobium. Mol Microbiol, 1995 May, 16(4), 657 - 67 Organization of host-inducible transcripts on the symbiotic plasmid of Rhizobium sp . NGR234; Fellay R et al.; In a systematic approach to identify genes involved in the early steps of the legume-Rhizobium symbiosis, we studied transcription patterns of symbiotic plasmid-borne loci . A competitive hybridization procedure was used to identify DNA restriction fragments carrying genes whose expression is enhanced by plant root exudates or by purified flavonoids . Fragments containing induced genes were then located on the physical map of the 500 kb pNGR234a . New inducible loci as well as previously described genes were identified and their time course of induction determined . After initial induction, transcription of loci such as nodABC and the host-specificity genes nodSU decreased to undetectable levels 24 h after incubation with purified flavonoids . In contrast, expression of other loci is detectable only after several hours of induction . Surprisingly, many genes remained transcribed in the nodD1- mutant suggesting the presence of other flavonoid-dependent activators in NGR234 . The hsnl region, which is involved in host specificity, was shown to carry several inducible but independently regulated transcripts . Sequencing analysis revealed several open reading frames whose products, based on sequence similarities, may be involved in L-fucose metabolism and its adjunction to the Nod factors. Proc Natl Acad Sci U S A, 1995 Apr 25, 92(9), 3759 - 63 Oxygen as a key developmental regulator of Rhizobium meliloti N2-fixation gene expression within the alfalfa root nodule; Soupene E et al.; The symbiotic pattern of expression of Rhizobium meliloti N2-fixation genes is tightly coupled with the histological organization of the alfalfa root nodule and thus is under developmental control . N2-fixation gene expression is induced very sharply at a particular zone of the nodule called interzone II-III that precedes the zone where N2 fixation takes place . We show here that this coupling can be disrupted, hereby resulting in ectopic expression of N2-fixation genes in the prefixing zone II of the nodule . Uncoupling was obtained either by using a R . meliloti strain in which a mutation rendered N2-fixation gene expression constitutive with respect to oxygen in free-living bacterial cultures or by placing nodules induced by a wild-type R . meliloti strain in a microoxic environment . These results implicate oxygen as a key determinant of the symbiotic pattern of N2-fixation gene expression. Proc Natl Acad Sci U S A, 1995 Apr 11, 92(8), 3498 - 501 Synthesis of "Nod"-like chitin oligosaccharides by the Xenopus developmental protein DG42; Semino CE et al.; The Xenopus DG42 gene is expressed only between the late midblastula and neurulation stages of embryonic development . Recent database searches show that DG42 has striking sequence similarity to the Rhizobium NodC protein . NodC catalyzes the synthesis of chitin oligosaccharides which subsequently are transformed into bacterium-plant root signaling molecules . We find that the DG42 protein made in an in vitro coupled transcription-translation system catalyzes the synthesis of an array of chitin oligosaccharides . The result suggests the intriguing possibility that a bacterium-plant type of "Nod" signaling system may operate during early stages of vertebrate embryonic development and raises issues about the use of chitin synthase inhibitors as fungal-specific drugs. Mol Gen Genet, 1995 Apr 10, 247(1), 39 - 47 The cycHJKL genes of Rhizobium meliloti involved in cytochrome c biogenesis are required for "respiratory" nitrate reduction ex planta and for nitrogen fixation during symbiosis; Kereszt A et al.; We report the genetic and biochemical analysis of Rhizobium meliloti mutants defective in symbiotic nitrogen fixation (Fix-) and "respiratory" nitrate reduction (Rnr-) . The mutations were mapped close to the ade-1 and cys-46 chromosomal markers and the mutated locus proved to be identical to the previously described fix-14 locus . By directed Tn5 mutagenesis, a 4.5 kb segment of the chromosome was delimited in which all mutations resulted in Rnr- and Fix- phenotypes . Nucleotide sequence analysis of this region revealed the presence of four open reading frames coding for integral membrane and membrane-anchored proteins . Biochemical analysis of the mutants showed that the four proteins were necessary for the biogenesis of all cellular c-type cytochromes . In agreement with the nomenclature proposed for rhizobial genes involved in the formation of c-type cytochromes, the four genes were designated cycH, cycJ, cycK, and cycL, respectively . The predicted protein product of cycH exhibited a high degree of similarity to the Bradyrhizobium japonicum counterpart, while CycK and CycL shared more than 50% amino acid sequence identity with the Rhodobacter capsulatus Cc11 and Cc12 proteins, respectively . cycJ encodes a novel membrane anchored protein of 150 amino acids . We suggest that this gene cluster codes for (parts of) a multisubunit cytochrome c haem lyase . Moreover, our results indicate that in R . meliloti c-type cytochromes are required for respiratory nitrate reduction ex planta, as well as for symbiotic nitrogen fixation in root nodules. Biochemistry, 1995 Apr 4, 34(13), 4467 - 77 Common links in the structure and cellular localization of Rhizobium chitolipooligosaccharides and general Rhizobium membrane phospholipid and glycolipid components; Cedergren RA et al.; Several common links between the structural chemistry of the chitolipooligosaccharides of Rhizobium and the general rhizobial membrane lipid and lipopolysaccharide chemistry of these bacteria have been uncovered . Aspects of common chemistry include sulfation, methylation, and the position and extent of fatty acyl chain unsaturation . We find that bacteria which are known to synthesize sulfated chitolipooligosaccharides (such as Rhizobium meliloti strains and the broad-host-range Rhizobium species strain NGR234) also have sulfated lipopolysaccharides . Their common origins of sulfation have been demonstrated by using mutants which are known to be impaired in sulfating their chitolipooligosaccharides . In such cases, there is a corresponding diminution or complete lack of sulfation of the lipopolysaccharides . The structural diversity of the fatty acids observed in the chitolipooligosaccharides is also observed in the other membrane lipids . For instance, the doubly unsaturated fatty acids which are known to be predominant components of R . meliloti chitolipooligosaccharides were also found in the usual phospholipids and glycolipids . Also, the known functionalization of the chitolipooligosaccharides of R . sp . NGR234 by O- and N-methylation was also reflected in the lipopolysaccharide of this organism . The common structural features of chitolipooligosaccharides and membrane components are consistent with a substantial degree of biosynthetic overlap and a large degree of cellular, spatial overlap between these molecules . The latter aspect is clearly demonstrated here since we show that the chitolipooligosaccharides are, in fact, normal membrane components of Rhizobium . This increases the importance of understanding the role of the bacterial cell surface chemistry in the Rhizobium/legume symbiosis and developing a comprehensive understanding of the highly integrated membrane lipid and glycolipid chemistry of Rhizobium. Plant Foods Hum Nutr, 1995 Apr, 47(3), 257 - 63 Effect of nitrogen fixation, nitrogen fertilization and viral infection on yield, tannin and protein contents and in vitro protein digestibility of faba bean; Babiker EE et al.; A field investigation of two faba bean cultivars (cv.), Agabat and Silaim, showed that bean yellow mosaic virus (BYMV) infection reduced (p < or = 0.001) yield (Kg/ha), protein content and in vitro protein digestibility (IVPD) but increased (p < or = 0.05) tannin content (mg/100 ml) . Nitrogen fertilization with viral infection significantly reduced yield and IVPD for cv . Silaim and increased (p < or = 0.05) protein and tannin contents . Nitrogen fertilization alone was found to increase (p < or = 0.05) yield, protein and tannin contents but slightly reduced IVPD . Rhizobium inoculation with viral infection significantly decreased yield per unit area, protein content and IVPD, but increased (p < or = 0.05) tannin content . Rhizobium inoculation alone significantly increased (p < or = 0.001) yield and tannin content and slightly increased protein content but decreased IVPD . The results indicated that nitrogen fertilization or nitrogen fixation increased yield, protein and tannin contents and decreased IVPD . Viral infection had an adverse effect on yield, protein content and IVPD but had no effect on tannin content. Mol Microbiol, 1995 Apr, 16(2), 191 - 203 Low-molecular-weight succinoglycan is predominantly produced by Rhizobium meliloti strains carrying a mutated ExoP protein characterized by a periplasmic N-terminal domain and a missing C-terminal domain; Becker A et al.; The membrane topology of the Rhizobium meliloti 2011 ExoP protein involved in polymerization and export of succinoglycan was analysed by translational fusions of lacZ and phoA reporter genes to the exoP gene . Based on this analysis, the ExoP protein could be divided into an N-terminal domain mainly located in the periplasmic space and a C-terminal domain located in the cytoplasm . Whereas the C-terminal domain of ExoP is characterized by a potential nucleotide-binding motif, the N-terminal ExoP domain contains the sequence motif 'PX2PX4SPKX11GXMXG', which is also present in proteins involved in the determination of O-antigen chain length . R . meliloti strains carrying mutated exoP* genes, exclusively encoding the N-terminal ExoP domain, produced a reduced amount of succinoglycan . This reduction could be suppressed by a mutation in the regulatory gene exoR . The ratio of low-molecular-weight to high-molecular-weight succinoglycan was significantly increased in the exoP* mutant strain . In the exoP*/exoR mutant strain only low-molecular-weight succinoglycan could be detected . Based on sequence homologies and similar hydropathic profiles, the N-terminal domain of ExoP was proposed to be a member of a protein family thought to be involved in polysaccharide chain-length determination. Proc Natl Acad Sci U S A, 1995 Mar 28, 92(7), 2706 - 9 In vitro sulfotransferase activity of Rhizobium meliloti NodH protein: lipochitooligosaccharide nodulation signals are sulfated after synthesis of the core structure; Schultze M et al.; The Rhizobium common nod gene products NodABC are involved in the synthesis of the core lipochitooligosaccharide (Nod factor) structure, whereas the products of the host-specific nod genes are necessary for diverse structural modifications, which vary in different Rhizobium species . The sulfate group attached to the Rhizobium meliloti Nod signal is necessary for activity on the host plant alfalfa, while its absence renders the Nod factor active on the non-host plant vetch . This substituent is therefore a major determinant of host specificity . The exact biosynthetic pathway of Nod factors has not been fully elucidated . In particular, it is not known why some chemical modifications are introduced with high fidelity whereas others are inaccurate, giving rise to a family of different Nod factor structures produced by a single Rhizobium strain . Using protein extracts and partially purified recombinant NodH protein obtained from Escherichia coli expressing the R . meliloti nodH gene, we demonstrate here NodH-dependent in vitro sulfotransferase activity . Kinetic analyses with Nod factors, chitooligosaccharides, and their deacetylated derivatives revealed that Nod factors are the preferred substrate for the sulfate transfer . Moreover, the tetrameric Nod factor, NodRm-IV, was a better substrate than the trimer, NodRm-III, or the pentamer, NodRm-V . These data suggest that the core lipochitooligosaccharide structure must be synthesized prior to its host-specific modification with a sulfate group . Since in R . meliloti tetrameric Nod factors are the most abundant and the most active ones, high affinity of NodH for the appropriate tetrameric substrate guarantees its modification and thus contributes to the fidelity of host-specific behavior. Biochemistry, 1995 Mar 21, 34(11), 3832 - 40 Structurally diverse chitolipooligosaccharide nod factors accumulate primarily in membranes of wild type Rhizobium leguminosarum biovar trifolii; Orgambide GG et al.; The general view on Rhizobium chitolipooligosaccharides (CLOS) is that they are made in very low levels as diffusible molecules and are primarily secreted by the bacteria into the extracellular milieu where they interact with the host . However, the structural and predicted physicochemical properties of these amphiphilic molecules led us to postulate that they should normally be targeted to bacterial membranes after synthesis . Thus, we analyzed membrane lipid extracts of Rhizobium leguminosarum bv . trifolii wild-type strain ANU843 cells and the corresponding culture supernatants for CLOS-type glycolipids . As predicted, fractionation of the membrane extracts from pelleted cells led to the isolation of a diverse family of CLOS in high yield (> or = 15 mg/L of culture), whereas all attempts to isolate CLOS from the corresponding culture supernatant failed . Structural analyses reveal that the membrane CLOS of ANU843 consist of a complex mixture of O-acetylated or non-O-acetylated chito- tri-, -tetra-, and pentasaccharides bearing an N-acyl moiety at the nonreducing glucosamine residue . cis-Vaccenic acid was the predominant acyl substituent (> 70%), but several other saturated, unsaturated, and 3-hydroxy fatty acids were found in the CLOS glycolipids . Membrane accumulation of CLOS in ANU843 is promoted by the presence of 4',7-dihydroxyflavone and pSym nod genes . Potential host-selective biological activity host-selective biological activity of the purified membrane CLOS fraction from ANU843 was indicated by its ability to elicit meristems resembling rudimentary nodule primordia in the root cortex of axenic seedlings of the host legume, white clover, but not of the nonhost legumes hairy vetch or alfalfa.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1995 Mar 17, 270(11), 6050 - 5 Wild type Rhizobium etli, a bean symbiont, produces acetyl-fucosylated, N-methylated, and carbamoylated nodulation factors; Poupot R et al.; Phaseolus vulgaris (common bean) can be nodulated by different Rhizobium species . A new species has been recently proposed: Rhizobium etli . Following transcriptional activation of the bacterial nodulation genes using naringenin or bean seed exudate, we have isolated, purified, and characterized R . etli extracellular nodulation factors . They are chitopentameric compounds that are N-methyl-N-vaccenoylated at their non-reducing end . At position 6 of the reducing N-acetyl-D-glucosamine, they are 4-O-acetyl-L-fucosylated . Minor compounds bear a carbamate group on the terminal non-reducing saccharidic residue. FEMS Microbiol Lett, 1995 Mar 15, 127(1-2), 1 - 9 Bradyrhizobium japonicum nodulation genetics; Stacey G; Studies of the genetics of nodulation by Bradyrhizobium japonicum have revealed many similar features with Rhizobium and Azorhizobium species, but also apparent differences . The regulation of nod gene expression in B . japonicum is complex, involving the interplay of the positive regulator, NodD1, as well as a repressor, No1A . A unique feature of B . japonicum is the involvement of a two-component regulatory system, NodV and NodW, in the control of nod gene expression . It is not clear why B . japonicum requires this level of complexity to control nod gene transcription . The nod gene products encode the biosynthesis of substituted lipo-chitin Nod signals that induce many of the early nodulation events . B . japonicum and B . elkanii produce a large variety of such Nod signals . The basic structure of the Nod signal, an acylated oligomer of N-acetylglucosamine, is synthesized through the action of NodA, NodB, and NodC . Various substitutions of this basic structure confer host specificity to the molecule . For example, in B . japonicum, the nodZ gene product is essential for fucosylation of the terminal, reducing N-acetylglucosamine residue . These observations argue for the interaction of a substituted Nod signal with a specific plant receptor molecule . However, structure/function studies using chemically synthesized Nod signal molecules suggest a more complex interaction between chain length and specific substitution . These findings leave open the possibility that a general chitin receptor may function in a unique way to elicit nodule formation . The novel features discovered through the study of B . japonicum contribute to our general understanding of nodulation and to the larger question of plant cell signal transduction. Genes Dev, 1995 Mar 15, 9(6), 714 - 29 The Rhizobium meliloti groELc locus is required for regulation of early nod genes by the transcription activator NodD; Ogawa J et al.; The molecular chaperones related to GroEL (hsp60, cpn60) interact with partially folded proteins and appear to assist them to attain active and correctly folded conformation . They are required for cell viability but are probably more important for some processes than for others . Through a random genetic search to find loci that are required for expression of the Rhizobium meliloti nod (nodulation) genes, we isolated a mutant (B4) defective in luteolin-dependent activation of nod gene expression, and found it carries a Tn5 insertion within a chromosomal groEL gene (groELc) located just downstream of a groESc gene . The groELc mutation affected activity of three related LysR-type activator proteins NodD1, NodD3, and SyrM; on plants, the mutants formed nodules late, and the nodules were Fix- . Hybridization and protein expression analysis show that a similar groESL locus (groESLa) maps to the Rm1021 megaplasmid pSyma . Southern blot analysis revealed additional, but less closely related sequences hybridizing to groELc and groESc probes elsewhere in the R . meliloti genome . Clones of groESLc and groESLa can each restore robust phage lambda growth on an Escherichia coli groE mutant . Likewise each clone can complement all of the phenotypes observed for B4 mutants; thus, the two appear to be functionally equivalent if expression is controlled . We determined that groELc is required for normal DNA binding of the NodD target sequence in R . meliloti . GroEL coimmunopurifies with NodD1 from R . meliloti, which suggests a direct physical association between these proteins . GroEL is thus probably involved in the folding or assembly of transcriptionally active NodD. J Biol Chem, 1995 Mar 10, 270(10), 5243 - 50 The oxygen sensor protein, FixL, of Rhizobium meliloti . Role of histidine residues in heme binding, phosphorylation, and signal transduction; Monson EK et al.; The two-component system sensor/response regulator pair, FixL/FixJ, controls the expression of Rhizobium meliloti nitrogen fixation (nif and fix) genes in response to changes in oxygen concentration . A truncated version of FixL, FixL*, is an oxygen-binding hemoprotein kinase that phosphorylates and dephosphorylates the nif and fix gene transcriptional activator, FixJ . Phosphorylation of FixJ is required for optimal transcriptional activation, and anaerobic conditions in vitro result in a substantial increase in the level of FixJ-phosphate . In this study, site-directed mutagenesis was carried out at histidine residues in FixL* . Mutant FixL* derivatives were purified and analyzed in vitro for their heme/oxygen binding properties and phosphorylation/dephosphorylation activities . Mutation of histidine 285, the putative autophosphorylation site, to glutamine results in the loss of FixL* phosphorylation activities . However, this mutant protein retains a substantial level of FixJ-phosphate dephosphorylation activity . Mutation of histidine 194 to asparagine results in the loss of heme binding and in the failure of FixL* to regulate its phosphorylation/dephosphorylation activities in response to changes in oxygen concentration . The FixL*H194N mutant protein also exhibits an increased FixJ phosphorylation activity under aerobic conditions . This study provides further evidence for the importance of the heme binding domain of FixL* in regulating FixJ phosphorylation and dephosphorylation activities in response to oxygen. J Bacteriol, 1995 Mar, 177(6), 1452 - 60 Molecular and expression analysis of the Rhizobium meliloti phosphoenolpyruvate carboxykinase (pckA) gene; Osteras M et al.; The pckA gene of Rhizobium meliloti, encoding phosphoenolpyruvate carboxykinase, was isolated from a genomic cosmid library by complementation of the succinate growth phenotype of a Pck- mutant . The gene region was mapped by subcloning and Tn5 insertion mutagenesis . The DNA sequence for a 2-kb region containing the structural gene and its promoter was determined . The pckA gene encodes as 536-amino-acid protein that shows homology with other ATP-dependent Pck enzymes . The promoter was identified following primer extension analysis and is similar to sigma 70-like promoters . Expression analysis with a pckA::lacZ gene fusion indicated that the pckA gene was strongly induced at the onset of stationary phase in complex medium . When defined carbon sources were tested, the expression level of the pckA gene was found to be high when cells were grown in minimal media with succinate or arabinose as the sole carbon source but almost absent when glucose, sucrose, or glycerol was the sole carbon source . Glucose and sucrose were not found to strongly repress pckA induction by succinate. Glycobiology, 1995 Mar, 5(2), 233 - 42 Rhizobial lipo-oligosaccharide nodulation factors: multidimensional chromatographic analysis of symbiotic signals involved in the development of legume root nodules; Price NP et al.; Nod factors are a group of biologically active oligosaccharide signals that are secreted by symbiotically competent bacteria of the family Rhizobiaceae . Their biosynthesis is determined by rhizobial nodulation (nod) genes, and is specifically induced in response to flavonoids secreted from the roots of host leguminous plants . The biological activity of Nod factors on these host legumes dramatically mimics the early developmental symptoms of the Rhizobium-legume symbiosis including, amongst other effects, root hair deformations and nodule initiation . Structurally, all Nod factors are short oligomers of beta-1,4-linked N-acetylglucosamine residues {usually degree of polymerization (dp) 4 or 5} that are N-acylated on the distal glucosamine . This common 'core' structure may be modified by a number of species-specific substituents on the distal or reducing sugars . These modifications are governed by rhizobial host specificity nod genes . The biological activity of purified Nod factors mirrors this host specificity, indicating that the symbiotic host range of individual Rhizobium species is, at least partially, determined by the variety of Nod factors they are able to produce . Here we describe techniques that are universally applicable to the extraction, chromatographic separation and identification of Nod factors . We have applied these techniques to Nod factors from the broad-host-range species Rhizobium fredii USDA257 and Rhizobium spp . NGR234, and the more narrow-host-range Bradyrhizobium japonicum USDA110, and have identified a group of novel, relatively hydrophilic Nod factors from the NGR234 species that may have implications for Nod factor biosynthesis. Curr Microbiol, 1995 Mar, 30(3), 177 - 82 Sensitivity of selected bacterial species to UV radiation; Gascon J et al.; The effect of exposure of bacterial suspensions to UV radiation by means of the dose-response curves was assessed . The D37 and D10 values were used for subsequent statistical analysis of the results . The aim of this article is to evaluate the sensitivity to UV radiation of several microorganisms of different habitats (Rhizobium meliloti, Rhodobacter sphaeroides, Escherichia coli, and Deinococcus radiodurans), two mutants with nonfunctional SOS DNA repair system (R . meliloti recA- and E . coli recA-), and a mutant in the synthesis of carotenoids (R . sphaeroides crtD) . The results reveal that D . radiodurans was an extremely resistant bacterium, R . meliloti was more resistant than R . sphaeroides, and E . coli was the most sensitive bacterium tested . The high sensitivity of recA- mutants was also verify . Moreover, it seems that the possession of pigments had no important effect in the sensitivity of R . sphaeroides to UV radiation. Anal Biochem, 1995 Mar 1, 225(2), 258 - 63 Study of parameters affecting poly(3-hydroxybutyrate) quantification by gas chromatography; Jan S et al.; Poly(3-hydroxybutyrate) (PHB) quantification has been developed mostly using acidic methanolysis followed by GC analysis of the 3-hydroxybutyrate methyl ester . However, under our experimental conditions, only 62% of the ester was detected by GC analysis . Following the study of the different steps involved in this method (i.e., hydrolysis, esterification, and recovery of the ester), the recovery was shown to be limiting . Addition of water to the organic phase, required for its purification before injection, led to the partition of the ester between the organic and the aqueous phase . The influence of the length of acidic methanolysis time on the amount of ester detected was also investigated . NMR analysis was used to show that secondary products were absent in both phases, regardless of heating time . Moreover, increasing acid concentration and the use of lyophilized cells were shown to lead to the decrease of the treatment time . Concerning internal standard choice, methyl benzoate was found to meet all the requirements to correct injection volume errors or to follow organic phase volume changes as a function of acid and water concentrations . The validity of the method was checked on Rhizobium meliloti M5N1 cells, which are shown to produce about 60% PHB (w/w) when cultivated with fructose as the carbon source. Microbiology, 1995 Mar, 141 ( Pt 3), 583 - 8 Synthesis of glycerophosphorylated cyclic (1,2)-beta-glucans in Rhizobium meliloti strain 1021 after osmotic shock; Breedveld MW et al.; The transfer of phosphoglycerol moieties from phosphatidylglycerol to the cyclic (1,2)-beta-glucans in growing cultures of Rhizobium meliloti strain 1021 was investigated using pulse-chase experiments with {3H}glycerol and/or {14C}glucose . No transfer occurred when cells were grown and pulse-chased in a medium containing 0.4 M NaCl . However, radiolabelled glycerophosphorylated cyclic (1,2)-beta-glucans could be detected within 30 min after transfer of these cultures to a low-osmolarity medium . Conversely, when low-osmolarity cultures were shifted to a high-osmolarity medium containing 0.4 M NaCl or 0.8 M sucrose, the transfer of phosphoglycerol substituents to the cyclic (1,2)-beta-glucans was inhibited . Further experiments revealed that the transfer of phosphoglycerol substituents to the cyclic (1,2)-beta-glucans occurs within the periplasmic compartment. Microbiology, 1995 Mar, 141 ( Pt 3), 573 - 81 Surface polysaccharide mutants of Rhizobium sp . (Acacia) strain GRH2: major requirement of lipopolysaccharide for successful invasion of Acacia nodules and host range determination; Lopez-Lara IM et al.; Two transposon Tn5-induced mutants of wild-type broad-host-range Rhizobium sp . GRH2 were isolated and found to harbour different alterations in surface polysaccharides . These mutants, designated GRH2-14 and GRH2-50, induced a few, empty nodules on Acacia and lost the ability to nodulate most host herbaceous legumes . Whereas mutant GRH2-14 produces an acidic exopolysaccharide (EPS) similar to the wild-type, the acidic EPS of mutant GRH2-50 lacks galactose and the pyruvyl and 3-hydroxybutyryl substituents attached to this sugar moiety . In addition, both mutants GRH2-50 and GRH2-14 were altered in smooth lipopolysaccharides (LPS) . DNA sequence analyses of the corresponding Tn5 insertions revealed that strain GRH2-50 was mutated in a DNA locus homologous to galE, and in vitro enzyme assays indicated that the UDPglucose 4-epimerase (GalE) activity was missing in this mutant strain . DNA hybridization studies showed that the GRH2-50 mutant DNA has homologous sequences within the different biovars of Rhizobium leguminosarum . However, no DNA homology to GRH2-14 altered DNA was found in those rhizobial strains, indicating that it represents a new chromosomal lps locus in Rhizobium sp . (Acacia) involved in symbiotic development. Microbiol Rev, 1995 Mar, 59(1), 124 - 42 The Rhizobium-plant symbiosis; van Rhijn P et al.; Rhizobium, Bradyrhizobium, and Azorhizobium species are able to elicit the formation of unique structures, called nodules, on the roots or stems of the leguminous host . In these nodules, the rhizobia convert atmospheric N2 into ammonia for the plant . To establish this symbiosis, signals are produced early in the interaction between plant and rhizobia and they elicit discrete responses by the two symbiotic partners . First, transcription of the bacterial nodulation (nod) genes is under control of the NodD regulatory protein, which is activated by specific plant signals, flavonoids, present in the root exudates . In return, the nod-encoded enzymes are involved in the synthesis and excretion of specific lipooligosaccharides, which are able to trigger on the host plant the organogenic program leading to the formation of nodules . An overview of the organization, regulation, and function of the nod genes and their participation in the determination of the host specificity is presented. Mol Microbiol, 1995 Mar, 15(6), 989 - 1000 Analysis of a chemotaxis operon in Rhizobium meliloti; Greck M et al.; Genes controlling chemotaxis towards L-amino acids and D-mannitol in Rhizobium meliloti have been identified by Tn5 insertions that lead to chemotaxis-deficient mutants . The tagged genes span an 8.7 kbp region that has been sequenced . These genes are part of a large operon containing three novel open reading frames, orf1, orf2 and orf9, and six familiar chemotaxis (che) genes, cheY1-cheA-cheW-cheR-cheB-cheY2, that have been assigned by their similarity to known Escherichia coli genes . The second copy of cheY may be part of a second signalling chain; orf1 and orf2 encode sequence motifs that resemble the signalling domain of E . coli MCPs (methyl-accepting chemotaxis proteins), while the product of orf9 may contain a transmembrane domain . No protein methylation has been observed in Rhizobium meliloti in response to L-amino acids . However, the presence of cheR (methyltransferase gene) and cheB (methylesterase gene) suggested that MCPs are likely components of the chemotactic response in R . meliloti . Therefore, it is postulated that two chemotaxis pathways are functional in R . meliloti: one responds to L-amino acids via ORF1-ORF2, whereas the other (probably responding to specific plant exudates) acts via MCP-like receptors, and both interact with the central components CheW-CheA-CheY1 and/or CheY2. Biochem J, 1995 Feb 15, 306 ( Pt 1), 259 - 64 Enzymatic radiolabelling to a high specific activity of legume lipo-oligosaccharidic nodulation factors from Rhizobium meliloti; Bourdineaud JP et al.; In this paper we describe the two-step coupled 35S-radiolabelling of the lipo-oligosaccharidic nodulation (Nod) factors of the bacterium Rhizobium meliloti to a specific radioactivity of 800 Ci/mmol . These radiolabelled Nod factors bind to a particulate fraction from roots of the bacterium's symbiotic host, Medicago truncatula, with an equilibrium dissociation constant (KD) of 117 nM, similar to that observed with a synthetic tritiated ligand . The first step of the 35S-labelling involves the synthesis of 3'-phosphoadenosine 5'-phospho{35S}sulphate ({35S}PAPS) from ATP and {35S}sulphate using yeast enzymes . The second step exploits the sulphotransferase activity of the R . meliloti NodH protein, which has been expressed in Escherichia coli, to transfer the labelled sulphate group from PAPS to non-sulphated Nod factors . This enzyme was found to be active in E . coli cultured at 18 degrees C but not 37 degrees C . NodH could also transfer the sulphate group from PAPS to a model substrate, tetra-N-acetyl chitotetraose, with apparent Km values of 56 and 70 microM respectively, and exhibited an apparent Km value for non-sulphated Nod factors of 28 microM . Coupling the two steps of the radiolabelling resulted in an efficiency of 35S incorporation from inorganic sulphate to the Nod factors of approximately 10% . These labelled factors will be a valuable tool in the search for high-affinity receptors for the lipo-oligosaccharidic nodulation factors. Gene, 1995 Feb 3, 153(1), 105 - 9 Analysis of DNA flanking the xlnB locus of Streptomyces lividans reveals genes encoding acetyl xylan esterase and the RNA component of ribonuclease P; Shareck F et al.; Nucleotide sequencing revealed the gene (axeA) encoding acetyl xylan esterase (AxeA) downstream from xlnB in the Streptomyces lividans DNA insert of plasmid pIAF42 . AxeA consists of a catalytic- and a substrate-binding domain separated by a Gly-rich linker . The N terminus showed no significant homology with published esterases and acetyl xylan esterases, but some homology was found with the xylanases XylA and XylD and the NodB protein of Rhizobium species which is involved in the biosynthesis of root nodulation factors . The C terminus of AxeA is highly homologous to the C-termini of xylanases XlnB and TFXA, corresponding to the xylan-binding domain of these enzymes . Furthermore, the RNaseP RNA component was found immediately upstream from xlnB gene. FEMS Microbiol Lett, 1995 Feb 1, 126(1), 25 - 30 Signal transduction in the Rhizobium meliloti dicarboxylic acid transport system; Giblin L et al.; The gene products of the Rhizobium meliloti dctB and dctD genes, which control the expression of the C4-dicarboxylic acid transporter DctA, were overproduced in Escherichia coli and purified . The purified sensor protein, DctB, was shown to have autophosphorylation activity in vitro and could subsequently phosphorylate the transcriptional activator, DctD . The presence of C4-dicarboxylic acids did not affect either reaction . In vitro experiments aimed at investigating 'crosstalk' between cognate components demonstrated that the phospho-transfer activity was specific between DctB and DctD . Studies on truncated versions of the DctB protein in vitro revealed that the cytoplasmic domain of DctB had strong autophosphorylation activity . Data from gel retardation experiments demonstrated that once the activator protein, DctD, was phosphorylated it had increased affinity for binding to the dctA promoter DNA. J Bacteriol, 1995 Feb, 177(4), 973 - 80 Discrete amplifiable regions (amplicons) in the symbiotic plasmid of Rhizobium etli CFN42; Romero D et al.; Frequent tandem amplification of defined regions of the genome, called amplicons, is a common characteristic in the genomes of some Rhizobium species, such as Rhizobium etli . In order to map these zones in a model Rhizobium replicon, we undertook an analysis of the plasticity patterns fostered by amplicons in the pSym (390 kb) of R . etli CFN42 . Data presented in this article indicate the presence of four amplicons in pSym, used for the generation of tandem amplifications and deletions . The amplicons are large, ranging from 90 to 175 kb, and they are overlapping . Each amplicon is usually flanked by specific reiterated sequences . Formation of amplifications and deletions requires an active recA gene . All the amplicons detected are concentrated in a zone of roughly one-third of pSym, covering most of the symbiotic genes detected in this plasmid . No amplicons were detected in the remaining two-thirds of pSym . These data support the idea that most of the known symbiotic genes in this plasmid are located in a genomic region that is prone to the formation of frequent tandem amplification. Mol Microbiol, 1995 Feb, 15(4), 733 - 47 NolR controls expression of the Rhizobium meliloti nodulation genes involved in the core Nod factor synthesis; Cren M et al.; The synthesis of Rhizobium meliloti Nod signal molecules, encoded by the nod gene products, is finely regulated . A negative control of plasmid-borne nod gene expression is provided by the NolR repressor encoded by the chromosomal nolR gene . NolR was previously shown to downregulate the expression of the activator nodD1 gene and the common nodABC operon by binding to an overlapping region of the two promoters adjacent to the n1 nod-box (Kondorosi et al., 1989) . We demonstrate here that NolR also controls the expression of two additional genes, nodD2 and nodM, but does not directly regulate the expression of the host-specific nod genes located downstream of the n2, n3 and n5 nod-boxes . Thus, the nod genes are differentially regulated by NolR and only those providing common nodulation functions, by determining the synthesis of the core Nod factor structure, are subjected to this negative regulation . Furthermore, NolR has a strong negative effect on the production of Nod metabolites, the level of which may serve as a fine-tuning mechanism for optimal nodulation, specific to host-plant genotypes . In addition, it elicits preferential synthesis of Nod factors carrying unsaturated C16 fatty acids . Expression of nolR was high both in the free-living bacterium and in the bacteroid and it was downregulated by its own product and by the nod gene inducer luteolin. Mol Microbiol, 1995 Feb, 15(4), 627 - 38 Structural identification of the lipo-chitin oligosaccharide nodulation signals of Rhizobium loti; Lopez-Lara IM et al.; Rhizobium loti is a fast-growing Rhizobium species that has been described as a microsymbiont of plants of the genus Lotus . Nodulation studies show that Lotus plants are nodulated by R . loti, but not by most other Rhizobium strains, indicating that R . loti produces specific lipo-chitin oligosaccharides (LCOs) which are necessary for the nodulation of Lotus plants . The LCOs produced by five different Rhizobium loti strains have been purified and were shown to be N-acetylglucosamine pentasaccharides of which the non-reducing residue is N-methylated and N-acylated with cis-vaccenic acid (C18:1) or stearic acid (C18:O) and carries a carbamoyl group . In one R . loti strain, NZP2037, an additional carbamoyl group is present on the non-reducing terminal residue . The major class of LCO molecules is substituted on the reducing terminal residue with 4-O-acetylfucose . Addition of LCOs to the roots of Lotus plants results in abundant distortion, swelling and branching of the root hairs, whereas spot inoculation leads to the formation of nodule primordia. J Ind Microbiol, 1995 Feb, 14(2), 94 - 104 Long-term effects of metals in sewage sludge on soils, microorganisms and plants; McGrath SP et al.; This paper reviews the evidence for impacts of metals on the growth of selected plants and on the effects of metals on soil microbial activity and soil fertility in the long-term . Less is known about adverse long-term effects of metals on soil microorganisms than on crop yields and metal uptake . This is not surprising, since the effects of metals added to soils in sewage sludge are difficult to assess, and few long-term experiments exist . Controlled field experiments with sewage sludges exist in the UK, Sweden, Germany and the USA and the data presented here are from these long-term field experiments only . Microbial activity and populations of cyanobacteria, Rhizobium leguminosarum bv . trifolii, mycorrhizae and the total microbial biomass have been adversely affected by metal concentrations which, in some cases, are below the European Community's maximum allowable concentration limits for metals in sludge-treated soils . For example, N2-fixation by free living heterotrophic bacteria was found to be inhibited at soil metal concentrations of (mg kg-1): 127 Zn, 37 Cu, 21 Ni, 3.4 Cd, 52 Cr and 71 Pb . N2-fixation by free-living cyanobacteria was reduced by 50% at metal concentrations of (mg kg-1): 114 Zn, 33 Cu, 17 Ni, 2.9 Cd, 80 Cr and 40 Pb . Rhizobium leguminosarum bv . trifolii numbers decreased by several orders of magnitude at soil metal concentrations of (mg kg-1): 130-200 Zn, 27-48 Cu, 11-15 Ni, and 0.8-1.0 Cd . Soil texture and pH were found to influence the concentrations at which toxicity occurred to both microorganisms and plants . Higher pH, and increased contents of clay and organic carbon reduced metal toxicity considerably . The evidence suggests that adverse effects on soil microbial parameters were generally found at surprizingly modest concentrations of metals in soils . It is concluded that prevention of adverse effects on soil microbial processes and ultimately soil fertility, should be a factor which influences soil protection legislation. Plant Cell, 1995 Feb, 7(2), 213 - 23 Symbiotic and nonsymbiotic hemoglobin genes of Casuarina glauca; Jacobsen-Lyon K et al.; Casuarina glauca has a gene encoding hemoglobin (cashb-nonsym) . This gene is expressed in a number of plant tissues . Casuarina also has a second family of hemoglobin genes (cashb-sym) expressed at a high level in the nodules that Casuarina forms in a nitrogen-fixing symbiosis with the actinomycete Frankia . Both the nonsymbiotic and symbiotic genes retained their specific patterns of expression when introduced into the legume Lotus corniculatus . We interpret this finding to mean that the controls of expression of the symbiotic gene in Casuarina must be similar to the controls of expression of the leghemoglobin genes that operate in nodules formed during the interaction between rhizobia and legumes . Deletion analyses of the promoters of the Casuarina symbiotic genes delineated a region that contains nodulin motifs identified in legumes; this region is critical for the controlled expression of the Casuarina gene . The finding that the nonsymbiotic Casuarina gene is also correctly expressed in L . corniculatus suggests to us that a comparable non-symbiotic hemoglobin gene will be found in legume species. Trends Microbiol, 1995 Feb, 3(2), 58 - 64 Plant isoflavonoids, pathogens and symbionts; Phillips DA et al.; It has recently been discovered that when symbiotic Rhizobium and Bradyrhizobium cells are outside the plant they are also exposed to the isoflavonoid phytoalexins that are normally associated with pathogenic infections . How the symbionts elicit and respond to isoflavonoids may help to define the mechanisms that are used by other beneficial soil microorganisms to colonize plant roots. Plant J, 1995 Feb, 7(2), 253 - 60 Characterization of a binding site for chemically synthesized lipo-oligosaccharidic NodRm factors in particulate fractions prepared from roots; Bono JJ et al.; This paper describes the characteristics of a binding site for the major, lipo-oligosaccharide Nod factor of Rhizobium meliloti in roots of the symbiotic host plant, Medicago truncatula . Chemically synthesized NodRm-IV(Ac, S, C16:2) was labelled by tritiation to a specific activity of 56 Ci mmol-1 and this ligand was shown to be biologically active in the root hair deformation assay at 10(-11) M . Binding of the ligand to a particulate fraction from roots of M . truncatula was found to be saturable and reversible with an affinity (Kd) of 86 nM and the binding characteristics were consistent with a single class of binding sites . Competition with modified Nod factors showed that the binding was independent of both the O-acetyl and the sulphyl group and did not depend on the unsaturation of the fatty acid . However, both moieties of the lipo-oligosaccharide are required for high-affinity binding since tetra-N-acetyl-chitotetraose and palmitate were found to be poor competitors of ligand binding . A binding site with analogous characteristics was also found in a similarly prepared particulate fraction of tomato roots . This binding site for Nod factors, termed NFBS1, which is present in both a leguminous and a non-leguminous plant, may have a more general role than symbiosis. Appl Environ Microbiol, 1995 Feb, 61(2), 507 - 12 Species limits in Rhizobium populations that nodulate the common bean (Phaseolus vulgaris); Eardly BD et al.; Evolutionary genetic relationships among 146 bean-nodulating Rhizobium strains, including 94 field isolates from three localities in Colombia and 36 strains from Mexico, were examined by multilocus enzyme electrophoresis and restriction fragment length polymorphism analysis of a PCR-amplified 260-bp segment of the 16S rRNA gene . Seventy-five electrophoretic types (ETs), corresponding to multilocus enzyme genotypes, were identified, including a genotypically diverse group of 18 ETs in Colombia that is strongly differentiated from the ETs of R . etli, which occur in Mexico, Colombia, and Brazil . Most strains of the distinctive Colombian ETs carried the same 16S rRNA allele as did strains of R . etli, but, surprisingly, 17 isolates of two of these ETs had the allele that is characteristic of R . leguminosarum, and strains of two other divergent groups of ETs were also polymorphic for the two alleles . No fully satisfactory explanation for the occurrence of the R . leguminosarum 16S rRNA allele in three distantly related groups of strains is available, but horizontal transfer and recombination of the gene, in whole or in part, would seem to be more plausible than convergence in nucleotide sequence. Glycoconj J, 1995 Feb, 12(1), 45 - 50 Localization of peanut (Arachis hypogaea) root lectin (PRA II) on root surface and its biological significance; Kalsi G et al.; The glucose-specific peanut root lectin, PRA II, is localized on the surface of 7-day-old peanut seedling root and in root cortical parenchymatous cells . The lectin is eluted from intact roots upon washing with buffer containing glucose . Rabbit erythrocytes bind to the root surface and the cortical cells; the binding is inhibited by antibodies raised against PRA II, peanut-specific Rhizobium cells and by glucose . Lipopolysaccharides isolated from host-specific Rhizobium strain inhibit the haemagglutinating activity of PRA II and are precipitated by the lectin . Our results suggest that PRA II might be involved in recognition of Rhizobium by peanut roots. J Biol Chem, 1995 Jan 27, 270(4), 1624 - 8 ADP-ribosylation of Rhizobium meliloti glutamine synthetase III in vivo; Liu Y et al.; The control of glutamine synthetase (GS), the first enzyme in the main pathway used by Rhizobium meliloti to assimilate ammonia, is central to cellular nitrogen metabolism . R . meliloti is unusual in having three distinct types of GS, including a unique GS, GSIII, that differs considerably from both GSI, which resembles other bacterial GS proteins and GSII, which resembles the GS found in eukaryotes . We show here that GSIII can be post-translationally modified in vivo by ADP-ribosylation at an arginine residue . 32PO4 attached to GSIII during bacterial growth as part of the modifying group could be removed by treatment with snake venom phosphodiesterase or by turkey erythrocyte ADP-ribosylarginine hydrolase . Treatment of modified GSIII with hydroxylamine at neutral pH releases a chromophore that has the retention time of ADP-ribose when analyzed by reversed-phase high performance liquid chromatography . ADP-ribosylation inhibits GSIII activity. Gene, 1995 Jan 11, 152(1), 65 - 7 Similarity between the Rhizobium meliloti fliP gene and pathogenicity-associated genes from animal and plant pathogens; Finan TM et al.; The nucleotide sequence of the Rhizobium meliloti (Rm) fliP gene was determined . Rm strains carrying insertions within this gene were non-motile, lacked flagella and formed normal N2-fixing root nodules on alfalfa . The FliP protein showed similarity to several bacterial gene products involved in pathogenicity in both plant and animal pathogens . It is likely that all of these proteins share a common functional role in the secretion of specific proteins from bacterial cells. Annu Rev Genet, 1995, 29, 107 - 29 The plant response in pathogenesis, symbiosis, and wounding: variations on a common theme? Baron C, Zambryski PC. Upon interaction with pathogenic Pseudomonads or symbiotic Rhizobia, or after wounding by abrasion or insects, the plant reacts in superficially different ways, leading to either a close association or defense against the intruder . However, a closer examination reveals that similar genes and metabolic pathways are induced . This raises the possibility that signal perception and transduction proceed via similar pathways, leading to overlaps in the response reaction . This review compares current knowledge of the plant reaction to apparently different biotic and abiotic stimuli, and highlights that within the course of evolution similar response mechanisms were adapted to specific needs. Acta Biol Hung, 1995, 46(1), 17 - 30 Some environmental factors influencing the survival of Rhizobium leguminosarum bv . viceae; Bayoumi HE et al.; Investigations were carried out to monitor the sensitivity of Rhizobium leguminosarum bv . viceae strains to various environmental stress-factors (salinity, pH, Al3+, Ca2+, Mg2+) and select them as potential candidates for Vicia faba inoculation . In the range between pH 5.0 and 10.0, the salt effect of 10-500 mM NaCl, Ca2+, Mg2+ (as chlorides and sulphates), Al2O3 and KAl (SO4)2 (25-400 microM) were tested in modified yeast-mannitol (YEM) liquid medium . Cell density of the suspensions inoculated with R . leguminosarum bv . viceae strains (initial cell-number 10(6) CFU/ml) were measured spectrophotometrically after 48 h incubation in a rotary shaker (rpm 150) at 28 degrees C . Data of optical density (OD550) are shown as percent of control (inoculated, free from treatments, pH 7.0) tubes . It was established, that results of tolerancy were in agreement with those found earlier for Rhizobium sensitivity . Strain of Lobab Z (isolated in Hungary) however proved to have an especially outstanding survival in any media tested in vitro . Lowest observed effect concentrations (LOECs) were: 500 mM NaCl, 200 microM Al3+ (as Al2O3 or KAl (SO4)2), 50-100 mM Mg2+, and 200-300 mM Ca2+ . For the Al3+, Ca2+ and Mg2+ cations, there was no variation between the stress-effect of sulphate and chloride anions . Both forms of Ca2+, however significantly reduced the growth potential of R . leguminosarum bv . viceae strains. Acta Biol Hung, 1995, 46(1), 9 - 16 Metal sensitivity of some symbiotic N2-fixing bacteria and Pseudomonas strains; Biro B et al.; An investigation was carried out to determine the sensitivity of different soil microbes (Rhizobium, Bradyrhizobium and Pseudomonas) to various metals (Cu2+, Zn2+, Co2+, Mn2+, Mo2+ and Fe2+) in vitro . Sulphate and chloride forms of these microelements were used (except Mo2+ as Na2Mo04) in 0.1, 1.0 and 10 micrograms/ml concentrations in modified YEM and nutrient broth . Growth (optical density, OD550 and OD640) of bacterium inoculated (approx . 10(6) CFU/ml) tubes, was measured spectrophotometrically after 48 h of incubation of 28 degrees C in a rotary shaker (150 rpm) . Data of triplicate samples are shown as percent of control tubes (inoculated, free from treatments) and after an analysis of variance SE was calculated . Strains of Rhizobium leguminosarum proved to be the most sensitive to Cu2+, Zn2+ and Co2+ . The slow growing Bradyrhizobium and plant growth promoting (PGPR) Pseudomonas isolates, however, were affected only at the highest (10 micrograms/ml) dose of these elements . In contrast Mn2+, Mo2+ and Fe2+ microelements were stimulatory for the growth of all investigated soil microbes . Sulphate forms of the most harmful Cu2+ and Zn2+ cations were more toxic than the chloride forms . An especially high diversity was found among the R . leguminosarum bv . viceae isolates . Monitoring the sensitivity of these microbes has a primary importance for selection of ecologically diverse isolates, as potential inocula in heavy-metal affected soils. Microbiology, 1995 Jan, 141 ( Pt 1), 103 - 11 Rhizobium leguminosarum nodulation gene (nod) expression is lowered by an allele-specific mutation in the dicarboxylate transport gene dctB; Mavridou A et al.; To identify host genes that might influence nod (nodulation) gene expression in Rhizobium leguminosarum, a nodC-phoA reporter plasmid (carrying nodD) was introduced into a chemically mutagenized population of a R . leguminosarum strain lacking a symbiotic plasmid . The transconjugants were screened for expression of alkaline phosphatase (PhoA) on plates containing hesperetin, an inducer of nod genes, and a mutant with reduced expression was identified . When the nodC-phoA plasmid was cured from the mutant and the symbiotic plasmid pRL1Jl introduced, the mutant formed nodules, but symbiotic nitrogen fixation was less than 20% of normal . When the nodC-phoA allele was introduced on pRL1Jl a low level of nod gene induction was found . The reduced nodC expression appeared to be caused by a decrease in expression of the regulatory gene nodD, since expression of a nodD-lacZ fusion was also lower in the mutant than in the control . These mutant phenotypes and the low nitrogen fixation were complemented with a plasmid (plJ1848) from a R . leguminosarum cosmid library . DNA hybridization confirmed that plJ1848 was not from the symbiotic plasmid and showed that a DNA insertion was present in the mutant . The complementing region of plJ1848 was defined by transposon mutagenesis; DNA sequencing revealed that it carried the dicarboxylic acid transport (dct) genes . However, the mutant grew well with succinate as sole C-source . Genetic analysis revealed that the mutant appeared to contain IS50 in the regulatory gene dctB and that this mutation caused the reduction in nod gene expression . The effect was allele-specific since other mutations in dctB did not influence nod gene expression . Surprisingly, the mutant had a constitutive high level of succinate transport, indicating that the mutation caused unregulated expression of dctA the structural gene for dicarboxylic acid transport . This in some way appears to have lowered the expression of nodD, indicating that the nodD promoter may be influenced by the metabolic status of the cells or by expression of dctD in the absence of dctB. Proc Soc Exp Biol Med, 1995 Jan, 208(1), 131 - 8 Endocrine activity of plant-derived compounds: an evolutionary perspective; Baker ME; Although plants have long been known to have important pharmacological effects in humans, the mechanism by which plant-derived compounds act in humans is still being elucidated . Two important pathways for the biological actions of plant-derived compounds involved binding either to hormone receptors or to enzymes that metabolize hormones . What are the origins of this interaction between plant-derived compounds and animals? And what insights can we gain from investigating this question? Some answers come from recent sequence analyses, revealing that 17 beta-hydroxysteroid dehydrogenase, which regulates estrogen and androgen levels in humans, and 15-hydroxyprostaglandin dehydrogenase, which regulates prostaglandin E2 and F2 alpha levels in humans, have a common ancestor with proteins in rhizobia that are important in forming nitrogen-fixing nodules in legume roots, and 3 beta-hydroxysteroid dehydrogenase, which regulates progestin and androgen levels in humans, has a common ancestor with enzymes important in the synthesis of anthocyanins . This evolutionary kinship, when combined with the structural similarities between flavonoids, licorice-derived compounds, and steroid hormones, provides another perspective on the hormone-like activity of flavonoids and other plant-derived compounds in humans: some of the hormone-like activity of plant-derived compounds is due to binding to steroid and prostaglandin dehydrogenases. Int J Syst Bacteriol, 1995 Jan, 45(1), 153 - 9 Characteristics of Rhizobium tianshanense sp . nov., a moderately and slowly growing root nodule bacterium isolated from an arid saline environment in Xinjiang, People's Republic of China; Chen W et al.; We performed a numerical analysis of 148 phenotypic characteristics of 20 strains of root nodule bacteria isolated from an arid saline desert soil in the Xinjiang region of northwestern People's Republic of China and compared these organisms with 28 Rhizobium and Bradyrhizobium strains obtained from different regions of the People's Republic of China and from other countries, including nine type strains of different species . All of the strains examined clustered into two groups at a similarity level of more than 63% . Group I included all of the previously described Rhizobium species and was divided into eight subgroups, which corresponded to previously described Rhizobium species, at a similarity level of more than 82% . Group II was divided into the following three subgroups at a similarity level of more than 80% Bradyrhizobium japonicum, a cluster containing 17 moderately and slowly growing strains isolated in the Xinjiang region, and a small subgroup containing three fast-growing strains . The generation times of the moderately and slowly growing strains were 5 to 15 h, and these organisms produced acid in medium containing mannitol . The DNA G+C contents of the members of this group ranged from 59 to 63 mol% . DNA-DNA hybridization experiments revealed that the levels of DNA homology among all of the moderately and slowly growing strains obtained from Xinjiang were more than 70% and that the levels of DNA homology between representative strains of this group and the type strains of all previously described species of root- and stem-nodulating bacteria were low.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1995 Jan, 177(2), 468 - 72 Phylogeny of Sym plasmids of rhizobia by PCR-based sequencing of a nodC segment; Ueda T et al.; To understand the host specificity of rhizobia and the relationship between the evolution of Sym plasmids and that of host plants, we determined partial nodC sequences of 10 representative rhizobium strains and then constructed an evolutionary tree for the deduced amino acid sequences with four published sequences . These coding sequences yield a phylogenetic tree similar to that for leghemoglobin of host plants, suggesting that the evolution of common nodulation genes may be lin