Rhizobacterium

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 linked to host legume evolution and speciation.

Mol Plant Microbe Interact, 1995 Jan-Feb, 8(1), 147 - 54
The nodS gene of Rhizobium tropici strain CIAT899 is necessary for nodulation on Phaseolus vulgaris and on Leucaena leucocephala; Waelkens F et al.; Rhizobium tropici strain CIAT899 induces nitrogen-fixing nodules on the roots of a wide range of tropical legumes, including Phaseolus vulgaris and Leucaena leucocephala . Previously, a DNA region of the CIAT899 pSym plasmid containing the common nodulation genes nodABC and one of the nodD alleles was characterized (P . van Rhijn, B . Feys, C . Verreth, and J . Vanderleyden, J . Bacteriol . 175: 438-447, 1993) . As reported here, the region immediately downstream of nodC contains the nodSU genes . The nucleotide sequence of these genes is presented . CIAT899 nodS and nodU mutants were constructed . The nodS mutant was completely deficient in nodulation on the host plants P . vulgaris and L . leucocephala . The nodU mutation caused a decrease in nodulation on Leucaena but resorted no effect on Phaseolus . Introduction of the CIAT899 nodABCSU region in R . etli CE-3, a strain that only nodulates P . vulgaris, caused an extension of the host range of strain CE-3 to L . leucocephala.

Plant Cell, 1995 Jan, 7(1), 43 - 55
Transient induction of a peroxidase gene in Medicago truncatula precedes infection by Rhizobium meliloti; Cook D et al.; Although key determinative events of the Rhizobium-legume symbiosis are likely to precede bacterial infection, no plant genes have been identified that are expressed strongly prior to infection and nodule morphogenesis . A subtractive hybridization-polymerase chain reaction technique was used to enrich for genes induced during the early phases of the R . meliloti-Medicago truncatula symbiosis . One gene so identified encodes a putative plant peroxidase protein, which we have named Rip1 for Rhizobium-induced peroxidase . The accumulation of rip1 transcript was rapidly and transiently induced by R . meliloti and by the corresponding lipooligosaccharide signal molecule Nod factor RmIV, which was both necessary and sufficient for rip1 induction . The duration of maximal rip1 expression coincided with the preinfection period: transcript levels for rip1 were near maximal by 3 hr postinoculation and declined by 48 hr, coincident with early infection events and the onset of nodule morphogenesis . Furthermore, although rip1 induction preceded bacterial infection by at least 24 hr, the transcript was localized to epidermal cells in the differentiating root zone that was subsequently infected by Rhizobium . Thus, a defining feature of the Rhizobium infection court is the prior induction of rip1 expression.

Acta Microbiol Immunol Hung, 1995, 42(1), 61 - 9
Effect of some environmental factors on Rhizobium and Bradyrhizobium strains; Bayoumi HE et al.; Effects of different abiotic factors (acidity, salinity, nitrate and temperature) on growth rate of root-nodule bacteria (Rhizobium and Bradyrhizobium) strains were investigated in vitro . Strains isolated from Vicia faba L., Coronilla varia L . and Lupinus albus L . exhibited a large variation in tolerance of the above-mentioned factors . These bacteria should be screened under stimulated conditions for enhanced survival before selection to be used for commercial inoculant production . Linear correlation matrix data were useful to find the appropriate concentrations for the selection of the tolerant strains.

Mol Gen Mikrobiol Virusol, 1995 Jan-Mar, (1), 14 - 21
{Cloning and heterologous expression of the Escherichia coli proBosm proA operon in the bacterial symbiont of soy, Rhizobium fredii}; Neumyvakin LV et al.; Effects of recombinant bacteria Rhizobium fredii/pNSA, content of proBosm proA operon, and R.fredii/pLVA, content of proBA wild type operon, on soybean plants were studied . R.fredii BD32/pNSA were found to synthesize more proline and to express higher osmotolerance than R.fredii BD32/pLVA or wild type bacteria . Plants infected with R.fredii Bd32/pNSA were characterized by lower water consumption before blooming . Moreover, these bacteria were found to promote more rapid sprout of seeds under unfavorable climatic conditions.

J Basic Microbiol, 1995, 35(4), 217 - 27
The fix Escherichia coli region contains four genes related to carnitine metabolism; Eichler K et al.; Anaerobic carnitine metabolism in Escherichia coli was recently shown to involve six genes organized in the cai operon and located at the first minute on the chromosome . The DNA sequence lying at the 5' end of the cai locus was further investigated . It contains four open reading frames organized as an operon . In vivo overexpression of this DNA region revealed four polypeptides with apparent molecular masses of 27, 33, 45 and 6 kDa . These proteins displayed significant amino acid sequence homologies with polypeptides encoded by the fixABCX operons from Azorhizobium caulinodans and Rhizobium meliloti . The four ORFs were thus named fixABCX . The first two gene products were also found to share a high degree of sequence similarity with the subunits beta and alpha, respectively, of mammalian electron transfer flavoproteins, suggesting a role for these proteins in a redox reaction . A singly polycistronic 5 kb mRNA transcript was detected in Northern blots under anaerobic conditions in the presence of DL-carnitine . Expression of a fixA-lacZ transcriptional fusion was induced by L(-)-carnitine and crotonobetaine but not by D(+)-carnitine, gamma-butyrobetaine, glycinebetaine and choline as found previously for the carnitine pathway . Similarly, the fix operon was repressed by glucose and nitrate . Moreover, expression of the fix operon was induced by the global regulatory proteins CRP and FNR and repressed by the histone-like protein H-NS . All these regulatory proteins have been shown also to control expression of carnitine enzymes . Results from Northern blots and lacZ fusion studies indicate a common regulation of expression of fix and cai operons, which implies a physiological linkage between these two loci.

Gene, 1994 Dec 2, 150(1), 201 - 2
Nucleotide sequence of the Rhizobium etli nodS gene; Villalobos MA et al.; The complete nucleotide sequence of the nodS gene from the bean-nodulating Rhizobium etli, presumably encoding a methyltransferase, was determined . A phylogenetic analysis of five different NodS proteins from three genera of Gram- soil bacteria, Azorhizobium, Bradyrhizobium and Rhizobium, was performed.

Gene, 1994 Dec 2, 150(1), 111 - 6
The pss4 gene from Rhizobium leguminosarum by viciae VF39: cloning, sequence and the possible role in polysaccharide production and nodule formation; Ivashina TV et al.; The Tn5 insertion into the genome of Rhizobium leguminosarum bv viciae VF39, resulting in non-mucoid growth and formation of non-N2-fixing nodule-like structures on Vicia faba plants, was mapped within a 1.4-kb EcoRV-SacI fragment . Nucleotide sequence analysis revealed an ORF (pss4) of 263 amino acids (aa) . Three transcription start points (tsp) were determined . Two of them were localized upstream from the first GTG codon; the third tsp was mapped in front of the second putative start codon (GTG) corresponding to Val64 of the Pss4 aa sequence . The expression of pss4 in a T7 RNA polymerase/promoter system produced a single approx . 29-kDa protein . Pss4 reveals similarity to several proteins involved in polysaccharide biosynthesis in various Rhizobium species . A nearly complete homology was found with PssA from Rl biovar phaseoli 8002 {Borthakur et al., Mol . Gen . Genet . 213 (1988) 155-162}, except that Pss4 has an additional 63 aa on its N terminus.

Gene, 1994 Dec 2, 150(1), 105 - 9
Construction of a bidirectional promoter probe vector and its use in analysing nod gene expression in Rhizobium loti; Parry SK et al.; A broad-host-range bidirectional promoter reporter vector, pSPV4, has been constructed to analyse the activity of cloned divergent regulatory regions . Plasmid pSPV4 contains a pair of divergent promoterless reporter genes (lacZ and gusA) that are bisected by an extensive multiple cloning site . {corrected} The transcriptional fusion vector pSPV4 has a distinct advantage over unidirectional promoter probe vectors in that it can determine the activity of a cloned bidirectional regulatory region simultaneously in both directions using the same set of cells . The relative activity of each of the divergent promoters, and hence the reporter genes, is therefore not a function of the particular growth state of the culture . To demonstrate the utility of pSPV4, the promoter activity of two Rhizobium loti nod regulatory regions were examined . Although the nod gene organisation in this species is unusual, the promoter activity of these two divergent nod regulatory regions is consistent with the conventional mode of nod expression (constitutive towards the regulatory nodD gene and inducible in the divergent nod gene direction) . The construction of the bidirectional reporter vector, pSPV4, involved two intermediary plasmids, pSPV1 and pSPV2 . Both of these constructs will also be useful for other researchers, especially given the trend towards the utilisation of gusA as a reporter gene.

J Bacteriol, 1994 Dec, 176(24), 7740 - 3
Serine residue 45 of nodulation protein NodF from Rhizobium leguminosarum bv . viciae is essential for its biological function; Ritsema T et al.; A system for testing the role of the Rhizobium nodF gene in the production of host-specific lipochitin oligosaccharides and in nodulation was developed . We show that a mutant nodF gene, in which the codon for serine residue 45 was changed to that for threonine, still expresses NodF, which, however, is no longer functional.

Plant Mol Biol, 1994 Dec, 26(5), 1413 - 22
Role of rhizobial lipo-chitin oligosaccharide signal molecules in root nodule organogenesis; Spaink HP et al.; The role of oligosaccharide molecules in plant development is discussed . In particular the role of the rhizobial lipo-chitin oligosaccharide (LCO) signal molecules in the development of the root nodule indicates that oligosaccharides play an important role in organogenesis in plants . Recent results of the analyses of structures and of the biosynthesis of the LCO molecules are summarized in this paper . The knowledge and technologies that resulted from these studies will be important tools for further studying the function of LCO signals in the plant and in the search for analogous signal molecules produced by plants.

Plant Mol Biol, 1994 Dec, 26(5), 1305 - 13
Signal molecules involved in plant embryogenesis; Schmidt ED et al.; In plant embryogenesis, inductive interactions mediated by diffusable signal molecules are most likely of great importance . Evidence has been presented that at late globular stages in plant embryogenesis, perturbation of the polar auxin transport results in abberrant embryo morphology . Rhizobium lipooligosaccharides or Nod factors are a newly discovered class of bacterial molecules that are able to trigger initial steps in root nodule development in legumes . Part of the activity of Nod factors may be directed towards alteration of endogenous plant growth regulator balance . The same bacterial Nod factors promoted the formation of globular embryos in the carrot cell line ts11 . Whether there exist plant analogues of the Nod factors and whether these molecules are active as a more universal control system perhaps designed to initiate and or mediate gradients in auxin and cytokinin remains to be determined.

Microbiologia, 1994 Dec, 10(4), 371 - 84
Genetic regulation of nitrogen fixation in Rhizobium meliloti; Cebolla A et al.; The soil bacterium Rhizobium meliloti fixes dinitrogen when associated with root nodules formed on its plant host, Medicago sativa (alfalfa) . The expression of most of the known genes required for nitrogen fixation (nif and fix genes), including the structural genes for nitrogenase, is induced in response to a decrease in oxygen concentration . Induction of nif and fix gene expression by low oxygen is physiologically relevant because a low-oxygen environment is maintained in root nodules to prevent inactivation of the highly oxygen-sensitive nitrogenase enzyme . The genes responsible for sensing and transducing the low oxygen signal, fixL and fixJ, encode proteins (FixL and FixJ, respectively) that are homologous to a large family of bacterial proteins involved in signal transduction, the two component regulatory system proteins . The two components consist of a sensor protein, to which FixL is homologous, and a response regulator protein, to which FixJ is homologous . The sensor protein respond to an activating signal by autophosphorylating and then transferring the phosphate to its cognate response regulator protein . The phosphorylated response regulator, which is often a transcriptional activator, is then able to activate its target . A cascade model of nif and fix gene regulation in R . meliloti has been proposed, whereby FixL acts as an oxygen sensor as the initial event in the cascade and transmits this information to FixJ . FixJ, which possesses a putative helix-turn-helix DNA-binding motif, then activates transcription of the nifA and fixK genes . The nifA and fixK gene products, are transcriptional activators of at least 14 other nif and fix genes.

Int J Biol Macromol, 1994 Dec, 16(6), 301 - 5
Physicochemical properties of extracellular (1-->4)-beta-D-glucuronan produced by the Rhizobium meliloti M5N1CS strain during fermentation: evidence of degradation by an exoenzyme activated by Mg2+; Michaud P et al.; The mutant Rhizobium meliloti M5N1CS (NCIMB 40472) produced a partially acetylated (1-->4)-beta-D-glucuronan during fermentation . The polysaccharide (EPS) extracted from broth during fermentation by microfiltration and ultrafiltration (using a 100 kDa cut-off membrane) was characterized by size exclusion chromatography . The weight-average molecular weight (Mw) of the EPS decreased from 7.5 x 10(5) to 5 x 10(5) between 27 and 75 h of fermentation . When MgSO4.7H2O (0.8 gl-1 per day) was present in the medium during the same period, the Mw of the EPS decreased to 1.5 x 10(5) . Since EPS degradation was detected after microfiltration of the fermentation medium supplemented with magnesium, the Mw decrease of the EPS extracted during fermentation may be explained by enzymic degradation of the polymer activated by Mg2+ ions.

Carbohydr Res, 1994 Nov 15, 264(2), 271 - 80
Structure of the unusual trisaccharide lipopolysaccharide component produced by a symbiotically defective mutant of Rhizobium leguminosarum biovar viciae; Hollingsworth RI et al.; The structure of an unusual trisaccharide component isolated from the lipopolysaccharide (LPS) of a Tn5 mutant of Rhizobium leguminosarum biovar viciae VF39 which is defective in infection of its host plant has been elucidated . This mutant also appears to be defective in the synthesis of a tetrasaccharide component normally synthesized by the wild-type organism . The three glycosyl components are galactose, mannose, and 3-deoxy-D-manno-2-octulosonic acid (Kdo) . Mannose is linked to the 5-position and galactose to the 7-position of the 3-deoxy-2-octulosonic acid residue (Kdo) . Both hexosyl components are in the alpha-pyranosyl form . In the isolated molecule the octulosonic acid appears to be present as its gamma-lactone . However, in the lipopolysaccharide molecule, it is most likely present in the pyranosyl form . The structure was determined by 1H NMR spectroscopy and methylation analysis as well as by fast-atom-bombardment mass spectrometry of the peracetylated and per(trideuterio) acetylated oligosaccharides . Small amounts of the methylation analysis product of another tetrasaccharide different to normal tetrasaccharide component made by the wild-type organisms were detected . This indicates that in this mutant, there is a block in the synthesis of the normal tetrasaccharide component in addition to a switch in the synthesis of the LPS type.

Carbohydr Res, 1994 Nov 15, 264(2), 253 - 69
Synthetic studies toward pyruvate acetal-containing saccharides: en route to the efficient synthesis of Rhizobium-related exopolysaccharide fragments; Eckhardt E et al.; The disaccharide building block benzyl O-(2,3-di-O-benzoyl-4,6-O-{(R)-1-(methoxycarbonyl) ethylidene}-beta-D-galactopyranosyl)-(1-->3)-2-O-benzoyl-4,6-O-{(S)-1- (methoxycarbonyl)ethylidene}-alpha-D-glucopyranoside (13), related to a Rhizobium exopolysaccharide, was prepared by coupling various 4,6-O-{(R)-1-(methoxycarbonyl)ethylidene}-D-galactosyl donors (benzoyl-protected chloride 1, pivaloyl-protected chloride 2, and benzoyl-protected fluorides 3 and 4, and trichloroacetimidate 5) with benzyl 2-O-benzoyl-4,6-O-{(S)-1- (methoxycarbonyl)ethylidene}-alpha-D-glucopyranoside (10) and the corresponding 2,3-O-tetraisopropyldisiloxane-protected glucoside 12 . The best results, with respect to beta-selectivity and yield of the coupling, were obtained with 5 and 10 in dichloromethane . The beta-linked (13) and alpha-linked (14) disaccharides were efficiently converted via the 1-OH derivatives 17 and 21 into the corresponding trichloroacetimidates 18 and 22 . The latter were used for the synthesis of the disaccharide ligands 4,6-(R)-pyruvate-beta-D-Galp-(1-->3)-4,6-(S)-pyruvate-beta-D-Glcp-O(CH2) 5NH2 (20), and 4,6-(R)-pyruvate-alpha-D-Galp-(1-->3)-4,6-(S)-pyruvate-beta-D-Glcp-O (CH2)5NH2 (24) . The corresponding tri- and tetra-saccharide derivatives 4,6-(R)-pyruvate-beta-D-Galp-(1-->3)-4,6-(S)-pyruvate-beta-D-Glcp-(1-->4 )-beta- D-Glcp-O(CH2)5NH2 (28) and 4,6-(R)-pyruvate-beta-D-Galp-(1-->3)-4,6-(S)-pyruvate-beta-D-Glcp-(1-->4 )-beta- D-Glcp-(1-->4)-beta-D-Glcp-O(CH2)25NH2 (36) were obtained similarly.

J Bacteriol, 1994 Nov, 176(22), 7055 - 64
Rhizobium meliloti NodP and NodQ form a multifunctional sulfate-activating complex requiring GTP for activity; Schwedock JS et al.; The nodulation genes nodP and nodQ are required for production of Rhizobium meliloti nodulation (Nod) factors . These sulfated oligosaccharides act as morphogenic signals to alfalfa, the symbiotic host of R . meliloti . In previous work, we have shown that nodP and nodQ encode ATP sulfurylase, which catalyzes the formation of APS (adenosine 5'-phosphosulfate) and PPi . In the subsequent metabolic reaction, APS is converted to PAPS (3'-phosphoadenosine 5'-phosphosulfate) by APS kinase . In Escherichia coli, cysD and cysN encode ATP sulfurylase; cysC encodes APS kinase . Here, we present genetic, enzymatic, and sequence similarity data demonstrating that nodP and nodQ encode both ATP sulfurylase and APS kinase activities and that these enzymes associate into a multifunctional protein complex which we designate the sulfate activation complex . We have previously described the presence of a putative GTP-binding site in the nodQ sequence . The present report also demonstrates that GTP enhances the rate of PAPS synthesis from ATP and sulfate (SO4(2-)) by NodP and NodQ expressed in E . coli . Thus, GTP is implicated as a metabolic requirement for synthesis of the R . meliloti Nod factors.

J Bacteriol, 1994 Nov, 176(21), 6717 - 29
Identification and characterization of a novel Bradyrhizobium japonicum gene involved in host-specific nitrogen fixation; Chun JY et al.; To understand the genetic mechanism of host specificity in the interaction between rhizobia and their hosts, it is important to identify genes that influence both early and late steps in symbiotic development . This paper focuses on the little-understood genetics of host-specific nitrogen fixation . A deletion mutant of Bradyrhizobium japonicum, strain NAD163, was found to induce effective, nitrogen-fixing nodules on soybean and siratro plants but produced ineffective nodules on cowpea plants . Additional transposon and deletion mutants defined a small region that conferred this phenotype, and this region was sequenced to identify two putative open reading frames (ORFs) . Data indicate that only one of these ORFs is detectable in bacteroids . This ORF was termed hsfA, with a predicted protein product of 11 kDa . The transcriptional start site of hsfA was determined and found to coincide with a predicted RpoN-dependent promoter . Microscopic studies of nodules induced by the wild type and hsfA mutants on cowpea and soybean plants indicate that the cowpea mutant nodules are slow to develop . The data indicate that hsfA appears to play a crucial role in bacteroid development on cowpea but does not appear to be essential for nitrogen fixation on the other hosts tested.

Mol Plant Microbe Interact, 1994 Nov-Dec, 7(6), 758 - 65
The regulation of exopolysaccharide production is important at two levels of nodule development in Rhizobium meliloti; Ozga DA et al.; We show that two exopolysaccharide overproducing Tn5 mutants of Rhizobium meliloti, exoR and exoS, have distinct symbiotic defects . While the exoR mutant is unable to colonize nodules, the exoS mutant retains that ability but varies in its ability to produce nitrogen-fixing nodules . We correlate these defects with different degrees of exopolysaccharide overproduction and growth impairment . We further show that the exoR mutant is able to enter developing infection threads but is unable to invade nodule cells . The exoR mutant gives rise to spontaneous pseudorevertants containing second-site suppressor mutations that decrease exopolysaccharide synthesis . These pseudorevertants form nitrogen-fixing nodules . Although the suppressor mutations have the opposite effect on exopolysaccharide production compared to the exoS::Tn5 mutation, they consistently map to the exoS::Tn5 region and belong to the same genetic complementation group as defined by transposon insertion mutations . The effect of the suppressor mutations on exopolysaccharide production is correlated with effects on the expression of exo genes involved in exopolysaccharide synthesis . Finally, we provide evidence that the exoR gene is not required for the regulation of exopolysaccharide synthesis by ammonia.

Mol Plant Microbe Interact, 1994 Nov-Dec, 7(6), 684 - 95
The biosynthesis of rhizobial lipo-oligosaccharide nodulation signal molecules; Carlson RW et al.; While a great deal has been learned concerning the biosynthesis of Nod factors, there is much that remains to be determined . The functions of many Nod proteins involved in adding the host-specific modifications to the Nod factors remain to be unequivocally identified . Some of the genes required for these modifications have not yet been isolated, e.g., those involved in carbamylation, or addition of D-Ara . Additionally the cellular location of most of the Nod proteins and, concomitantly, the modifications they determine are not known . The actual in vivo substrates for the NodABC proteins have not been identified, and the enzyme activities of purified NodA and NodC have not been demonstrated . The synthesis and export of the Nod factors most probably involves some type of carrier/anchor which remains unidentified . Analysis of GlcNAc metabolites from various mutants, e.g., nodA-, nodB-, or nodC- mutants, should facilitate the identification of the in vivo substrates involved in the synthesis of the "common" Nod factor and, thereby, lead to a greater understanding of Nod factor biosynthesis and transport . Finally, comparison of Nod factor biosynthesis to other examples of polysaccharide or glycolipid biosynthetic pathways suggest that several key enzymes remain to be identified . It is hoped that this discussion will be helpful in designing strategies for the detection and isolation of such novel enzymes.

Cell Mol Biol (Noisy-le-grand), 1994 Nov, 40(7), 1029 - 37
The in vitro biosynthesis of functional nodulation factors (Nod Rm) produced by Rhizobium meliloti 1021; Semino CE et al.; Rhizobium meliloti associates symbiotically with alfalfa by forming root nodules in which the bacteria reduce atmospheric N2 into products useful to both organisms . Nod factors are signal molecules, lipooligosaccharides, produced by the bacteria that trigger nodule formation in the plant host . Nod Rm-1 consists of a beta-1,4-N-acetyl glucosamine tetrasaccharide from which the N-acetyl group at the non reducing end is replaced by a fatty acid and the N-acetyl glucosamine at the reducing end is sulfated at position 6 . By in vitro incubation of electroporated cells in the presence of {35S}PAPS or UDP-{14C}GlcNAc a labelled compound has been obtained with the properties of the in vivo produced Nod Rm-1 factor, as judged by HPLC, TLC and HPTLC techniques . The {14C}GlcNAc labelled compound has also hair root deformation activity on alfalfa plantlets indicating that a functional Nod Rm-1 factor has been synthesized in vitro.

Mol Gen Genet, 1994 Nov 1, 245(3), 313 - 22
Oxygen sensitivity and metal ion-dependent transcriptional activation by NIFA protein from Rhizobium leguminosarum biovar trifolii; Screen S et al.; The NIFA protein from Rhizobium leguminosarum biovar trifolii (R . trifolii) strain ANU843 lacks an N-terminal domain present in homologous NIFA proteins from other diazotrophs . The R . trifolii nifA gene product is unstable when expressed in Escherichia coli under both aerobic and microaerobic conditions . Stability is increased by fusion of additional amino acids to the N-terminus of the protein or by expression of nifA in sno mutant (presumed protease deficient) strains of E . coli . Transcriptional activation in vivo by R . trifolii NIFA decreases under aerobic growth conditions, or when cultures are depleted of metal ions . In sno mutant strains this decrease in activity reflects a loss of specific activity rather than proteolytic degradation, implying that R . trifolii NIFA requires metal ions for activity and is oxygen sensitive . The addition of 30 amino acids to the amino-terminus of R . trifoli NIFA results in an oxygen-tolerant protein, with metal ion-dependent activity . Metal ions are therefore not only required for oxygen sensing by R . trifolii NIFA but may play an additional role in determining NIFA structure or activity.

FEBS Lett, 1994 Oct 31, 354(1), 89 - 92
DNA binding activity of NtrC from Rhizobium grown on different nitrogen sources; Patriarca EJ et al.; The DNA-binding activity of the NtrC protein can be demonstrated in gel retardation assays with concentrated protein extracts of Rhizobium etli . Using extracts from either the wild type or a ntrC mutant strain and an antiserum raised against the NtrC protein, we demonstrate specific binding of NtrC to the upstream regulatory region of the glnII gene, where two putative NtrC-binding sites are present . KNO3-grown bacteria contain less NtrC protein and more NtrC-binding activity than NH4Cl-grown bacteria, thus showing that with this protocol it is possible to detect changes in NtrC-binding activity . The advantages of this assay system in comparison with that using pure proteins is discussed.

Mol Gen Genet, 1994 Oct 17, 245(1), 11 - 24
Molecular and genetic characterization of the rhizopine catabolism (mocABRC) genes of Rhizobium meliloti L5-30; Rossbach S et al.; Rhizopine (L-3-O-methyl-scyllo-inosamine, 3-O-MSI) is a symbiosis-specific compound, which is synthesized in nitrogen-fixing nodules of Medicago sativa induced by Rhizobium meliloti strain L5-30 . 3-O-MSI is thought to function as an unusual growth substrate for R . meliloti L5-30, which carries a locus (mos) responsible for its synthesis closely linked to a locus (moc) responsible for its degradation . Here, the essential moc genes were delimited by Tn5 mutagenesis and shown to be organized into two regions, separated by 3 kb of DNA . The DNA sequence of a 9-kb fragment spanning the two moc regions was determined, and four genes were identified that play an essential role in rhizopine catabolism (mocABC and mocR) . The analysis of the DNA sequence and the amino acid sequence of the deduced protein products revealed that MocA resembles NADH-dependent dehydrogenases . MocB exhibits characteristic features of periplasmic-binding proteins that are components of high-affinity transport systems . MocC does not share significant homology with any protein in the database . MocR shows homology with the GntR class of bacterial regulator proteins . These results suggest that the mocABC genes are involved in the uptake and subsequent degradation of rhizopine, whereas mocR is likely to play a regulatory role.

Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 9891 - 5
Differential gene expression in an actinorhizal symbiosis: evidence for a nodule-specific cysteine proteinase; Goetting-Minesky MP et al.; Nodules formed on the roots of actinorhizal plants as a consequence of nitrogen-fixing symbioses with the actinomycete Frankia appear to result from modification of the developmental pathway that leads to lateral root formation . Presently no information exists about factors that control this developmental switch or, until now, about genes that are differentially expressed as a result of an altered developmental pathway . Differential screening of an Alnus glutinosa nodule cDNA library revealed altered levels of gene expression in nodules as compared with roots and allowed isolation of host plant nodule-specific cDNA sequences . The deduced amino acid sequence of one full-length cDNA, AgNOD-CP1, represents a nodule-specific cysteine proteinase similar to cysteine proteinases of the papain superfamily . Residues critical to catalysis, active site, and disulfide bridges are conserved . Suggested roles for this enzyme are as a defense response to Frankia invasion, as a component of tissue remodeling in root and nodule tissues, as a cell cycle component, or as an element of protein turnover . Complexity of hybridization patterns revealed by Southern blot analysis suggests that the gene for AgNOD-CP1 is a member of a multigene family . Northern hybridization results indicate that this gene may have been recruited for a role specific to this symbiosis, a phenomenon observed in the Rhizobium-legume symbioses, perhaps common to many microbe-plant interactions.

Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 9990 - 4
The nodulation-signaling protein NodO from Rhizobium leguminosarum biovar viciae forms ion channels in membranes; Sutton JM et al.; The secreted nodulation-signaling protein NodO was purified from the supernatant of cultures of Rhizobium leguminosarum biovar viciae . The native protein has a M(r) of approximately 67,000, suggesting that it exists as a dimer since the DNA sequence predicts a M(r) of 30,002 . Pure NodO protein had no protease, pectinase, or cellulase activity, and no binding was observed to lipooligosaccharide nodulation factors . Although NodO is relatively hydrophilic, it appeared to insert into liposomes and was protected by liposomes from proteolytic cleavage . When added to planar lipid bilayers, NodO formed cation-selective channels that allowed the movement of monovalent cations (K+ and Na+) across the membrane . NodO is a Ca(2+)-binding protein; in the presence of high concentrations of Ca2+, channel activity was reduced . We hypothesize that NodO plays a role in nodulation signaling by stimulating uptake of nodulation factors or by forming cation-specific channels that function synergistically with the proposed lipooligosaccharide-induced depolarization of the plasma membrane of leguminous plants.

Biochemistry, 1994 Oct 4, 33(39), 11782 - 8
Structures of nodulation factors from the nitrogen-fixing soybean symbiont Rhizobium fredii USDA257; Bec-Ferte MP et al.; We have isolated and characterized the extracellular Nod factors of Rhizobium fredii USDA257, a nitrogen-fixing symbiont of soybean {Glycine max (L.) Merr.} and several other legume species . These signals are produced upon exposure to the isoflavone genistein and consist of a series of substituted, beta 1,4-linked tri-, tetra-, and pentamers of N-acetylglucosamine . N-Vaccenic acid replaces acetate on the nonreducing residue, and the reducing residue contains alpha-linked 2-O-methylfucose on carbon 6 . Small amounts of a fucose-containing tetramer also were present . The Nod factors elicit root-hair deformations on soybean and two other plants at concentrations ranging from 10(-6) to 10(-12) M.

Can J Microbiol, 1994 Oct, 40(10), 873 - 9
Expression of the symbiotic plasmid from Rhizobium leguminosarum biovar trifolii in Sphingobacterium multivorum; Fenton M et al.; An inoculant strain of Rhizobium leguminosarum biovar trifolii containing a Tn5 marked symbiotic plasmid transferred this plasmid by conjugation to Sphingobacterium multivorum, an organism that can be found in soil . The transconjugant bacteria nodulated the roots of white clover (Trifolium repens) seedlings but did not fix atmospheric nitrogen . Microscopic examination revealed abnormal nodule structures . Bacteria isolated from the nodules were shown to be closely related to the recipient S . multivorum and Southern blots of genomic digests probed with nodA DNA confirmed that the transconjugants contained symbiotic genes . This is the first report of the spontaneous transfer, by conjugation, of a symbiotic plasmid from R . leguminosarum biovar trifolii to S . multivorum.

Microbiology, 1994 Oct, 140 ( Pt 10), 2797 - 809
Identification of chromosomal genes located downstream of dctD that affect the requirement for calcium and the lipopolysaccharide layer of Rhizobium leguminosarum; Poole PS et al.; In Rhizobium leguminosarum both the C4-dicarboxylate transport system and wild-type lipopolysaccharide layer (LPS) are essential for nitrogen fixation . A Tn5 mutant (RU301) of R . leguminosarum bv . viciae 3841, was isolated that is only able to synthesize LPS II, which lacks the O-antigen . Strain RU301 exhibits a rough colony morphology, flocculates in culture and is unable to swarm in TY agar . It also fails to grow on organic acids, sugars or TY unless the concentration of calcium or magnesium is elevated above that normally required for growth . The defects in the LPS and growth in strain RU301 were complemented by a series of cosmids from a strain 3841 cosmid library (pRU3020-pRU3022) and a cosmid from R . leguminosarum bv . phaseoli 8002 (pIJ1848) . The transposon insertion in strain RU301 was shown to be located in a 3 kb EcoRI fragment by Southern blotting and cloning from the chromosome . Sub-cloning of pIJ1848 demonstrated that the gene disrupted by the transposon in strain RU301 is located on a 2.4 kb EcoRI-PstI fragment (pRU74) . R . leguminosarum bv . viciae VF39-C86, which is one of four LPS mutants previously isolated by U . B . Priefer (1989, J Bacteriol 171, 6161-6168), was also complemented by sub-clones of pIJ1848 but not by pRU74, suggesting the mutation is in a gene adjacent to that disrupted in strain RU301 . Complementation and Southern analysis indicate that the region contained in pIJ1848 does not correspond to any other cloned Ips genes . Two dctA mutants, RU436 and RU437, were also complemented by pIJ1848 and pRU3020 . Mapping of pIJ1848 and Southern blotting of plasmid-deleted strains of R . leguminosarum revealed that dctD and the region mutated in strain RU301 are located approximately 10 kb apart on the chromosome . Analysis of homogenotes demonstrated that there is not a large region important in calcium utilization, organic acid metabolism or LPS biosynthesis located between the gene disrupted in strain RU301 and dctD . Strain VF39C-86 also required an elevated concentration of calcium for growth on succinate, while strains mutated in the alpha-chromosomal or beta-plasmid group of Ips genes grew at the same calcium concentrations as the wild type, demonstrating that the additional calcium requirement is not a property of all LPS rough mutants . Strain RU301 nodulates peas, but does not reduce acetylene, demonstrating that the gene mutated in this strain is essential for nitrogen fixation.

Plant Cell, 1994 Oct, 6(10), 1415 - 26
Rhizobium nod factors reactivate the cell cycle during infection and nodule primordium formation, but the cycle is only completed in primordium formation; Yang WC et al.; Rhizobia induce the formation of root nodules on the roots of leguminous plants . In temperate legumes, nodule organogenesis starts with the induction of cell divisions in regions of the root inner cortex opposite protoxylem poles, resulting in the formation of nodule primordia . It has been postulated that the susceptibility of these inner cortical cells to Rhizobium nodulation (Nod) factors is conferred by an arrest at a specific stage of the cell cycle . Concomitantly with the formation of nodule primordia, cytoplasmic rearrangement occurs in the outer cortex . Radially aligned cytoplasmic strands form bridges, and these have been called preinfection threads . It has been proposed that the cytoplasmic bridges are related to phragmosomes . By studying the in situ expression of the cell cycle genes cyc2, H4, and cdc2 in pea and alfalfa root cortical cells after inoculation with Rhizobium or purified Nod factors, we show that the susceptibility of inner cortical cells to Rhizobium is not conferred by an arrest at the G2 phase and that the majority of the dividing cells are arrested at the G0/G1 phase . Furthermore, the outer cortical cells forming a preinfection thread enter the cell cycle although they do not divide.

Plant Cell, 1994 Oct, 6(10), 1357 - 74
Rhizobium meliloti lipooligosaccharide nodulation factors: different structural requirements for bacterial entry into target root hair cells and induction of plant symbiotic developmental responses; Ardourel M et al.; Rhizobium meliloti produces lipochitooligosaccharide nodulation NodRm factors that are required for nodulation of legume hosts . NodRm factors are O-acetylated and N-acylated by specific C16-unsaturated fatty acids . nodL mutants produce non-O-acetylated factors, and nodFE mutants produce factors with modified acyl substituents . Both mutants exhibited a significantly reduced capacity to elicit infection thread (IT) formation in alfalfa . However, once initiated, ITs developed and allowed the formation of nitrogen-fixing nodules . In contrast, double nodF/nodL mutants were unable to penetrate into legume hosts and to form ITs . Nevertheless, these mutants induced widespread cell wall tip growth in trichoblasts and other epidermal cells and were also able to elicit cortical cell activation at a distance . NodRm factor structural requirements are thus clearly more stringent for bacterial entry than for the elicitation of developmental plant responses.

Appl Environ Microbiol, 1994 Oct, 60(10), 3815 - 32
Alfalfa yield response to inoculation with recombinant strains of Rhizobium meliloti with an extra copy of dctABD and/or modified nifA expression; Bosworth AH et al.; The construction of rhizobial strains which increase plant biomass under controlled conditions has been previously reported . However, there is no evidence that these newly constructed strains increase legume yield under agricultural conditions . This work tested the hypothesis that carefully manipulating expression of additional copies of nifA and dctABD in strains of Rhizobium meliloti would increase alfalfa yield in the field . The rationale for this hypothesis is based on the positive regulatory role that nifA plays in the expression of the nif regulon and the fact that a supply of dicarboxylic acids from the plant is required as a carbon and energy source for nitrogen fixation by the Rhizobium bacteroids in the nodule . These recombinant strains, as well as the wild-type strains from which they were derived, are ideal tools to examine the effects of modifying or increasing the expression of these genes on alfalfa biomass . The experimental design comprised seven recombinant strains, two wild-type strains, and an uninoculated control . Each treatment was replicated eight times and was conducted at four field sites in Wisconsin . Recombinant strain RMBPC-2, which has an additional copy of both nifA and dctABD, increased alfalfa biomass by 12.9% compared with the yield with the wild-type strain RMBPC and 17.9% over that in the uninoculated control plot at the site where soil nitrogen and organic matter content was lowest . These increases were statistically significant at the 5% confidence interval for each of the three harvests made during the growing season . Strain RMBPC-2 did increase alfalfa biomass at the Hancock site; however, no other significant increases or decreases in alfalfa biomass were observed with the seven other recombinant strains at that site . At three sites where this experiment was conducted, either native rhizobial populations or soil nitrogen concentrations were high . At these sites, none of the recombinant strains affected yield . We conclude that RMBPC -2 can increase alfalfa yields under field conditions of nitrogen limitation, low endogenous rhizobial competitors, and sufficient moisture.

Appl Environ Microbiol, 1994 Oct, 60(10), 3615 - 23
The NodD proteins of Rhizobium sp . strain BR816 differ in their interactions with coinducers and in their activities for nodulation of different host plants; van Rhijn P et al.; The early steps of symbiotic nodule formation by Rhizobium spp . on plants require coordinate expression of several nod gene operons, which is accomplished by the activating protein NodD . Rhizobium sp . strain BR816, isolated from Leucaena leucocephala, contains four nodD genes which differ in their interaction with flavonoids . Two of the four NodD proteins, namely, NodD1 and NodD2, obey the LysR rule regarding the need of a coinducer . NodD3 shows hardly any inducing activity, and NodD4 contains a high basal activity and no response to any of the flavonoids tested . Complementation experiments with the NGR234 nodD mutant by the different nodD genes of BR816, as well as the analysis of the nodulation phenotype of different nodD mutants of BR816, revealed that all the nodD genes of BR816 are functional, but differences can be noticed when different host plants are tested . Whereas the nodD2 and nodD4 genes of BR816 have a great impact on the nodulation of L . leucocephala, nodD3 and nodD4 appear to be important for the nodulation of Phaseolus vulgaris . It appears that NodD1 of BR816 can function as a transcriptional activator in bean nodulation but not in nodulation of L . leucocephala.

Plant Mol Biol, 1994 Oct, 26(1), 303 - 11
Repression of the L-asparaginase gene during nodule development in Lupinus angustifolius; Vincze E et al.; Upon the establishment of an effective nitrogen-fixing symbiosis in amide-transporting plants the enzymatic activity and transcript levels of L-asparaginase are dramatically decreased . This decrease in L-asparaginase activity is essential for the correct functioning of the Rhizobium-legume symbiosis in lupin in which asparagine, synthesized from recently fixed nitrogen, is exported to aerial parts of the plant for use in growth and development . Concomitant with this decrease in L-asparaginase transcript a DNA-binding protein was detected in the nodules . This binding protein was not detectable in ineffective nodules, in nodules treated with nitrate, or in root tips, mature roots, developing flowers or developing seeds . The DNA-binding activity was shown to interact with a 59 bp sequence proximal to the transcription start site . Within this sequence a CTAAAAT direct repeat and a ACTGT/TGTCA incomplete inverted repeat were implicated in the binding of protein to the DNA by DNase I protection experiments . Competitive binding studies with synthesized binding sites were consistent with the CTAAAAT/TGTCA sequence pair proximal to the transcription start site having the highest affinity for the DNA-binding protein . We postulate that this DNA-binding protein is associated with repression of L-asparaginase gene expression in mature lupin root nodules.

J Bacteriol, 1994 Oct, 176(19), 6066 - 73
Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis; Rey L et al.; The products of the Rhizobium leguminosarum hyp gene cluster are necessary for synthesis of a functional uptake {NiFe} hydrogenase system in symbiosis with pea plants, and at least for HypB and HypF, a role in hydrogenase-specific nickel metabolism has been postulated (L . Rey, J . Murillo, Y . Hernando, E . Hidalgo, E . Cabrera, J . Imperial, and T . Ruiz-Argueso, Mol . Microbiol . 8:471-481, 1993) . The R . leguminosarum hypB gene product has been overexpressed in Escherichia coli and purified by immobilized nickel chelate affinity chromatography in a single step . The purified recombinant HypB protein was able to bind 3.9 +/- 0.1 Ni2+ ions per HypB monomer in solution . Co2+, Cu2+, and Zn2+ ions competed with Ni2+ with increasing efficiency . Monospecific HypB antibodies were raised and used to show that HypB is synthesized in R . leguminosarum microaerobic vegetative cells and pea bacteroids but not in R . leguminosarum aerobic cells . HypB protein synthesized by R . leguminosarum microaerobic vegetative cells could also be isolated by immobilized nickel chelate affinity chromatography . A histidine-rich region at the amino terminus of the protein (23-HGHHHH DGHHDHDHDHDHHRGDHEHDDHHH-54) is proposed to play a role in nickel binding, both in solution and in chelated form.

Lett Appl Microbiol, 1994 Oct, 19(4), 240 - 3
Phosphodiesterase and phosphotriesterase in Rhizobium and Bradyrhizobium strains and their roles in the degradation of organophosphorus pesticides; Abd-Alla MH; Of 13 Rhizobium and Bradyrhizobium strains investigated for the production of cellular and extracellular phosphodiesterase and phosphotriesterase, all were found to produce both enzymes . Phosphodiesterase was produced at a much higher level than phosphotriesterase . Rhizobium meliloti TAL 1373 was the most productive . The extracellular enzymes were activated by inclusion in the assay mixture of Ca2+ or Mg2+ . The enzymes were inhibited by Zn2+ but not significantly affected by Cu2+, Co2+ and Mn2+ . Both hydrolases were inhibited by dithiothreitol but not by thiol-directed inhibitors, suggesting that sulphydryl groups are not directly involved in catalysis . The enzymes have the ability to hydrolyse some organophosphorus compounds, suggesting that Rhizobium and Bradyrhizobium strains play an important role in the degradation of organophosphorus pesticides.

J Biol Chem, 1994 Sep 23, 269(38), 23784 - 9
Phosphorylation of the Rhizobium meliloti FixJ protein induces its binding to a compound regulatory region at the fixK promoter; Galinier A et al.; The FixJ protein is a member of the regulator class of two-component systems involved in the transcriptional activation of nitrogen fixation genes in Rhizobium meliloti . Phosphorylation of FixJ was previously demonstrated to dramatically enhance its transcriptional activity at the nifA and fixK promoters . Here we show that the isolated carboxyl-terminal domain of FixJ, FixJC, binds the fixK promoter, whereas binding of the full-length FixJ protein requires its phosphorylation . By analyzing the DNase I and Exonuclease III protection patterns of the wild-type and a mutant fixK promoter, we have identified two overlapping binding regions for both phosphorylated FixJ and FixJC . A higher affinity region is located between positions -69 and -44 relative to the transcription start site, and a lower affinity region, between positions -57 and -31, overlaps the -35 region of the promoter.

J Bacteriol, 1994 Sep, 176(17), 5297 - 303
Nickel availability to pea (Pisum sativum L.) plants limits hydrogenase activity of Rhizobium leguminosarum bv . viciae bacteroids by affecting the processing of the hydrogenase structural subunits; Brito B et al.; Rhizobium leguminosarum bv . viciae UPM791 induces the synthesis of an {NiFe} hydrogenase in pea (Pisum sativum L.) bacteroids which oxidizes the H2 generated by the nitrogenase complex inside the root nodules . The synthesis of this hydrogenase requires the genes for the small and large hydrogenase subunits (hupS and hupL, respectively) and 15 accessory genes clustered in a complex locus in the symbiotic plasmid . We show here that the bacteroid hydrogenase activity is limited by the availability of nickel to pea plants . Addition of Ni2+ to plant nutrient solutions (up to 10 mg/liter) resulted in sharp increases (up to 15-fold) in hydrogenase activity . This effect was not detected when other divalent cations (Zn2+, Co2+, Fe2+, and Mn2+) were added at the same concentrations . Determinations of the steady-state levels of hupSL-specific mRNA indicated that this increase in hydrogenase activity was not due to stimulation of transcription of structural genes . Immunoblot analysis with antibodies raised against the large and small subunits of the hydrogenase enzyme demonstrated that in the low-nickel situation, both subunits are mainly present in slow-migrating, unprocessed forms . Supplementation of the plant nutrient solution with increasing nickel concentrations caused the conversion of the slow-migrating forms of both subunits into fast-moving, mature forms . This nickel-dependent maturation process of the hydrogenase subunits is mediated by accessory gene products, since bacteroids from H2 uptake-deficient mutants carrying Tn5 insertions in hupG and hupK and in hypB and hypE accumulated the immature forms of both hydrogenase subunits even in the presence of high nickel levels.

J Bacteriol, 1994 Sep, 176(17), 5210 - 7
Osmoadaptation in rhizobia: ectoine-induced salt tolerance; Talibart R et al.; After having shown that ectoine (a tetrahydropyrimidine) displays osmoprotective properties towards Escherichia coli (M . Jebbar, R . Talibart, K . Gloux, T . Bernard, and Blanco, J . Bacteriol . 174:5027-5035, 1992), we have investigated the involvement of this molecule in the osmotic adaptation of Rhizobium meliloti . Ectoine appeared almost as effective as glycine betaine in improving the growth of R . meliloti under adverse osmotic conditions (0.5 M NaCl) . Moreover, improvement of growth of rhizobial strains insensitive to glycine betaine was also observed . Ectoine transport proved inducible, periplasmic protein dependent, and, as shown by competition experiments, distinct from the transport of glycine betaine . Medium osmolarity had little effect on the uptake characteristics, since the rate of influx increased from 12 to only 20 nmol min-1 mg of protein-1 when NaCl concentrations were raised from 0 to 0.3 or 0.5 M, with a constant of transport of 80 microM . Natural-abundance 13C-nuclear magnetic resonance and radiolabelling assays showed that ectoine, unlike glycine betaine, is not intracellularly accumulated and, as a consequence, does not repress the synthesis of endogenous compatible solutes (glutamate, N-acetylglutaminylglutamine amide, and trehalose) . Furthermore, the strong rise in glutamate content in cells osmotically stressed in the presence of ectoine suggests that, instead of being involved in osmotic balance restoration, ectoine should play a key role in triggering the synthesis of endogenous osmolytes . Hence, we believe that there are at least two distinct classes of osmoprotectants: those such as glycine betaine or glutamate, which act as genuine osmolytes, and those such as ectoine, which act as chemical mediators.

Microbiol Rev, 1994 Sep, 58(3), 352 - 86
Genetic regulation of nitrogen fixation in rhizobia; Fischer HM; This review presents a comparison between the complex genetic regulatory networks that control nitrogen fixation in three representative rhizobial species, Rhizobium meliloti, Bradyrhizobium japonicum, and Azorhizobium caulinodans . Transcription of nitrogen fixation genes (nif and fix genes) in these bacteria is induced primarily by low-oxygen conditions . Low-oxygen sensing and transmission of this signal to the level of nif and fix gene expression involve at least five regulatory proteins, FixL, FixJ, FixK, NifA, and RpoN (sigma 54) . The characteristic features of these proteins and their functions within species-specific regulatory pathways are described . Oxygen interferes with the activities of two transcriptional activators, FixJ and NifA . FixJ activity is modulated via phosphorylation-dephosphorylation by the cognate sensor hemoprotein FixL . In addition to the oxygen responsiveness of the NifA protein, synthesis of NifA is oxygen regulated at the level of transcription . This type of control includes FixLJ in R . meliloti and FixLJ-FixK in A . caulinodans or is brought about by autoregulation in B . japonicum . NifA, in concert with sigma 54 RNA polymerase, activates transcription from -24/-12-type promoters associated with nif and fix genes and additional genes that are not directly involved in nitrogen fixation . The FixK proteins constitute a subgroup of the Crp-Fnr family of bacterial regulators . Although the involvement of FixLJ and FixK in nifA regulation is remarkably different in the three rhizobial species discussed here, they constitute a regulatory cascade that uniformly controls the expression of genes (fixNOQP) encoding a distinct cytochrome oxidase complex probably required for bacterial respiration under low-oxygen conditions . In B . japonicum, the FixLJ-FixK cascade also controls genes for nitrate respiration and for one of two sigma 54 proteins.

Mol Plant Microbe Interact, 1994 Sep-Oct, 7(5), 564 - 72
DNA sequence of the common nodulation genes of Bradyrhizobium elkanii and their phylogenetic relationship to those of other nodulating bacteria; Dobert RC et al.; A 6.6-kb BamHI fragment containing the common nodulation genes of Bradyrhizobium elkanii USDA94 was identified by southern hybridization using the common nod genes of B . japonicum as a probe . This fragment was cloned and sequenced . Analysis of the sequence showed open reading frames highly homologous to nolA, nodD2, nodD1, and nodKABC from other bradyrhizobial sources . The sequence showed higher homology to the common nod genes of Bradyrhizobium sp . (Parasponia) than to those from B . japonicum . The open reading frame identified between nodD1 and nodA in the B . elkanii sequence was far more similar to nodK from Bradyrhizobium sp . (Parasponia) than to nodY from B . japonicum . The molecular phylogeny of nodD and nodAB from many sources was analyzed . The genetic distance between the nod genes is far greater than the distance between the 16S rRNA and nifH genes . The differences between the nod genes among the species of Rhizobium is as great as that between Bradyrhizobium and Rhizobium . The host range of the microsymbiont was found to be a better predictor of the similarities of the common nod genes than the 16S rRNA or nifH genes . We propose two groups of nod genes among the rhizobia and bradyrhizobia, based on molecular phylogenetic analysis: those which nodulate legumes of temperate origin in the tribes Vicieae or Trifolieae and those which nodulate legumes of tropical origin in the tribe Phaseoleae.

Plant Cell, 1994 Sep, 6(9), 1265 - 75
A new proline-rich early nodulin from Medicago truncatula is highly expressed in nodule meristematic cells; Wilson RC et al.; We cloned and characterized MtPRP4, a new member of the repetitive proline-rich protein gene family in Medicago truncatula . The sequence of MtPRP4 predicts a 62-kD protein consisting of a 22-amino acid N-terminal signal peptide and a 527-amino acid repetitive proline-rich domain composed of three repetitive pentapeptide motifs arranged into two decapeptide repeats: PPVEKPPVHK and PPVEKPPVYK . MtPRP4 is the largest PRP described to date and contains repeated motifs that have not previously been found together in a single polypeptide . RNA gel blot experiments detected MtPRP4 transcripts in symbiotic root nodules, but not in roots, hypocotyls, or leaves . Accumulation of MtPRP4 transcript was an early response to Rhizobium inoculation and did not depend on nodule infection . In situ hybridization experiments demonstrated that MtPRP4 was expressed early in the development of the nodule meristem and that expression was highest in the meristematic cells of mature indeterminate nodules . These data support the proposition that an important early response of legume host roots to Rhizobium involves remodeling the host extracellular matrix and that proline-rich wall proteins play an important role in this architectural modification.

Mol Cell Biochem, 1994 Sep, 138(1-2), 123 - 9
Reversible ADP-ribosylation as a mechanism of enzyme regulation in procaryotes; Ludden PW; Several cases of ADP-ribosylation of endogenous proteins in procaryotes have been discovered and investigated . The most thoroughly studied example is the reversible ADP-ribosylation of the dinitrogenase reductase from the photosynthetic bacterium Rhodospirillum rubrum and related bacteria . A dinitrogenase reductase ADP-ribosyltransferase (DRAT) and a dinitrogenase reductase ADP-ribose glycohydrolase (DRAG) from R . rubrum have been isolated and characterized . The genes for these proteins have been isolated and sequences and show little similarity to the ADP-ribosylating toxins . Other targets for endogenous ADP-ribosylation by procaryotes include glutamine synthetase in R . rubrum and Rhizobium meliloti and undefined proteins in Streptomyces griseus and Pseudomonas maltophila.

Mol Microbiol, 1994 Sep, 13(5), 821 - 31
Structural identification of metabolites produced by the NodB and NodC proteins of Rhizobium leguminosarum; Spaink HP et al.; The Rhizobium nodulation genes nodABC are involved in the synthesis of lipo-chitin oligosaccharides . We have analysed the metabolites which are produced in vivo and in vitro by Rhizobium strains which express the single nodA, nodB and nodC genes or combinations of the three . In vivo radioactive labelling experiments, in which D-{1-14C}-glucosamine was used as a precursor, followed by mass spectrometric analysis of the purified radiolabelled metabolic products, showed that Rhizobium strains that only express the combination of the nodB and nodC genes do not produce lipo-chitin oligosaccharides but instead produce chitin oligomers (mainly pentamers) which are devoid of the N-acetyl group on the non-reducing terminal sugar residue (designated NodBC metabolites) . Using the same procedure we have shown that when the nodL gene is expressed in addition to the nodBC genes the majority of metabolites contain an additional O-acetyl substituent on the non-reducing terminal sugar residue (designated NodBCL metabolites) . The NodBC and NodBCL metabolites purified after in vivo labelling were compared with the radiolabelled metabolites produced in vitro by Rhizobium bacterial cell lysates to which UDP-N-acetyl-D-{U-14C}-glucosamine was added using thin-layer chromatography . The results show that the lysates of strains which expressed the nodBC or nodBCL genes can also produce NodBC and NodBCL metabolites . The same results were obtained when the NodB and NodC proteins were produced separately in two different strains . On the basis of these and other recent results, we propose that NodB is a chitin oligosaccharide deacetylase, NodC an N-acetylglucosaminyltransferase and, by default, NodA is involved in lipid attachment.

Trends Microbiol, 1994 Sep, 2(9), 318 - 24
Signalling strategies for nodulation of legumes by rhizobia; Downie JA; During the formation of nitrogen-fixing root nodules, the establishment of the symbiotic relationship between rhizobia and leguminous plants depends on a highly specific exchange of signals . The products of several of the rhizobial nodulation (nod) genes are involved in the biosynthesis of host-specific lipo-oligosaccharide signalling molecules that can induce nodule morphogenesis on legume roots . Such signalling may point to a more widespread cell-to-cell signalling system in plants.

Lett Appl Microbiol, 1994 Sep, 19(3), 142 - 5
Construction of a single-transposon-insertion mutant in Rhizobium sp . strain TAL1145 from a double-insertion mutant; Parveen N et al.; A method for developing a single-transposon-insertion mutant from a double-insertion mutant in Rhizobium is described . An exopolysaccharide (EPS)-defective mutant containing two Tn5-lacZ insertions was complemented with cloned wild-type DNA for EPS synthesis . One of the Tn5-lacZ insertions from the mutant was transferred to the complementing plasmid by homologous recombination . The plasmid containing the Tn5-lacZ insertion in the gene involved in EPS synthesis was transferred into the wild-type strain and the Tn5-lacZ was homogenized to obtain an EPS-defective mutant with a single Tn5-lacZ insertion.

Proc Natl Acad Sci U S A, 1994 Aug 30, 91(18), 8418 - 22
Biosynthesis of Rhizobium meliloti lipooligosaccharide Nod factors: NodA is required for an N-acyltransferase activity; Atkinson EM et al.; Rhizobium bacteria synthesize N-acylated beta-1,4-N-acetylglucosamine lipooligosaccharides, called Nod factors, which act as morphogenic signal molecules to legume roots during development of nitrogen-fixing nodules . The biosynthesis of Nod factors is genetically dependent upon the nodulation (nod) genes, including the common nod genes nodABC . We used the Rhizobium meliloti NodH sulfotransferase to prepare 35S-labeled oligosaccharides which served as metabolic tracers for Nod enzyme activities . This approach provides a general method for following chitooligosaccharide modifications . We found nodAB-dependent conversion of N-acetylchitotetraose (chitotetraose) monosulfate into hydrophobic compounds which by chromatographic and chemical tests were equivalent to acylated Nod factors . Sequential incubation of labeled intermediates with Escherichia coli containing either NodA or NodB showed that NodB was required before NodA during Nod factor biosynthesis . The acylation activity was sensitive to oligosaccharide chain length, with chitotetraose serving as a better substrate than chitobiose or chitotriose . We constructed a putative Nod factor intermediate, GlcN-beta 1,4-(GlcNAc)3, by enzymatic synthesis and labeled it by NodH-mediated sulfation to create a specific metabolic probe . Acylation of this oligosaccharide required only NodA . These results confirm previous reports that NodB is an N-deacetylase and suggest that NodA is an N-acyltransferase.

Carbohydr Res, 1994 Aug 17, 261(2), 279 - 95
Molecular architecture of a galactoglucan from Rhizobium meliloti; Chandrasekaran R et al.; Rhizobium meliloti mutants produce a linear, acidic exopolysaccharide with alternating galactopyranosyl and glucopyranosyl units having (1-->3) linkages . It has a 4,6-O-pyruvic cyclic acetal group on the alpha-galactosyl and 6-O-acetyl ester group on the beta-glucosyl units . X-ray diffraction patterns from polycrystalline and well oriented specimens of its potassium salt indicate that the polymer forms a 2-fold helix of pitch 15.89 A . The three-dimensional structure has been determined and refined by using the X-ray intensities and the linked-atom least-squares technique . The details of the antiparallel packing arrangement of two helices in an orthorhombic unit cell, a = 14.49, b = 9.79, and c = 15.89 A, reveal that each disaccharide repeating unit is associated with one potassium and three water molecules . The helices are interconnected by a series of ...COO- ...K+ ...W ...COO- .. . interactions . Both pyruvyl and acetyl groups, which are on the periphery of the helix, are involved in the association of the polysaccharide chains and thus appear to be an integral component of the galactoglucan in the nodule invasion process.

J Biol Chem, 1994 Aug 12, 269(32), 20401 - 9
Constitutive ATP hydrolysis and transcription activation by a stable, truncated form of Rhizobium meliloti DCTD, a sigma 54-dependent transcriptional activator; Lee JH et al.; The dctD gene product (DCTD) activates transcription from dctA by the sigma 54-holoenzyme form of RNA polymerase in Rhizobium meliloti . We have purified a constitutively active form of R . meliloti DCTD that lacks 142 amino acid residues from the N terminus (designated DCTDL143) . Purified DCTDL143 recognized the DCTD-binding sites at the dctA promoter region and catalyzed the isomerization of closed complexes between sigma 54-holoenzyme and the dctA promoter to open complexes . Like the related sigma 54-dependent activators NTRC and NIFA, a purine nucleoside triphosphate with a hydrolyzable beta-gamma bond was required prior to transcription initiation for this isomerization . DCTDL143 hydrolyzed purine nucleoside triphosphates but not pyrimidine nucleoside triphosphates . As observed with NTRC-phosphate, the specific activity for the ATPase of DCTDL143 was strongly dependent on the enzyme concentration and was stimulated by DNA fragments bearing the binding sites for the protein . These DNA fragments increased the Vmax for MgATP hydrolysis but did not significantly lower the apparent Km for MgATP . These data are consistent with the idea proposed for related activators that DCTDL143 must assemble into an active, oligomeric form before it can hydrolyze MgATP and presumably activate transcription.

J Biol Chem, 1994 Aug 5, 269(31), 19777 - 86
Cloning and sequencing of ATP sulfurylase from Penicillium chrysogenum . Identification of a likely allosteric domain; Foster BA et al.; Fungal (Penicillium chrysogenum) and yeast (Saccharomyces cerevisiae) ATP sulfurylases were shown to have very similar kinetic and chemical properties except that the fungal enzyme (a) contains a highly reactive Cys residue (SH-1) whose modification results in sigmoidal velocity curves (Renosto, F., Martin, R . L., and Segel, I . H . (1987) J . Biol . Chem . 262, 16279-16288) and (b) is allosterically inhibited by 3'-phosphoadenosine 5'-phosphosulfate (PAPS), while the yeast enzyme displays neither of these properties . The fungal enzyme subunit (64.3 kDa, 572 amino acids) is also larger than the yeast enzyme subunit (59.3 kDa, 521 amino acids) . To correlate the unique allosteric properties of the fungal enzyme with specific structural features, we cloned and sequenced the ATP sulfurylase gene (aps) from P . chrysogenum . The yeast and fungal enzymes are homologous over the first 400 amino acids and contain two regions high in basic residues which are conserved in sulfurylases from Arabidopsis and the Riftia pachyptila (hydrothermal vent tube worm) chemolithotrophic symbiont . These regions may participate in forming the binding sites for MgATP2- and SO4(2-) . The fungal enzyme has no sites for MgATP2- and SO4(2-) . The fungal enzyme has no significant sequence homology to the yeast enzyme in the C-terminal 172 amino acids . This C-terminal region contains SH-1 (Cys-508) and has homology to MET14 (S . cerevisiae), CYSC (E . coli), and NODQ (Rhizobium meliloti), i.e . adenosine 5'-phosphosulfate (APS) kinase . The cumulative results suggest that (a) the allosteric PAPS binding site of P . chrysogenum ATP sulfurylase is located in the C-terminal domain of the protein and (b) that this domain may have evolved from APS kinase . In spite of the homology, this C-terminal region does not account for the APS kinase activity of P . chrysogenum . Fungal ATP sulfurylase has no significant homology to (or regulatory properties in common with) CYSD or CYSN, proteins reported to comprise E . coli ATP sulfurylase (Leyh, T., Vogt, T . F., and Suo, Y . (1992) J . Biol . Chem . 267, 10405-10410).

J Bacteriol, 1994 Aug, 176(15), 4646 - 55
Biosynthesis of a structurally novel lipid A in Rhizobium leguminosarum: identification and characterization of six metabolic steps leading from UDP-GlcNAc to 3-deoxy-D-manno-2-octulosonic acid2-lipid IVA; Price NP et al.; Lipopolysaccharides (LPSs) are prominent structural components of the outer membranes of gram-negative bacteria . In Rhizobium spp . LPS functions as a determinant of the nitrogen-fixing symbiosis with legumes . LPS is anchored to the outer surface of the outer membrane by the lipid A moiety, the principal lipid component of the outer bacterial surface . Several notable structural differences exist between the lipid A of Escherichia coli and that of Rhizobium leguminosarum, suggesting that diverse biosynthetic pathways may also exist . These differences include the lack of phosphate groups and the presence of a 4'-linked GalA residue in the latter . However, we now show that UDP-GlcNAc plays a key role in the biosynthesis of lipid A in R . leguminosarum, as it does in E . coli . 32P-labeled monosaccharide and disaccharide lipid A intermediates from E . coli were isolated and tested as substrates in cell extracts of R . leguminosarum biovars phaseoli and viciae . Six enzymes that catalyze the early steps of E . coli lipid A biosynthesis were also present in extracts of R . leguminosarum . Our results show that all the enzymes of the pathway leading to the formation of the intermediate 3-deoxy-D-manno-2-octulosonic acid (Kdo2)-lipid IVA are functional in both R . leguminosarum biovars . These enzymes include (i) UDP-GlcNAc 3-O-acyltransferase; (ii) UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase; (iii) UDP-3-O-(R-3-hydroxymyristoyl)-GlcN N-acyltransferase; (iv) disaccharide synthase; (v) 4'-kinase; and (vi) Kdo transferase . Our data suggest that the early steps in lipid A biosynthesis are conserved and that the divergence leading to rhizobial lipid A may occur at a later stage in the pathway, presumably after the attachment of the Kdo residues.

Trends Microbiol, 1994 Aug, 2(8), 277 - 83
Host-plant invasion by Rhizobium: the role of cell-surface components; Kannenberg EL et al.; Rhizobia are soil bacteria that can become endosymbionts, reducing atmospheric nitrogen within nodules formed on the roots of legume plants . During tissue and cell invasion, bacterial cell-surface components adapt the bacterium to survive as an endophyte without eliciting host-defence responses . The structures of many of these components have been established recently, allowing their possible roles in invasion to be defined more clearly.

Plant J, 1994 Aug, 6(2), 241 - 9
Rhizobium meliloti Nod factors elicit cell-specific transcription of the ENOD12 gene in transgenic alfalfa; Journet EP et al.; Extracellular lipo-oligosaccharides of Rhizobium, known as Nod factors, play a key role in the molecular signal exchange which leads to the specific nitrogen-fixing symbiotic association between the soil microbe and its host legume . The biological activity of Nod factors and their perception by the host plant during the earliest stages of the Rhizobium/legume interaction have been studied using transgenic alfalfa carrying a fusion between the promoter of the early nodulin gene MtENOD12 and the beta-glucuronidase (GUS) reporter gene . Histochemical staining has shown that GUS accumulates specifically in the differentiating root epidermis, prior to and during root hair emergence, within 2-3 h following the addition of purified Rhizobium meliloti Nod factors . This precocious transcriptional activation of the MtENOD12 gene, reminiscent of that observed after inoculation with intact Rhizobium, implies that the Nod factor signal can be perceived at a developmental stage preceding root hair formation . GUS activity can be detected following treatment with a wide range of R . meliloti Nod factor concentrations down to 10(-13) M, and furthermore, this rapid response to the bacterial elicitor appears to be non-systemic . Significantly, MtENOD12-GUS expression is not observed after inoculation with a R . meliloti nodH mutant which synthesizes exclusively non-sulphated Nod factors . Indeed purified Nod factors which lack the sulphate substituent are approximately 1000-fold less active than their sulphated counterparts . Thus, the triggering of ENOD12 transcription in the alfalfa root epidermis is a rapid molecular response which is subject to the same host-specificity determinant (Nod factor sulphation) that governs the interaction between alfalfa and its bacterial symbiont.

Gene, 1994 Jul 22, 145(1), 87 - 90
Two inverted repeats in the nodD promoter region are involved in nodD regulation in Rhizobium leguminosarum; Mao C et al.; In Rhizobium leguminosarum (R.l.) biovar viciae, the nodulation gene nodD encodes a transcriptional activator (NodD) which binds to highly conserved DNA sequences (nod-boxes) in the promoters of other nod operons . In addition, NodD represses nodD transcription and this occurs at the divergent and overlapping nodA-nodD promoters . We mutagenised this region with hydroxylamine, and by cloning the mutagenised DNA into a vector carrying the lacZ reporter gene downstream from the cloning site identified mutations affecting nodD expression and repression . The resulting plasmids were transferred to R . l . viciae strains containing or lacking nodD . Two classes of promoter mutants were identified: those in which nodD transcription was altered and those in which NodD-dependent repression was altered . The nucleotide (nt) changes in the promoter region were found to be located within two inverted repeat sequences (A2 and A3) which are about 70 bp apart . A2 is important for nodD transcription and A3 (which is upstream from A2) is involved in NodD-dependent repression . The nt sequence at A3 shows some homology to the nod-box region of the nodA promoter . It is proposed that the NodD-dependent repression occurs as a result of NodD binding to both A3 and the nodA nod-box, forming a loop which prevents transcription of nodD from its promoter, A2, which lies between A3 and the nod-box . This model is supported by the observation that there are at least three sites for NodD binding in the nodA-nodD promoter region.

Carbohydr Res, 1994 Jul 16, 260(2), 305 - 17
The structure of the O-antigenic chain of the lipopolysaccharide of Rhizobium trifolii 4s; Wang Y et al.; The structure of the O-antigen chain of the lipopolysaccharide (LPS) of Rhizobium trifolii 4s has been determined by a combination of chemical and spectroscopic methods . The glycosyl components were found to be L-rhamnose, N-acetyl-D-glucosamine, and N-acetyl-D-mannosamine in 3:1:1 molar proportion, as determined by gas chromatography and gas chromatography-mass spectrometry of alditol acetate and persilylated (R)-2-hydroxybutyl glycoside derivatives . The linkage positions and configurations of the glycosyl residues were obtained by 1D and 2D NMR spectroscopy . The polymer has a pentasaccharide repeating-unit containing rhammose and N-acetylglucosamine in the main chain and N-acetylmannosamine as the sole-side chain component . This latter residue is linked to a main-chain rhamnose residue . This result was suggested by NMR spectroscopy and confirmed by periodate oxidation . The sequence was deduced by 1D and 2D NMR NOE experiments and by partial hydrolysis studies . The repeating unit of the polysaccharide is shown . This constitutes the first complete structure of an O-antigenic chain of the lipopolysaccharide of any strain of Rhizobium trifolii.

J Biol Chem, 1994 Jul 8, 269(27), 17931 - 8
Specific, high affinity binding of chitin fragments to tomato cells and membranes . Competitive inhibition of binding by derivatives of chitooligosaccharides and a Nod factor of Rhizobium; Baureithel K et al.; Suspension-cultured tomato cells have a sensitive perception system for chitin fragments with a degree of polymerization (DP) > or = 4 and react to these compounds with a transient alkalinization of their culture medium (Felix, G., Regenass, M., and Boller, T . (1993) Plant . J . 4, 307-316) . A chitin fragment with DP 5 was aminated at the reducing end and coupled to t-butoxycarbonyl-L-{35S}methionine via an amidoglycine spacer . The radiolabeled chitin fragment (approximately 1000 Ci/mmol) exhibited specific, saturable, reversible binding to whole tomato cells as well as to tomato microsomal membranes with dissociation constants of 1.4 and 23 nM, respectively . Binding of the radioligand was competed by chitin fragments of different DP with IC50 values (50% inhibition of binding) that closely paralleled the concentrations inducing the alkalinization response half-maximally . Deacetylated chitooligosaccharides and N-propanoyl chitooligosaccharides were weak elicitors of the alkalinization response as well as weak competitors of radioligand binding . A lipochitooligosaccharide (Nod factor) from Rhizobium leguminosarum stimulated the alkalinization response in tomato cells half-maximally at 3 nM and competed radioligand binding to the cells with an IC50 of 8 nM . These results demonstrate the presence of a high affinity binding site for chitin fragments on the tomato cell membrane that may function as a receptor.

Biochemistry, 1994 Jul 5, 33(26), 8067 - 73
Heme-based sensors, exemplified by the kinase FixL, are a new class of heme protein with distinctive ligand binding and autoxidation; Gilles-Gonzalez MA et al.; FixL's are chimeric heme protein kinases from symbiotic nitrogen-fixing Rhizobia . We have overexpressed three FixL variants in Escherichia coli . Bradyrhizobium japonicum FixL, a soluble dimeric protein, is the first full-length FixL to be purified . The other two proteins are soluble truncations of Rhizobium meliloti FixL, which is a membrane protein . One contains both heme and kinase domains and is dimeric; the other has only the heme domain and is monomeric . We find that all the FixL's bind oxygen and carbon monoxide non-cooperatively, with very low affinities due entirely to slow association rates . FixL P50's for oxygen are 17-76 mmHg . FixL's may sense nitric oxide and carbon monoxide in addition to oxygen, especially at the low oxygen pressures encountered in vivo . Autoxidation rates are about 50 times faster than that of sperm whale myoglobin . The carbon monoxide affinity of FixL's is about 300 times lower than that of myoglobin, resulting in the unusually low values of 7.5-17 for the partition constant, M = P50(O2)/P50(CO), between carbon monoxide and oxygen . Met-FixL's have their Soret absorption maximum at 395 nm instead of the typical 408 nm and a steep hydroxymet transition at pH > or = 9.3; these properties indicate a pentacoordinated high-spin ferric heme and suggest a sterically hindered hydrophobic heme pocket lacking a distal (E7) histidine . FixL is the first member of a new class of heme proteins, the heme-based sensors, distinct from the oxygen carriers and electron transporters . We expect that some of the novel properties of FixL will be characteristic of the class.

Mol Plant Microbe Interact, 1994 Jul-Aug, 7(4), 498 - 507
Rhizobium inoculation and physical wounding result in the rapid induction of the same chalcone synthase copy in Trifolium subterraneum; Lawson CG et al.; The gene or genes encoding chalcone synthase (CHS) in the legume Trifolium subterraneum (subterranean clover) were induced within 6 hr after inoculation with Rhizobium leguminosarum bv . trifolii strain ANU843 . No induction was found in uninoculated controls or plants inoculated with either the nodulation-deficient R . l . bv . trifolii strain ANU845 (pSym-) or R . meliloti strain 1021, which is capable of nodulating alfalfa but not clover . Morphological examination of the interaction between the legume and bacteria in this system showed that root hair distortion (a marker of the early events in the interaction) was apparent within 10 hr after inoculation . This indicated that CHS induction could occur before any detectable sign of rhizobial penetration of root hairs . The addition of a crude preparation of R . l . bv . trifolii lipooligosaccharide signals (Nod metabolites) to the plant growth medium had no effect on the expression of CHS over 36 hr, although root hair distortion was apparent over this time . These treatments were then contrasted with physical wounding . Wounding the plants led to a rapid induction of CHS, occurring within 2 hr . Sequence analysis of cloned CHS cDNA from pools sampled after Rhizobium inoculation or wounding treatments showed the gene designated CHS5 was the major CHS species in both treatments . Conserved sequences were found in promoters of CHS5 and soybean Gmchs7, a gene which has overlapping expression patterns . These findings support the view that the induction of the phenylpropanoid pathway is involved in the very early events of the Rhizobium infection of legumes.

J Bacteriol, 1994 Jul, 176(14), 4338 - 47
Flavone-enhanced accumulation and symbiosis-related biological activity of a diglycosyl diacylglycerol membrane glycolipid from Rhizobium leguminosarum biovar trifolii; Orgambide GG et al.; Rhizobium leguminosarum bv . trifolii is the bacterial symbiont which induces nitrogen-fixing root nodules on the leguminous host, white clover (Trifolium repens L.) . In this plant-microbe interaction, the host plant excretes a flavone, 4',7-dihydroxyflavone (DHF), which activates expression of modulation genes, enabling the bacterial symbiont to elicit various symbiosis-related morphological changes in its roots . We have investigated the accumulation of a diglycosyl diacylglycerol (BF-7) in wild-type R . leguminosarum bv . trifolii ANU843 when grown with DHF and the biological activities of this glycolipid bacterial factor on host and nonhost legumes . In vivo labeling studies indicated that wild-type ANU843 cells accumulate BF-7 in response to DHF, and this flavone-enhanced alteration in membrane glycolipid composition was suppressed in isogenic nodA::Tn5 and nodD::Tn5 mutant derivatives . Seedling bioassays performed under microbiologically controlled conditions indicated that subnanomolar concentrations of purified BF-7 elicit various symbiosis-related morphological responses on white clover roots, including thick short roots, root hair deformation, and foci of cortical cell divisions . Roots of the nonhost legumes alfalfa and vetch were much less responsive to BF-7 at these low concentrations . A structurally distinct diglycosyl diacylglycerol did not induce these responses on white clover, indicating structural constraints in the biological activity of BF-7 on this legume host . In bioassays using aminoethoxyvinylglycine to suppress plant production of ethylene, BF-7 elicited a meristematic rather than collaroid type of mitogenic response in the root cortex of white clover . These results indicate an involvement of flavone-activated nod expression in membrane accumulation of BF-7 and a potent ability of this diglycosyl diacylglycerol glycolipid to perform as a bacterial factor enabling R . leguminosarum bv . trifolii to activate segments of its host's symbiotic program during early development of the root nodule symbiosis.

J Bacteriol, 1994 Jul, 176(13), 4117 - 23
Identification of a gene encoding a thioredoxin-like product necessary for cytochrome c biosynthesis and symbiotic nitrogen fixation in Rhizobium leguminosarum; Vargas C et al.; A Tn5-induced mutant of Rhizobium leguminosarum bv . viciae could not form nitrogen-fixing nodules on pea or vetch because of a lesion in electron transport to oxygen . The mutant lacked spectroscopically detectable cytochromes c and aa3 . No proteins containing c-type cytochrome could be identified in the mutant by heme staining of proteins fractionated on polyacrylamide gels, indicating that the mutant was defective in maturation of all c-type cytochromes . The Tn5 mutation was determined to be located in a gene that was called cycY . The cycY gene product is homologous to the thioredoxin-like protein HelX involved in the assembly of c-type cytochromes in Rhodobacter capsulatus and to an open reading frame from a Bradyrhizobium japonicum gene cluster containing other genes involved in cytochrome c biogenesis . Our observations are consistent with CycY functioning as a thioredoxin that reduces cysteine residues in apocytochromes c before heme attachment.

Plasmid, 1994 Jul, 32(1), 46 - 54
IS1032 from Acetobacter xylinum, a new mobile insertion sequence; Iversen T et al.; IS1031 elements constitute a family of related insertion sequences (IS) in Acetobacter xylinum strains . A new IS1031-related element, IS1032, was isolated from A . xylinum ATCC 23770 . Southern hybridization analysis showed that one or more sequences similar to IS1032 are present in most of the A . xylinum strains examined . In addition, one copy was detected in Acetobacter aceti ATCC 15973 . The transposition of IS1032 was evident from the appearance of an extra insertion in a spontaneous exopolysaccharide-negative mutant of A . xylinum ATCC 23770 . IS1032 consists of 916 bp and has imperfect terminal inverted repeats of 14 bp (IR-Left) and 16 bp (IR-Right) . A 3-bp target sequence is duplicated upon insertion . IS1032 displays a single open reading frame, encoding a putative 276-amino-acid protein sharing 58% identity with the corresponding protein encoded by IS1031 . Thus, IS1032 is a member of the IS1031 family in A . xylinum . A striking degree of nucleotide sequence similarity between IS1032 and ISRm4 from Rhizobium meliloti was found . Furthermore, the IS1031-family transposases also display stretches of amino acid sequence similarities with putative transposases encoded by IS elements from other species.

Mol Microbiol, 1994 Jul, 13(1), 51 - 66
Rhizobium meliloti DctD, a sigma 54-dependent transcriptional activator, may be negatively controlled by a subdomain in the C-terminal end of its two-component receiver module; Gu B et al.; Rhizobium meliloti DctD is believed to have three functional domains: an N-terminal, two-component receiver domain; and like other sigma 54-dependent activators, C-terminal and central domains for DNA binding and transcription activation . We have characterized a progressive series of N-terminal deletions of R . meliloti DctD . The N-terminal domain was not needed for binding the dctA upstream activation sequence . Only 25% of the C-terminal end of the receive domain was needed to significantly inhibit the central domain, and proteins lacking up to 60% of the N-terminal end of the receiver domain were 'inducible' in R . meliloti cells . We hypothesize that the N-terminal two-thirds of the DctD receiver domain augments and controls an adjacent subdomain for inhibiting the central domain.

Mol Microbiol, 1994 Jul, 13(1), 171 - 8
Nod factors of Rhizobium are a key to the legume door; Relic B et al.; Symbiotic interactions between rhizobia and legumes are largely controlled by reciprocal signal exchange . Legume roots excrete flavonoids which induce rhizobial nodulation genes to synthesize and excrete lipo-oligosaccharide Nod factors . In turn, Nod factors provoke deformation of the root hairs and nodule primordium formation . Normally, rhizobia enter roots through infection threads in markedly curled root hairs . If Nod factors are responsible for symbiosis-specific root hair deformation, they could also be the signal for entry of rhizobia into legume roots . We tested this hypothesis by adding, at inoculation, NodNGR-factors to signal-production-deficient mutants of the broad-host-range Rhizobium sp . NGR234 and Bradyrhizobium japonicum strain USDA110 . Between 10(-7) M and 10(-6) M NodNGR factors permitted these NodABC- mutants to penetrate, nodulate and fix nitrogen on Vigna unguiculata and Glycine max, respectively . NodNGR factors also allowed Rhizobium fredii strain USDA257 to enter and fix nitrogen on Calopogonium caeruleum, a nonhost . Detailed cytological investigations of V . unguiculata showed that the NodABC- mutant NGR delta nodABC, in the presence of NodNGR factors, entered roots in the same way as the wild-type bacterium . Since infection threads were also present in the resulting nodules, we conclude that Nod factors are the signals that permit rhizobia to penetrate legume roots via infection threads.

Appl Biochem Biotechnol, 1994 Jul, 48(1), 37 - 43
Biological control of fungal pathogens; Chet I et al.; Biological control of soil-borne plant pathogens is a potential alternative to the use of chemical pesticides, which have already been proved to be harmful to the environment . Several strains of the fungus Trichoderma have been isolated and found to be effective biocontrol agents of various soil-borne plant pathogenic fungi under greenhouse and field conditions . Different application approaches have been used including integration of Trichoderma with reduced doses of chemical agents . Biochemical and molecular biology studies carried out to explore the mechanisms involved in biological control revealed that Trichoderma is a rather specific mycoparasite . Lectins were found to be involved in the recognition between Trichoderma and its host fungi, whereas chitinase is involved in the degradation of the host cell wall . Genetic engineering techniques were employed in order to increase the effectiveness, stability, and biocontrol capacity of Trichoderma spp . as well as other biocontrol agents, such as Pseudomonass spp . and Rhizobium.

Microb Releases, 1994 Jul, 2(4), 231 - 7
Isolation of a Rhizobium galegae strain-specific DNA probe; Tas E et al.; We have isolated a strain-specific DNA probe from the strain Rhizobium galegae HAMBI 1174 by a subtraction hybridization procedure followed by PCR amplification and DNA cloning . The specificity of the 342-bp DNA probe (P3) was tested in dot blot or Southern blot hybridizations against total genomic DNA of 41 bacterial strains (21 of them belong to R . galegae, 15 to other Rhizobium species and five to other bacterial species) . Only the samples from four R . galegae strains, which are different isolates but identical to the strain HAMBI 1174, hybridized with the probe . The P3 probe was sequenced and PCR primers were designed based on its sequence . PCR amplification from purified total genomic DNA of 52 strains and subsequent hybridization with the P3 probe proved that the primers are strain specific.

Int J Syst Bacteriol, 1994 Jul, 44(3), 392 - 403
Photosynthetic symbionts of Aeschynomene spp . form a cluster with bradyrhizobia on the basis of fatty acid and rRNA analyses; So RB et al.; The relationship between photosynthetic rhizobia that nodulate 10 Aeschynomene species (Aeschynomene afraspera, Aeschynomene denticulata, Aeschynomene evenia, Aeschynomene indica, Aeschynomene nilotica, Aeschynomene pratensis, Aeschynomene rudis, Aeschynomene scabra, Aeschynomene schimperi, and Aeschynomene sensitiva) and reference strains of the genera Bradyrhizobium, Rhizobium, and Azorhizobium was investigated by analyzing cellular fatty acid methyl esters (FAME) and 16S rRNA sequences . The members of each genus produced very distinct FAME patterns, and the photosynthetic rhizobia formed a subcluster in the Bradyrhizobium cluster . The absence of the cyc C19:0 type of fatty acid in all of the photosynthetic rhizobium strains isolated from 10 Aeschynomene species distinguished these microorganisms from other known rhizobia, including strain BTAi 1, a photosynthetic symbiont of A . indica . We sequenced a 264-base segment of the 16S rRNA genes of selected strains after amplification by the PCR and compared the results with previously published sequences for species of rhizobia and related photosynthetic bacteria . Photosynthetic strains IRBG 2 (from A . afraspera), IRBG 230 (from A . nilotica), and ORS 322 (from A . afraspera) had identical sequences but were distinct from strain BTAi (from A . indica) and from strain IRBG 231 (from A . denticulata), which is similar to the type strain (DNA homology group Ia) of Bradyrhizobium japonicum . Nonphotosynthetic strain IRBG 274 (from A . afraspera) was closely related to Bradyrhizobium elkanii (DNA homology group II) . All of the photosynthetic rhizobia clearly fell into the Bradyrhizobium cluster . Although the results of the FAME and 16S rRNA analyses were in excellent agreement, our placement of the photosynthetic rhizobia is in apparent conflict with phenotypic data, as determined by numerical taxonomy (Ladha and So, Int . J . Syst . Bacteriol., in press) which placed the photosynthetic rhizobia in a coherent cluster that is as far from the genus Bradyrhizobium as the genera Rhizobium and Azorhizobium are . While the FAME and 16S rRNA data probably provide a more reliable indication of phylogeny, the degree of phenotypic divergence observed raises questions concerning the polyphasic approach to bacterial systematics.

Mutat Res, 1994 Jul 1, 308(1), 99 - 107
Autoregulation and kinetics of induction of the Rhizobium phaseoli recA gene; Fernandez de Henestrosa AR et al.; A fusion between the recA gene of Rhizobium phaseoli and the lacZ gene was constructed in vitro and cloned in a mini-Tn5 transposon derivative to obtain chromosomal insertions which make it possible to quantitatively examine their transcriptional regulation in both R . phaseoli and E . coli . Likewise, and by insertion of a spectinomycin-resistance gene cassette into the recA gene of R . phaseoli and subsequent marker exchange, a RecA- derivative of this bacterial species has been obtained . Analysis of this recA-lacZ fusion showed that it was inducible by DNA damage in the RecA+ strain of R . phaseoli but not in the RecA- mutant . On the other hand, the recA-lacZ fusion of R . phaseoli was not induced in DNA-damaged RecA+ cells of E . coli . Furthermore, the range of UV doses which give rise to dose dependence in the induction of its respective recA genes is different in R . phaseoli from that in E . coli.

Gene, 1994 Jun 24, 144(1), 87 - 91
Identification of a Rhizobium leguminosarum gene homologous to nodT but located outside the symbiotic plasmid; Rivilla R et al.; An open reading frame (ORF471), homologous to nodT from Rhizobium leguminosarum, has been found outside the symbiotic plasmid . The deduced amino-acid sequence indicates that the gene product is an outer membrane lipoprotein similar to the NodT proteins . This ORF seems to be widespread among R . leguminosarum bv . viciae strains and its presence in the genetic background of different strains may explain the lack of a nodulation-deficient phenotype found in such strains carrying a nod mutation.

Gene, 1994 Jun 24, 144(1), 17 - 24
Construction of multipurpose gene cartridges based on a novel synthetic promoter for high-level gene expression in gram-negative bacteria; Giacomini A et al.; A series of gene cartridges containing a novel synthetic promoter (Psyn) was constructed . The Psyn sequence is based on the consensus of a number of naturally occurring promoters and displays strong activity in Escherichia coli and Rhizobium leguminosarum . In a direct comparison, Psyn proved to be about twice as strong as the tac promoter in E . coli, while the difference in Rhizobium was about tenfold . A small Psyn cartridge was constructed by adding a Shine-Dalgarno sequence, an ATG codon, and a removable lac operator, whose excision can convert the regulated cartridge into a constitutively expressed unit . A second cassette was obtained by the addition of a lacIq gene in order to provide autonomous regulation also in hosts lacking lacI functions, such as R . leguminosarum . A promoterless lacZ gene was inserted to monitor the activity . This gene can be either replaced with genes of interest, or used for gene fusions by means of conveniently positioned restriction sites . A third cassette was generated by adding a mercury-resistance determinant as a selectable marker, suitable for monitoring tagged bacteria released into environments . In such cases, where a non-antibiotic-resistant marker is preferable, the use of mercury chloride adds the advantage of inhibiting fungal growth when plating soil suspensions . The presence of the second marker, lacZ driven by the strong Psyn, facilitates the selection . Furthermore, the Psyn fragment can be used as a specific probe for the detection of released bacteria engineered with any of the above constructs.

J Bacteriol, 1994 Jun, 176(12), 3723 - 9
Characterization of the gene encoding the autotrophic ATP sulfurylase from the bacterial endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila; Laue BE et al.; ATP sulfurylase is a key enzyme in the energy-generating sulfur oxidation pathways of many chemoautotrophic bacteria . The utilization of reduced sulfur compounds to fuel CO2 fixation by the still-uncultured bacterial endosymbionts provides the basis of nutrition in invertebrates, such as the tubeworm Riftia pachyptila, found at deep-sea hydrothermal vents . The symbiont-containing trophosome tissue contains high levels of ATP sulfurylase activity, facilitating the recent purification of the enzyme . The gene encoding the ATP sulfurylase from the Riftia symbiont (sopT) has now been cloned and sequenced by using the partial amino acid sequence of the purified protein . Characterization of the sopT gene has unequivocally shown its bacterial origin . This is the first ATP sulfurylase gene to be cloned and sequenced from a sulfur-oxidizing bacterium . The deduced amino acid sequence was compared to those of ATP sulfurylases reported from organisms which assimilate sulfate, resulting in the discovery that there is substantial homology with the Saccharomyces cerevisiae MET3 gene product but none with the products of the cysDN genes from Escherichia coli nor with the nodP and nodQ genes from Rhizobium meliloti . This and emerging evidence from other sources suggests that E . coli may be atypical, even among prokaryotic sulfate assimilators, in the enzyme it employs for adenosine 5'-phosphosulfate formation . The sopT gene probe also was shown to specifically identify chemoautotrophic bacteria which utilize ATP sulfurylase to oxidize sulfur compounds.

J Bacteriol, 1994 Jun, 176(11), 3286 - 94
Infection of soybean and pea nodules by Rhizobium spp . purine auxotrophs in the presence of 5-aminoimidazole-4-carboxamide riboside; Newman JD et al.; Purine auxotrophs of various Rhizobium species are symbiotically defective, usually unable to initiate or complete the infection process . Earlier studies demonstrated that, in the Rhizobium etli-bean symbiosis, infection by purine auxotrophs is partially restored by supplementation of the plant medium with 5-amino-imidazole-4-carboxamide (AICA) riboside, the unphosphorylated form of the purine biosynthetic intermediate AICAR . The addition of purine to the root environment does not have this effect . In this study, purine auxotrophs of Rhizobium fredii HH303 and Rhizobium leguminosarum 128C56 (bv . viciae) were examined . Nutritional and genetic characterization indicated that each mutant was blocked in purine biosynthesis prior to the production of AICAR . R . fredii HH303 and R . leguminosarum 128C56 appeared to be deficient in AICA riboside transport and/or conversion into AICAR, and the auxotrophs derived from them grew very poorly with AICA riboside as a purine source . All of the auxotrophs elicited poorly developed, uninfected nodules on their appropriate hosts . On peas, addition of AICA riboside or purine to the root environment led to enhanced nodulation; however, infection threads were observed only in the presence of AICA riboside . On soybeans, only AICA riboside was effective in enhancing nodulation and promoting infection . Although AICA riboside supplementation of the auxotrophs led to infection thread development on both hosts, the numbers of bacteria recovered from the nodules were still 2 or more orders of magnitude lower than in fully developed nodules populated by wild-type bacteria . The ability to AICA riboside to promote infection by purine auxotrophs, despite serving as a very poor purine source for these strains, supports the hypothesis that AICAR plays a role in infection other than merely promoting bacterial growth.

Plant Physiol, 1994 Jun, 105(2), 585 - 92
Alfalfa Enod12 genes are differentially regulated during nodule development by Nod factors and Rhizobium invasion; Bauer P et al.; MsEnod12A and MsEnod12B are two early nodulin genes from alfalfa (Medicago sativa) . Differential expression of these genes was demonstrated using a reverse transcription-polymerase chain reaction approach . MsEnod12A RNA was detected only in nodules and not in other plant tissues . In contrast, MsEnod12B transcripts were found in nodules and also at low levels in roots, flowers, stems, and leaves . MsEnod12B expression was enhanced in the root early after inoculation with the microsymbiont Rhizobium meliloti and after treatment with purified Nod factors, whereas MsEnod12A induction was detected only when developing nodules were visible . In situ hybridization showed that in nodules, MsEnod12 expression occurred in the infection zone . In empty Fix- nodules the MsEnod12A transcript level was much reduced, and in spontaneous nodules it was not detectable . These data indicate that MsEnod12B expression in roots is related to the action of Nod factors, whereas MsEnod12A expression is associated with the invasion process in nodules . Therefore, alfalfa possesses different mechanisms regulating MsEnod12A and MsEnod12B expression.

Eur J Cell Biol, 1994 Jun, 64(1), 88 - 94
Cell wall degradation during infection thread formation by the root nodule bacterium Rhizobium leguminosarum is a two-step process; van Spronsen PC et al.; In the nitrogen-fixing root-nodule symbiosis of Rhizobium leguminosarum biovar viciae and its host plants pea and vetch, the bacteria enter one root cortical cell after another via a tip-growing structure, the infection thread . Rhizobial Nod (nodulation) factors induce the formation of preinfection thread structures (Van Brussel, A.A.N., R . Bakhuizen, P.C . van Spronsen, H.P . Spaink, T . Tak, B.J.J . Lugtenberg, J.W . Kijne, Science 257, 70-72 (1992)), but formation of infection threads requires the presence of bacterial cells . Passing of an infection thread from cell to cell requires local cell wall degradation . We compared at the ultrastructural level local cell wall changes in the outer root cortex of pea and vetch related to preinfection thread formation and infection thread formation, respectively . Cell wall modifications in the outer periclinal walls of root cortical cells induced by Nod factors appeared to be similar to those induced by rhizobia . These modifications take place opposite cytoplasmic bridges and are probably related to induction of tip growth . However, complete cell wall degradation was never observed in the absence of rhizobia . We propose a two-step cell wall degradation process for infection thread formation . The first step is a local cell wall modification by plant enzymes, induced by rhizobial Nod factors . The second step is complete cell wall degradation in the presence of rhizobia.

Res Microbiol, 1994 Jun-Aug, 145(5-6), 454 - 9
Oxygen regulation of expression of nitrogen fixation genes in Rhizobium meliloti; Agron PG et al.; The sensor kinase FixL and the response regulator FixJ induce the expression of the nitrogen fixation genes of Rhizobium meliloti in response to microaerobiosis, which is a characteristic feature of the plant root nodule interior where the bacteria fix nitrogen . The kinase activity of a purified, soluble derivative of the membrane-bound hemoprotein FixL, designated FixL*, is stimulated under low oxygen conditions, thus increasing FixJ-phosphate levels . FixJ-phosphate is a potent transcriptional activator of the nifA and fixK genes, the products of which, in turn, induce the expression of most if not all of the remaining nitrogen fixation genes . FixL* and FixL*-phosphate also dephosphorylate FixJ-phosphate, and this activity is depressed by low oxygen concentrations . In the current model, gene expression is reciprocally coordinated by the kinase and phosphatase activities of FixL according to changes in oxygen tension.

J Biol Chem, 1994 May 20, 269(20), 14402 - 10
Structure of lipid A component of Rhizobium leguminosarum bv . phaseoli lipopolysaccharide . Unique nonphosphorylated lipid A containing 2-amino-2-deoxygluconate, galacturonate, and glucosamine; Bhat UR et al.; The structure of lipid A from the lipopolysaccharide of Rhizobium leguminosarum bv . phaseoli (wild type strain CE3) was investigated by alkylation analysis, nuclear magnetic resonance spectroscopy, and electrospray and fast atom bombardment mass spectrometry of the de-O-acylated lipid A . The lipid A carbohydrate backbone was shown to be a trisaccharide containing galacturonic acid, glucosamine, and the unique sugar 2-amino-2-deoxygluconic acid, previously unreported in lipopolysaccharides . Nuclear magnetic resonance spectroscopy and ethylation analyses revealed that the galacturonic acid is alpha-1,4-linked to the glucosamine, while the amino aldonic acid residue, which may exist as the 1,5-lactone, is attached as an aglycone to the glucosamine and, thus, occupies the reducing end of the molecule . The resulting backbone is hydrophilic and analogous to the commonly observed bisphosphorylated glucosamine disaccharide from enteric bacterial lipopolysaccharides in that both the nonreducing and reducing ends carry negatively charged substituents . The fatty acids of the R . leguminosarum lipid A are attached both as O- and N-acyl substituents to glucosamine and 2-aminogluconate . All fatty acids are hydroxylated consisting of 3-hydroxymyristate (3-OH-C14.0), 3-hydroxypentadecanoate (3-OH-C15.0), 3-hydroxypalmitate (3-OH-C16.0), 3-hydroxystearate (3-OH-C18.0), and 27-hydroxyoctacosanoate (27-OH-C28.0) in the approximate mole ratio 3:0.2:1:0.6:1 . Unlike lipid As from enteric bacteria, the R . leguminosarum lipid A lacks 3-acyloxyacyl substituents; however, the long chain 27-hydroxy fatty acid carries ester-linked beta-hydroxybutyrate at the 27-hydroxy position . Fast atom bombardment mass spectrometry of the de-O-acylated lipid A demonstrated the presence of 2 molecular species that differ by 28 mass units due to fatty acid heterogeneity at the two amide linkages . One species carries amide-linked 3-OH-C14.0 and 3-OH-C16.0; the second species carries 3-OH-C14.0 and 3-OH-C18.0 . Each molecular species also exists as the aldonolactone, yielding molecular ions at ((M+H)+)-18 . The heterogeneity in the amide-linked fatty acids further distinguishes the Rhizobium lipid A from enteric lipid As.

Nucleic Acids Res, 1994 May 11, 22(9), 1555 - 61
Intramolecular signal transduction within the FixJ transcriptional activator: in vitro evidence for the inhibitory effect of the phosphorylatable regulatory domain; Da Re S et al.; FixJ is a phosphorylatable 'response regulator' controlling the transcription of the key nitrogen fixation genes nifA and fixK in Rhizobium meliloti . Sequence and genetic analyses indicated that FixJ comprises an N-terminal phosphorylatable regulatory domain, FixJN, and a C-terminal transcriptional activator domain, FixJC . We have now overexpressed and purified the FixJC protein and show that it is fully active in an in vitro transcription system with purified RNA polymerase . FixJC appeared to act synergistically with RNA polymerase at the nifA promoter . Furthermore FixJC was more active in vitro than the full-length dephosphorylated FixJ protein . Therefore activity of FixJC is inhibited by FixJN within the FixJ protein . This inhibition is relieved by phosphorylation of FixJN . Such a negative mode of intramolecular signal transduction may be generalizable to other response regulators.

EMBO J, 1994 May 1, 13(9), 2139 - 49
The Rhizobium meliloti regulatory nodD3 and syrM genes control the synthesis of a particular class of nodulation factors N-acylated by (omega-1)-hydroxylated fatty acids; Demont N et al.; Rhizobia elicit the formation of nitrogen-fixing nodules on specific legume hosts . Rhizobium meliloti nodulation (nod) genes control a signal exchange between the two symbiotic partners during infection and the early steps of nodulation . The regulatory nodD1, nodD2 and nodD3 genes are involved in the specific perception of different plant and environmental signals and activate the transcription of the nod operons . Once activated, the structural nod genes specify the synthesis of diffusible lipo-oligosaccharides, the Nod factors, which signal back to the plant . R . meliloti Nod factors are sulfated chito-oligosaccharides which are mono-N-acylated by unsaturated C16 fatty acids or by a series of C18 to C26 (omega-1)-hydroxylated fatty acids . In this paper we show that the regulatory nodD3 gene and another symbiotic regulatory gene, syrM, which mediate bacterial responses to plant signals that differ from those involving nodD1 and nodD2, determine the synthesis of Nod factors with different acyl moieties . nodD3 and syrM are required for the synthesis of Nod factors N-acylated by the (omega-1)-hydroxylated fatty acids . This regulatory mechanism makes possible the qualitative adaptation of bacterial Nod signal production to plant signals in the course of the symbiotic process.

J Bacteriol, 1994 May, 176(9), 2560 - 8
Functional analysis of the fixNOQP region of Azorhizobium caulinodans; Mandon K et al.; The deduced amino acid sequences of four open reading frames identified upstream of the fixGHI region in Azorhizobium caulinodans are very similar to the putative terminal oxidase complex coded by the fixNOQP operons from Rhizobium meliloti and Bradyrhizobium japonicum . The expression of the A . caulinodans fixNOQP genes, which was maximal under microaerobiosis, was positively regulated by FixK and independent of NifA . In contrast to the Fix- phenotype of B . japonicum and R . meliloti fixN mutants, an A . caulinodans fixNO-deleted mutant strain retained 50% of the nitrogenase activity of the wild type in the symbiotic state . In addition, the nitrogenase activity was scarcely reduced under free-living conditions . Analysis of membrane fractions of A . caulinodans wild-type and mutant strains suggests that the fixNOQP region encodes two proteins with covalently bound hemes, tentatively assigned to fixO and fixP . Spectral analysis showed a large decrease in the c-type cytochrome content of the fixN mutant compared with the wild type . These results provide evidence for the involvement of FixNOQP proteins in a respiratory process . The partial impairment in nitrogen fixation of the fixN mutant in planta may be due to the activity of an alternative terminal oxidase compensating for the loss of the oxidase complex encoded by fixNOQP.

Microbiology, 1994 May, 140 ( Pt 5), 1231 - 5
Effects of organic acids and low pH on Rhizobium meliloti 104A14; Perez-Galdona R et al.; In the symbiotic relationship between Rhizobium meliloti and alfalfa (Medicago sativa), the bacteria are enclosed within the plant cell by a membrane that may function like a plant vacuolar membrane and maintain a pH between 5.5 and 6.0 . Free-living Rhizobium meliloti 104A14 is sensitive to pH in this range and its sensitivity was influenced by the presence of acetate and other monocarboxylic acids . R . meliloti can grow at pH 6.0 in 3 mM succinate but does not grow at pH 6.2 if 10 mM acetate is added . The combination of low pH and acetate is bacteriostatic . Measurement of internal pH (pHi) using 14C-labelled benzoate as a permeant acid showed that growth inhibition occurs when pHi falls below 7.15.

J Hered, 1994 May-Jun, 85(3), 179 - 82
Chromosomal location of lectin genes indicates they are not the basis of Rhizobium strain specificity mutations identified in pea (Pisum sativum L.); Lu J et al.; A lectin gene family is located on linkage group 7 in pea . The lectin genes are arranged as a cluster, with no recombination observed within the multigene family . A lectin-like cDNA clone, pEA207, and eight DNA fragments generated by random priming also were mapped in the region of the lectin genes . None of the known pea mutants altering Rhizobium leguminosarum strain specificity map to this region of the genome, and therefore their altered specificities appear not to be directly produced by mutations in the lectin genes.

Mol Plant Microbe Interact, 1994 May-Jun, 7(3), 411 - 8
Accumulation of transcripts encoding a lipid transfer-like protein during deformation of nodulation-competent Vigna unguiculata root hairs; Krause A et al.; A cDNA library was constructed from RNA of Vigna unguiculata root hairs harvested 1 day and 4 days after inoculation with Rhizobium sp . NGR234 . A heterologous probe was used to identify a cDNA clone, the predicted 99-amino-acid sequence of which shares homology with a nonspecific lipid transfer protein (LTP) of Hordeum vulgare . Other characteristics, including an estimated molecular weight of 10.4 kD, an isoelectric point of 8.6, and a signal peptide with a hydrophobic region at the amino-terminal end, are shared by most LTPs . A transcript of 630 nt was found in all tissues tested, except nodules . Levels of mRNA increased in root hairs 24 hr after treatment with Rhizobium sp . NGR234, with different hormones, or with Nod factors . Amounts of transcripts were dependent on the concentration of Nod factors . Accumulation of transcripts during nodule development correlated with root hair deformation, the first visible step in the Rhizobium-legume symbiosis.

Can J Microbiol, 1994 May, 40(5), 345 - 54
Genotypic and phenotypic diversity of Rhizobium isolated from chickpea (Cicer arietinum L.); Nour SM et al.; The diversity of 16 strains of chickpea-infective rhizobia from various geographical origins was analysed using genotypic and phenotypic approaches . Multilocus enzyme electrophoresis was performed, and restriction fragment length polymorphisms of the amplified 16S + IGS (intergenic spacer) rRNA gene, assimilation of 147 carbon sources, antibiotic resistance, and tolerance to NaCl and extreme pH values and temperatures were tested . These approaches had different discriminating powers . Esterase polymorphisms gave a unique pattern for each strain, allowing this method to be used for strain fingerprinting . Genetic distances between strains were estimated . The three approaches used in this study yielded consistent results . They evidenced high heterogeneity among the strains, and made it possible to classify the strains into two clusters . Isozyme patterns for superoxide dismutase were particularly interesting, since they delineated the same two groups . The phenotypic tests clearly confirmed the existence of two genetic groups on the basis of 11 phenotypic characters . Owing to the large phylogenetic distance between the two groups of strains, the taxonomic status of chickpea-infective strains is discussed.

Microbiology, 1994 May, 140 ( Pt 5), 1223 - 9
The psi operon of Rhizobium leguminosarum biovar phaseoli: identification of two genes whose products are located at the bacterial cell surface; Mimmack ML et al.; We have delineated three short open reading frames, psiA, ORF-P and psiB within the psi operon of Rhizobium leguminosarum biovar phaseoli . psiA, in a multi-copy plasmid, causes inhibition of exopolysaccharide synthesis in R . leguminosarum . In addition, the suppression of exopolysaccharide synthesis due to the multi-copy psiA caused R . leguminosarum strains to stain with the dye calcofluor, a response that does not occur with wild-type strains of this species . Insertions of a defective phoA gene (lacking its promoter, ribosomal binding site and leader sequence) into psiA and psiB were isolated and the precise locations of the insertions were established . PsiA-PhoA and PsiB-PhoA protein fusions were found to express alkaline phosphatase activity indicating that PsiA and PsiB span the inner membrane or are translocated across it.

Nucleic Acids Res, 1994 Apr 25, 22(8), 1335 - 41
Subtraction hybridisation and shot-gun sequencing: a new approach to identify symbiotic loci; Perret X et al.; Traditionally, new loci involved in the Rhizobium-legume symbiosis have been identified by transposon mutagenesis and/or complementation . Wide dispersal of the symbiotic loci in Rhizobium species NGR234, as well as the large number of potential host-plants to be screened, greatly reduces the efficiency of these techniques . As an alternate strategy designed to identify new NGR234 genes involved in the early stages of the symbiosis, we combined data from competitive RNA hybridisation, subtractive DNA hybridisation and shot-gun sequencing . On the assumption that the expression of most nodulation genes is triggered by compounds released by the host-plant, we identified, in the ordered cosmid library of the large symbiotic plasmid pNGR234a, restriction fragments that carry transcripts induced by flavonoids . To target genes not present in the closely related strain R . fredii USDA257, we selected fragments that also carried sequences purified by subtractive DNA hybridisation . Shot-gun sequencing of this subset of fragments lead to the identification of sequences with strong homology to diverse prokaryotic genes/proteins . Amongst these, a symbiotically active ORF from pNGR234a, is highly homologous to the leucine responsive regulatory protein of Escherichia coli (Lrp), is induced by flavonoids, and is not present in USDA257.

J Biol Chem, 1994 Apr 15, 269(15), 11090 - 7
Phospholipids of Rhizobium contain nodE-determined highly unsaturated fatty acid moieties; Geiger O et al.; In Rhizobium leguminosarum the nodABC and nod-FEL operons are involved in the production of lipooligosaccharide signals, which mediate host specificity . A nodE-determined highly unsaturated C18:4 fatty acid (trans-2,trans-4,trans-6,cis-11-octadecatetraenoic acid) is essential for the ability of the signals to induce nodule primordia (Spaink, H . P., Sheeley, D . M., van Brussel, A . A . N., Glushka, J., York, W.S., Tak, T., Geiger, O., Kennedy, E . P., Reinhold, V . N., and Lugtenberg, B . J . J . (1991) Nature 354, 125-130) and preinfection thread structures (van Brussel, A . A . N., Bakhuizen, R., van Spronsen, P . C., Spaink, H . P., Tak, T., Lugtenberg, B . J . J., and Kijne, J . W . (1992) Science 257, 70-72) on the host plant Vicia sativa . We presently focus on the question of how these lipo-oligosaccharide signals are synthesized in Rhizobium . Here we show that after the induction of the nodFE genes, even in the absence of the nodABC genes, the trans-2,trans-4,trans-6,cis-11-octadecatetraenoic acid, which has a characteristic absorbance maximum of 303 nm, is synthesized; this shows that the biosynthesis of the unusual fatty acid is not dependent on the synthesis of the lipooligosaccharides . This finding also suggests that the biosynthesis of the unusual fatty acid is completed before it is linked to the sugar backbone of the lipooligosaccharide . In an attempt to identify the lipid fraction with which the unusual C18:4 fatty acid is associated, we found that it is linked to the sn-2 position of the phospholipids . Even when lipooligosaccharide signals are produced in a wild type Rhizobium cell, a fraction of the unusual fatty acid is still bound to all major phospholipids . These findings offer interesting possibilities . 1) The phospholipids might be biosynthetic intermediates for the synthesis of lipooligosaccharide signals, and 2) phospholipids, containing nodFE-derived fatty acids, might have a signal function of their own.

Proc Natl Acad Sci U S A, 1994 Apr 12, 91(8), 3122 - 6
Biosynthesis of lipooligosaccharide nodulation factors: Rhizobium NodA protein is involved in N-acylation of the chitooligosaccharide backbone; Rohrig H et al.; Rhizobium meliloti interacts symbiotically with alfalfa by forming root nodules in which the bacteria fix nitrogen . The Rhizobium nodulation genes nodABC are involved in the synthesis of lipooligosaccharide symbiotic signal molecules, which are mono-N-acylated chitooligosaccharides . These bacterial signals elicit nodule organogenesis in roots of legumes . To elucidate the role of the NodA protein in lipooligosaccharide biosynthesis, we prepared a radiolabeled tetrasaccharide precursor carrying an amino group as a potential attachment site for N-acylation at the nonreducing glucosamine residue . Various criteria demonstrate that NodA is involved in the attachment of a fatty acyl chain to this tetrasaccharide precursor, yielding a biologically active nodulation factor.

J Bacteriol, 1994 Apr, 176(8), 2454 - 7
Analysis of the lipid moiety of lipopolysaccharide from Rhizobium tropici CIAT899: identification of 29-hydroxytriacontanoic acid; Gil-Serrano AM et al.; The lipid moieties of two lipid A's isolated from the phenolic and aqueous fractions of lipopolysaccharide from Rhizobium tropici CIAT899 have been studied . Several 3-hydroxy fatty acids and two long-chain hydroxy fatty acids, 27-hydroxyoctacosanoic acid, and 29-hydroxytriacontanoic acid were identified; the ratios of these acids are the same in both lipid A's . These results can be used for chemotaxonomic purposes.

J Bacteriol, 1994 Apr, 176(7), 2044 - 54
Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa; Ochsner UA et al.; A mutant strain (65E12) of Pseudomonas aeruginosa that is unable to produce rhamnolipid biosurfactants and lacks rhamnosyltransferase activity was genetically complemented by using a P . aeruginosa PG201 wild-type gene library . A single complementing cosmid was isolated on the basis of surface tension measurements of subcultures of the transconjugants by using a sib selection strategy . The subcloning of the complementing cosmid clone yielded a 2-kb fragment capable of restoring rhamnolipid biosynthesis, rhamnosyltransferase activity, and utilization of hexadecane as a C source in mutant 65E12 . The nucleotide sequence of the complementing 2-kb fragment was determined, and a single open reading frame (rhlR) of 723 bp specifying a putative 28-kDa protein (RhlR) was identified . Sequence homologies between the RhlR protein and some regulatory proteins such as LasR of P . aeruginosa, LuxR of Vibrio fischeri, RhiR of Rhizobium leguminosarum, and the putative activator 28-kDa UvrC of Escherichia coli suggest that the RhlR protein is a transcriptional activator . A putative target promoter which is regulated by the RhlR protein has been identified 2.5 kb upstream of the rhlR gene . Multiple plasmid-based rhlR gene copies had a stimulating effect on the growth of the P . aeruginosa wild-type strain in hexadecane-containing minimal medium, on rhamnolipid production, and on the production of pyocyanin chromophores . Disruption of the P . aeruginosa wild-type rhlR locus led to rhamnolipid-deficient mutant strains, thus confirming directly that this gene is necessary for rhamnolipid biosynthesis . Additionally, such PG201::'rhlR' mutant strains lacked elastase activity, indicating that the RhlR protein is a pleiotropic regulator.

J Bacteriol, 1994 Apr, 176(7), 2033 - 43
Rhizobium meliloti contains a novel second homolog of the cell division gene ftsZ; Margolin W et al.; We have identified a second homolog of the cell division gene, ftsZ, in the endosymbiont Rhizobium meliloti . The ftsZ2 gene was cloned by screening a genomic lambda library with a probe derived from PCR amplification of a highly conserved domain . It encodes a 36-kDa protein which shares a high level of sequence similarity with the FtsZ proteins of Escherichia coli and Bacillus subtilis and FtsZ1 (Z1) of R . meliloti but lacks the carboxy-terminal region conserved in other FtsZ proteins . The identity of the ftsZ2 gene product was confirmed both by in vitro transcription-translation in an R . meliloti S-30 extract and by overproduction in R . meliloti cells . As with Z1, the overproduction of FtsZ2 in E . coli inhibited cell division and induced filamentation, although to a lesser extent than with Z1 . However, the expression of ftsZ2 in E . coli under certain conditions caused some cells to coil dramatically, a phenotype not observed during Z1 overproduction . Although several Tn3-GUS (glucuronidase) insertions in a plasmid-borne ftsZ2 gene failed to cross into the chromosome, one interruption in the chromosomal ftsZ2 gene was isolated, suggesting that ftsZ2 is nonessential for viability . The two ftsZ genes were genetically mapped to the R . meliloti main chromosome, approximately 100 kb apart.

J Bacteriol, 1994 Apr, 176(7), 1997 - 2002
Detailed structural characterization of succinoglycan, the major exopolysaccharide of Rhizobium meliloti Rm1021; Reinhold BB et al.; The detailed structure of the symbiotically important exopolysaccharide succinoglycan from Rhizobium meliloti Rm1021 was determined by mass spectrometry with electrospray ionization and collision-induced dissociation of the octameric oligosaccharide repeating unit . Previously undetermined locations of the succinyl and acetyl modifications were determined, in respect to both residue locations within the octamer and the carbon positions within the pyranose ring . Glycosidic linkages determined previously by methylation analysis were also verified.

J Bacteriol, 1994 Apr, 176(7), 1969 - 76
FixL of Rhizobium meliloti enhances the transcriptional activity of a mutant FixJD54N protein by phosphorylation of an alternate residue; Reyrat JM et al.; In Rhizobium meliloti, transcription of nitrogen fixation genes is induced in oxygen-depleted conditions under the control of the two-component regulatory system FixLJ . FixJ is a transcriptional activator whose activity is dramatically enhanced by phosphorylation, whereas FixL is a hemoprotein kinase that controls the level of phosphorylated FixJ in response to oxygen availability . We have found that a mutant FixJ protein, FixJD54N, in which the presumed site of phosphorylation (aspartate 54) was changed to an asparagine, is strongly affected for phosphorylation by FixL and is not detectably phosphorylated from the low-molecular-weight phosphate donor, acetyl-phosphate . Unexpectedly, FixL strongly enhances the transcriptional activity of the FixJD54N protein both in vivo and in vitro . We present evidence that FixJD54N transcriptional activity is enhanced by phosphorylation of an alternate residue in a reaction that requires FixL and ATP and is not affected by oxygen . We also demonstrate the key role of Asp-54 of FixJ in oxygen signal transduction.

Steroids, 1994 Apr, 59(4), 248 - 58
Sequence analysis of steroid- and prostaglandin-metabolizing enzymes: application to understanding catalysis; Baker ME; Amino acid sequence comparisons have revealed that mammalian 11 beta-hydroxysteroid and 17 beta-hydroxysteroid dehydrogenases and bacterial 3 alpha, 20 beta- and 3 beta-hydroxysteroid dehydrogenases are homologs; that is, these enzymes are descended from a common ancestor . These steroid dehydrogenases are also homologous to human 15-hydroxyprostaglandin dehydrogenase and to proteins found in Rhizobia, bacteria that form nitrogen-fixing nodules in the roots of legumes . We constructed a multiple sequence alignment of these proteins, which, when combined with the recently determined tertiary structure of Streptomyces hydrogenans 3 alpha, 20 beta-hydroxysteroid dehydrogenase and a homologous enzyme, rat dihydropteridine reductase, identifies segments and residues that are likely to be structurally important in the functioning of these enzymes especially regarding specificity for NADPH and NADH.

Glycobiology, 1994 Apr, 4(2), 127 - 34
Symbiotic host specificity between leguminous plants and rhizobia is determined by substituted and acylated glucosamine oligosaccharide signals; Lerouge P; Rhizobia are nitrogen-fixing bacteria which invade root hairs of leguminous plants and induce, in a specific manner, the formation of root nodules in which they fix nitrogen . The early steps of the symbiosis can be considered as a reciprocal molecular communication between the two partners . Initially, the plant excretes a gene inducer which stimulates the expression of bacterial nodulation genes . These nodulation genes are responsible for the synthesis of extracellular host-specific signals, called nodulation factors . The bacterial nodulation factors were isolated and structurally identified as substituted and N-acylated chitin oligosaccharides . These prokaryotic lipo-oligosaccharide signals play a key role in the symbiosis by controlling the host specificity of the bacteria . They constitute a new class of signalling molecules able to elicit nodule organogenesis in leguminous plants in the absence of bacteria.

Genetics, 1994 Apr, 136(4), 1233 - 43
Second site mutations specifically suppress the Fix- phenotype of Rhizobium meliloti ndvF mutations on alfalfa: identification of a conditional ndvF-dependent mucoid colony phenotype; Oresnik IJ et al.; Rhizobium meliloti mutants carrying ndvF insertion or deletion mutations induce nodules on alfalfa which contain very few infected cells and fail to fix N2 (Fix-) . We have characterized five independent second site mutations (designated sfx) which completely suppress the Fix- phenotype of ndvF mutants on Medicago sativa but not on another R . meliloti host Melilotus alba . Genetic mapping and phenotypic analysis revealed that the suppressor mutations sfx-1, sfx-4 and sfx-5 mapped to a single locus which was distinct from another locus defined by the sfx-2 and sfx-3 mutations . Tn5-mob-mediated conjugal mapping experiments showed that the sfx-1 locus was located clockwise from trp-33 on the R . meliloti chromosome and a detailed cotransduction map of this region was generated . To clone the sfx-1 locus, we prepared a cosmid library from total DNA obtained from an sfx-1, ndvF deletion strain . From this library, a cosmid pTH56, which converted Fix- ndvF mutants to Fix+, was isolated . Southern blot analysis provided direct physical evidence that the insert DNA in plasmid pTH56 was contiguous with the sfx-1 region . On low osmolarity glutamate-yeast extract-mannitol-salts medium (GYM) agar medium, ndvF insertion and deletion mutants were found to have a mucoid colony phenotype, as opposed to the dry colony phenotype of the wild-type strain . This phenotype was shown to be dependent on the exoB and expE genes required for synthesis of exopolysaccharide II in R . meliloti but not to be dependent on genes required exclusively for the synthesis of the succinoglycan or exopolysaccharide I . Transduction of either sfx-1 or sfx-2 or transfer of the cosmid pTH56 into the ndvF mutants restored them to a wild-type dry colony phenotype . The mucoid phenotype is not responsible for the Fix- phenotype of ndvF mutants as the Fix-, ndvF exp double mutants can be complemented to Fix+ by introducing plasmids which carry only the wild-type ndvF genes.

J Bacteriol, 1994 Apr, 176(7), 2021 - 32
Lipopolysaccharide epitope expression of Rhizobium bacteroids as revealed by in situ immunolabelling of pea root nodule sections; Kannenberg EL et al.; To investigate the in situ expression of lipopolysaccharide (LPS) epitopes on nodule bacteria of Rhizobium leguminosarum, monoclonal antibodies recognizing LPS macromolecules were used for immunocytochemical staining of pea nodule tissue . Many LPS epitopes were constitutively expressed, and the corresponding antibodies reacted in nodule sections with bacteria at all stages of tissue infection and cell invasion . Some antibodies, however, recognized epitopes that were only expressed in particular regions of the nodule . Two general patterns of regulated LPS epitope expression could be distinguished on longitudinal sections of nodules . A radial pattern probably reflected the local physiological conditions experienced by endosymbiotic bacteria as a result of oxygen diffusion into the nodule tissue . The other pattern of expression, which followed a linear axis of symmetry along a longitudinal section of the pea nodule, was apparently associated with the differentiation of nodule bacteria and the development of the nitrogen-fixing capacity in bacteroids . Basically similar patterns of LPS epitope expression were observed for pea nodules harboring either of two immunologically distinct strains of R . leguminosarum bv . viciae, although these epitopes were recognized by different sets of strain-specific monoclonal antibodies . Furthermore, LPS epitope expression of rhizobia in pea nodules was compared with that of equivalent strains in nodules of French bean (Phaseolus vulgaris) . From these observations, it is suggested that structural modifications of Rhizobium LPS may play an important role in the adaptation of endosymbiotic rhizobia to the surrounding microenvironment.

Proc Natl Acad Sci U S A, 1994 Mar 29, 91(7), 2669 - 73
The NodC protein of Azorhizobium caulinodans is an N-acetylglucosaminyltransferase; Geremia RA et al.; Nod factors are signal molecules produced by Azorhizobium, Bradyrhizobium, and Rhizobium species that trigger nodule formation in leguminous host plants . The backbone of Nod factors consists of a beta-1,4-N-acetylglucosamine oligosaccharide from which the N-acetyl group at the nonreducing end is replaced by a fatty acid . The nodABC gene products are necessary for backbone biosynthesis . By incubation of cell extracts from Azorhizobium caulinodans with radioactive uridine diphosphate-N-acetylglucosamine, Nod factor precursors were identified and characterized as beta-1,4-N-acetylglucosamine oligosaccharides . By analysis of different nod gene mutants and by expression of nodC in Escherichia coli, the N-acetylglucosaminyltransferase activity was ascribed to the NodC protein . The results suggest that the first step in biosynthesis of Nod factors is the assembly of the oligosaccharide chain.

Proc Natl Acad Sci U S A, 1994 Mar 15, 91(6), 2196 - 200
Perception of Rhizobium nodulation factors by tomato cells and inactivation by root chitinases; Staehelin C et al.; The bacterial genera Rhizobium and Bradyrhizobium, nitrogen-fixing symbionts of legumes, secrete specific lipo-chitooligosaccharides that induce the formation of nodules on their host plants . When preparations of such nodulation-inducing factors (Nod factors) were added to suspension-cultured tomato cells, a rapid and transient alkalinization of the culture medium occurred . Lipo-oligosaccharide preparations from Rhizobium or Bradyrhizobium treated with flavonoids, known inducers of Nod factor synthesis, were up to 100 times more potent in inducing alkalinization than the ones from untreated bacteria . The activity was absent from preparations of the mutant strain Rhizobium sp . NGR234 delta nodABC, unable to produce any Nod factors . Preparations of Nod factors from various bacteria as well as individual, highly purified Nod factors from Rhizobium sp . NGR(pA28) induced alkalinization in the tomato cell cultures at nanomolar concentrations . This demonstrates that Nod factors can be perceived by tomato, a nonhost of rhizobia . Using the alkalinization response as a sensitive bioassay, Nod factors were found to be inactivated by plant chitinases . Root chitinases purified from different legumes differed in their potential to inactivate differently substituted Nod factors produced by Rhizobium sp . NGR(pA28) . This indicates that the specificity of the bacterium-host plant interaction may be due, at least in part, to differential inactivation of Nod factors by root chitinases.

Biochemistry, 1994 Mar 15, 33(10), 3120 - 7
Rhodobacter capsulatus contains a novel cb-type cytochrome c oxidase without a CuA center; Gray KA et al.; The facultative phototrophic bacterium Rhodobacter capsulatus is capable of growth in a wide range of environmental conditions using a highly branched electron-transfer chain . During respiratory growth of this organism reducing equivalents are conveyed to oxygen via two terminal oxidases, previously called "cyt b410" (cytochrome c oxidase) and "cyt b260" (quinol oxidase) . The cytochrome c oxidase was purified to homogeneity from a semiaerobically grown R . capsulatus strain . The purified enzyme consumes oxygen at a rate of 600 s-1, oxidizes reduced equine cyt c and R . capsulatus cyt c2, and has high sensitivity to cyanide . The complex is composed of three major polypeptides of apparent molecular masses 45, 32, and 28 kDa on SDS-PAGE . The 32- and 28-kDa proteins also stain with tetramethylbenzidine, indicating that they are c-type cytochromes . Partial amino acid sequences obtained from each of the subunits reveal significant homology to the fixN, fixO, and fixP gene products of Bradyrhizobium japonicum and Rhizobium meliloti . The reduced enzyme has an optical absorption spectrum with distinct features near 550 and 560 nm and an asymmetric Soret band centered at 418 nm, indicating the presence of both c- and b-type cytochromes . Two electrochemically distinct cyt c are apparent, with redox midpoint potentials (Em7) of 265 and 320 mV, while the low-spin cyt b has an Em7 value of 385 mV . The enzyme binds carbon monoxide, and the CO difference spectrum indicates that CO binds to a high-spin cyt b . Pyridine hemochrome and HPLC analyses suggest that the complex contains 1 mol of heme C to 1 mol of protoheme and that neither heme O nor heme A is present.(ABSTRACT TRUNCATED AT 250 WORDS)

Plant Mol Biol, 1994 Mar, 24(5), 767 - 77
Isolation of chalcone synthase and chalcone isomerase cDNAs from alfalfa (Medicago sativa L.): highest transcript levels occur in young roots and root tips; McKhann HI et al.; Flavonoids are involved in several different interactions between plants and microorganisms . In the Rhizobium-legume symbiosis, they play an important role as inducers of rhizobial nodulation (nod) genes . We have identified from an alfalfa cDNA library four clones for chalcone synthase (CHS) and two clones for chalcone isomerase (CHI); CHS and CHI are key enzymes in flavonoid biosynthesis . In Medicago sp., CHS is encoded by 8-12 genes, and CHI is encoded by 1-2 genes . Here we report the DNA sequence of these clones as well as their relatedness to other legume CHS and CHI clones . In addition, we report on the expression patterns of two CHS gene family members as well as the CHI gene in M . sativa cv . Iroquois . While CHS and CHI transcript levels are high in root tips and entire young roots, they are low in effective nodules elicited by wild-type strains of Rhizobium meliloti and very low in aerial portions of the plant (stems, leaves, flowers) . However, wounding the cotyledons results in a rapid increase in transcript levels of both chalcone synthase and chalcone isomerase genes in these organs.

EMBO J, 1994 Mar 1, 13(5), 1093 - 102
Activation of the cell cycle machinery and the isoflavonoid biosynthesis pathway by active Rhizobium meliloti Nod signal molecules in Medicago microcallus suspensions; Savoure A et al.; We have shown that treatment of Medicago microcallus suspensions with the cognate Rhizobium meliloti Nod signal molecule NodRm-IV(C16:2,S) can modify gene expression both qualitatively and quantitatively . At concentrations of 10(-6) - 10(-9) M, this host specific plant morphogen but not the inactive non-sulfated molecule stimulated cell cycle progression as indicated by the significantly enhanced thymidine incorporation, elevated number of S phase cells, increase in kinase activity of the p34cdc2-related complexes and enhancement of the level of expression of several cell cycle marker genes, the histone H3-1, the cdc2Ms and the cyclin cycMs2 . The presented data suggest that at least part of the physiological role of the Nod factor may be linked to molecular events involved in the control of the plant cell division cycle . In situ hybridization experiments with antisense H3-1 RNA probe indicated that only certain cells of the calli were able to respond to the Nod factor . High (10(-6) M) but not low (10(-9) M) concentrations of the active Nod factors induced the expression of the isoflavone reductase gene (IFR), a marker gene of the isoflavonoid biosynthesis pathway in most callus cells . Our results indicate that Medicago cell responses to the Nod signal molecules can be investigated in suspension cultures.

Mol Gen Genet, 1994 Mar, 242(5), 505 - 16
The new class II transposon Tn163 is plasmid-borne in two unrelated Rhizobium leguminosarum biovar viciae strains; Ulrich A et al.; Tn163 is a transposable element identified in Rhizobium leguminosarum bv . viciae by its high insertion rate into positive selection vectors . The 4.6 kb element was found in only one further R . leguminosarum bv . viciae strain out of 70 strains investigated . Both unrelated R . leguminosarum bv . viciae strains contained one copy of the transposable element, which was localized in plasmids native to these strains . DNA sequence analysis revealed three large open reading frames (ORFs) and 38 bp terminal inverted repeats . ORF1 encodes a putative protein of 990 amino acids displaying strong homologies to transposases of class II transposons . ORF2, transcribed in the opposite direction, codes for a protein of 213 amino acids which is highly homologous to DNA invertases and resolvases of class II transposons . Homology of ORF1 and ORF2 and the genetic structure of the element indicate that Tn163 can be classified as a class II transposon . It is the first example of a native transposon in the genus Rhizobium . ORF3, which was found not to be involved in the transposition process, encodes a putative protein (256 amino acids) of unknown function . During transposition Tn163 produced direct repeats of 5 bp, which is typical for transposons of the Tn3 family . However, one out of the ten insertion sites sequenced showed a 6 bp duplication of the target DNA; all duplicated sequences were A/T rich . Insertion of Tn163 into the sacB gene revealed two hot spots . Chromosomes of different R . leguminosarum bv . viciae strains were found to be highly refractory to the insertion of Tn163.

Can J Microbiol, 1994 Mar, 40(3), 208 - 15
Genetic characterization of Rhizobium sp . strain TAL1145 that nodulates tree legumes; George ML et al.; Rhizobium sp . strain TAL1145 nodulates Leucaena leucocephala and Phaseolus vulgaris, in addition to a wide range of tropical tree legumes . Six overlapping clones that complemented nodulation defects in leucaena and bean rhizobia were isolated and a 40-kb map of the symbiosis region was constructed . The common nod and nifA genes were situated approximately 17 kb apart, with the nodIJ genes in between . These clones enabled a derivative of TAL1145 carrying a partially deleted pSym to form ineffective nodules on both leucaena and bean, and a similar derivative of Rhizobium etli TAL182 to form ineffective nodules on bean . When two representative clones, pUHR9 and pUHR114, were each transferred to wild-type rhizobial strains, they allowed ineffective nodulation by Rhizobium meliloti on both leucaena and bean and by Rhizobium leguminosarum bv . vicia on bean . Transconjugants of R . leguminosarum bv . trifolii formed effective nodules on leucaena and ineffective nodules on bean . Tn5 mutagenesis of the symbiosis region resulted in a variety of nodulation and fixation phenotypes on leucaena and bean . On the basis of 16S rRNA sequences, TAL1145 was found to be distinct from both R . tropici and NGR234, the two groups of leucaena symbionts that were previously described.

Microbiology, 1994 Mar, 140 ( Pt 3), 455 - 61
Sequence and regulation of psrA, a gene on the Sym plasmid of Rhizobium leguminosarum biover phaseoli which inhibits transcription of the psi genes; Mimmack ML et al.; The psr region of Rhizobium leguminosarum biovar phaseoli had originally been recognized on the basis of its ability to repress the transcription of the psi genes, one of which, psiA, inhibits exopolysaccharide synthesis when cloned in multi-copy plasmids . Both psr and psi are located on the symbiotic plasmid pRP2JI . The psrA gene was localized and sequenced . The deduced amino acid sequence of PsrA was shown to have similarity to the DNA-binding region of a family of other transcriptional regulators, consistent with its known effects on the expression of psi . The transcription of psrA itself appears to be constitutive in free-living Rhizobium, but is regulated by another gene on the Sym plasmid pRP2JI.

Microbiology, 1994 Mar, 140 ( Pt 3), 443 - 53
Analysis of the expression from Rhizobium meliloti fix-promoters in other Rhizobium backgrounds; Cebolla A et al.; Using translational fusions to lacZ, we have measured expression from the promoters of Rhizobium meliloti regulatory genes, nifA and fixK, and structural genes, nifH and fixA, in other fast-growing rhizobia whose nitrogen fixation regulation is less known . Neither nifA nor fixK promoters were activated under both free-living microaerobic and symbiotic conditions, except in R . tropici, where clear symbiotic activation of either nifA or fixK expression could be observed . Both nifH and fixA promoters showed strong heterologous activation during symbiosis and weak activation under free-living nitrogen starvation conditions . Only when the nifH promoter was in R . tropici and R . leguminosarum bv . phaseoli, was clear induction observed in the microaerobic free-living state . Deletion analysis of these promoters suggested that a NifA binding site (UAS) was needed for full heterologous activation of nifHp, either in microaerobiosis or symbiosis . In contrast, the UAS region seemed to be unnecessary for fixA activation . However, a region containing a potential integration host factor (IHF) binding site was observed to be needed for complete heterologous symbiotic induction from fixAp . The moderate induction observed in nitrogen-free medium only required the sigma 54 holoenzyme recognition sequence; this may be indicative of the existence of non-specific activation by NtrC-like proteins . Our results suggest possible common and different features in the control mechanisms of the nitrogen fixation gene expression among Rhizobium species.

Mol Plant Microbe Interact, 1994 Mar-Apr, 7(2), 240 - 7
Production of cell-associated polysaccharides of Rhizobium fredii USDA205 is modulated by apigenin and host root extract; Reuhs BL et al.; Rhizobium fredii USDA205 cells were cultured in the presence of 4',5,7-trihydroxyflavone (apigenin), a compound that has been shown to induce the nod genes and other symbiosis-related genes in R . fredii . The cell-associated polysaccharides were then extracted with hot phenol/water, separated by repetitive gel filtration chromatography, and analyzed by polyacrylamide gel electrophoresis, nuclear magnetic resonance spectrometry, high-performance anion-exchange chromatography, and gas chromatography . These analyses showed that apigenin effects a modulation in the production of some cell-associated bacterial polysaccharides: 1) The production of a glucan is severely attenuated; 2) the lipopolysaccharide O antigen is modified in composition and M(r) distribution; and 3) the ratio of two extracted polysaccharides, which are structurally analogous to group II K antigens (capsular polysaccharides), is altered . Similar effects resulted from the inclusion of host plant root extract in the growth medium.

Biochem Biophys Res Commun, 1994 Feb 28, 199(1), 1 - 10
The Streptococcus pyogenes hyaluronan synthase: sequence comparison and conservation among various group A strains; DeAngelis PL et al.; We recently cloned the hyaluronan synthase gene (hasA) from Streptococcus pyogenes (DeAngelis et al., J . Biol . Chem . 268, 19181, 1993) . Since this is the first glycosaminoglycan synthase gene to be cloned and these enzymes have also not been purified, nothing is yet known at the molecular level about the similarity or relatedness of hyaluronan synthase to other gene products . We found several proteins in the sequence database with substantial similarities to hyaluronan synthase (HasA), including NodC from Rhizobium, DG42 from Xenopus, and the three chitin synthases (Chs) from Saccharomyces . Like HasA, NodC and the Chs proteins all have at least an N-acetylglucosaminyl transferase activity in common and are membrane-associated . The polymerase chain reaction was also used to show that HasA, and probably the two gene operon for hyaluronan biosynthesis (hasA/hasB), is highly conserved among Group A streptococcal strains . In nine strains, isolated from 1917 through 1993, only five silent mutations were detected . The results show that hyaluronan synthase is highly conserved within Group A strains, although another virulence factor, the M protein, varies considerably in these same strains.

J Mol Biol, 1994 Feb 11, 236(1), 81 - 90
The domain structure of sigma 54 as determined by analysis of a set of deletion mutants; Wong C et al.; Escherichia coli sigma 54 was analyzed by making a series of 16 internal deletions within its gene and analyzing the properties of the mutant proteins . All of the mutant proteins except one were strongly defective in a growth test that relied on sigma 54 function . Additional assays were applied to determine the causes of these defects . The assays monitored the following properties: the level of protein expression; ability to bind to the -24 promoter element of the glnAP2 promoter in vivo; the ability to bind to the -12 promoter element in vivo; ability to melt the promoter start site in vivo; ability to bind the Rhizobium meliloti nifH promoter in vitro; and the ability to form a sigma 54-core RNA polymerase complex (E sigma 54 holoenzyme) in vitro . The analysis shows a modular structure in that certain regions of the protein predominate in contributing to each of these properties . A large carboxyl region of the protein is essential for promoter binding . A smaller amino-terminal segment is essential for DNA melting . An element essential for the forming the E sigma 54 holoenzyme lies between these two regions . None of these domains resemble those of sigma 70 and this difference is discussed in view of the different transcription mechanisms directed by the two proteins.

Gene, 1994 Feb 11, 139(1), 133 - 4
A protein involved in stabilization of a large non-symbiotic plasmid of Rhizobium meliloti shows homology to eukaryotic cytoskeletal proteins and DNA-binding proteins; Mercado-Blanco J et al.; An open reading frame, denoted ORF2, present in the replication and stabilization region of plasmid pRmeGR4a of Rhizobium meliloti GR4, was identified by sequence analysis . This 1068-bp ORF2 potentially codes for a 356-amino-acid protein that seems to play a role in pRmeGR4a stabilization . Similarities of the ORF2-encoded protein with eukaryotic cytoskeletal proteins and DNA-binding proteins were found.

J Bacteriol, 1994 Feb, 176(3), 620 - 33
nodZ, a unique host-specific nodulation gene, is involved in the fucosylation of the lipooligosaccharide nodulation signal of Bradyrhizobium japonicum; Stacey G et al.; The nodulation genes of rhizobia are regulated by the nodD gene product in response to host-produced flavonoids and appear to encode enzymes involved in the production of a lipo-chitose signal molecule required for infection and nodule formation . We have identified the nodZ gene of Bradyrhizobium japonicum, whose product is required for the addition of a 2-O-methylfucose residue to the terminal reducing N-acetylglucosamine of the nodulation signal . This substitution is essential for the biological activity of this molecule . Mutations in nodZ result in defective nodulation of siratro . Surprisingly, although nodZ clearly codes for nodulation function, it is not regulated by NodD and, indeed, shows elevated expression in planta . Therefore, nodZ represents a unique nodulation gene that is not under the control of NodD and yet is essential for the synthesis of an active nodulation signal.

Mol Microbiol, 1994 Feb, 11(4), 793 - 804
Nodulation protein NodL of Rhizobium leguminosarum O-acetylates lipo-oligosaccharides, chitin fragments and N-acetylglucosamine in vitro; Bloemberg GV et al.; Upon induction of their nodulation genes, the root nodule-inducing Rhizobium bacteria produce lipo-oligosaccharide signal molecules . All lipo-oligosaccharides identified from Rhizobium leguminosarum bv . viciae carry an O-acetyl group at the C-6 position of the non-reducing terminal sugar, the presence of which is important for biological activity and host specificity . Previously we showed that a functional nodL gene product is required for the presence of this O-acetyl moiety . The production of polyclonal antibodies against isolated NodL protein, using a NodL-overproducing Escherichia coli strain is described . These antibodies were used (i) to elucidate the subcellular localization of the NodL protein, which appeared to be present in the cytosol, and (ii) for the purification of native NodL protein from E . coli . Here we provide biochemical proof that purified NodL protein has transacetylating activity in vitro with acetyl-CoA as the acetyl donor . NodL protein appeared to be able to acetylate various substrates, such as lipo-oligosaccharides, chitin fragments and N-acetylglucosamine . For chitinpentaose as the substrate we have shown, using mass spectrometry and NMR spectroscopy, that NodL protein substitutes one O-acetyl group at the C-6 position of the non-reducing terminal sugar.

Trends Genet, 1994 Feb, 10(2), 63 - 7
Exopolysaccharides of Rhizobium: synthesis, regulation and symbiotic function; Leigh JA et al.; Exopolysaccharides of Rhizobium have long been suspected, and are now known, to function in the Rhizobium-legume root nodule symbiosis . Recent studies have enhanced our knowledge of these extracellular polymers as symbiotic signals and have elucidated their biosynthesis and regulation.

Plant Cell, 1994 Feb, 6(2), 201 - 13
ENOD12, an early nodulin gene, is not required for nodule formation and efficient nitrogen fixation in alfalfa; Csanadi G et al.; To demonstrate the importance of an extensively studied early nodulin gene ENOD12 in symbiotic nodule development, plants of different Medicago sativa subspecies were tested for the presence or absence of ENOD12 alleles . In M . s . ssp coerulea w2 (Mcw2), two ENOD12 genes were detected, whereas in M . s . ssp quasifalcata k93 (Mqk93) only one gene was present . In both plants, the ENOD12 genes were expressed in nodules induced by Rhizobium meliloti . The nucleotide sequence of the ENOD12 genes showed that the two Mcw2-specific genes were similar to the ENOD12A and ENOD12B genes of the tetraploid M . s . ssp sativa . ENOD12 from Mqk93 was similar to the corresponding gene found in M . truncatula . From the aligned ENOD12 sequences, an evolutionary tree was constructed . Genetic analysis of the progenies of a cross between Mqk93 and Mcw2 showed that several offspring in F1 carried a null allele originating from Mcw2, and among the F2 progenies, plants with the null allele only lacking the ENOD12 gene appeared . Surprisingly, the ENOD12-deficient plants were similar to their wild-type parents in viability, nodule development, nodule structure, and nitrogen fixation efficiency . Therefore, we concluded that in Medicago the ENOD12 gene is not required for symbiotic nitrogen fixation . Furthermore, we proposed that the heterozygous nature of these legumes can be exploited for the identification of mutated alleles of other known nodulin genes; this will permit the construction of plant mutants deficient in these genes.

J Bacteriol, 1994 Feb, 176(4), 1047 - 51
Synthesis of glycerophosphorylated cyclic beta-(1,2)-glucans by Rhizobium meliloti ndv mutants; Breedveld MW et al.; The periplasmic cyclic beta-(1,2)-glucans of Rhizobium spp . are believed to provide functions during hypoosmotic adaptation and legume nodulation . In Rhizobium meliloti, cyclic beta-(1,2)-glucans are synthesized at highest levels when cells are grown at low osmolarity, and a considerable fraction (> or = 35%) of these glucans may become substituted with phosphoglycerol moieties . Thus far, two chromosomally encoded proteins, NdvA and NdvB, have been shown to function during cyclic beta-(1,2)-glucan biosynthesis; however, the precise roles for these proteins remain unclear . In the present study, we show that R . meliloti mutants lacking up to one-third of the downstream region of ndvB synthesize cyclic beta-(1,2)-glucans similar to those produced by wild-type cells with respect to size and phosphoglycerol substituent profile . In contrast, no phosphoglycerol substituents were detected on the cyclic beta-(1,2)-glucans synthesized by an R . meliloti ndvA mutant.

Mol Microbiol, 1994 Feb, 11(4), 685 - 93
Regulation of nitrogen metabolism is altered in a glnB mutant strain of Rhizobium leguminosarum; Amar M et al.; We isolated a Rhizobium leguminosarum mutant strain altered in the glnB gene . This event, which has never been described in the Rhizobiaceae, is rare in comparison to mutants isolated in the contiguous gene, glnA . The glnB mutation removes the glnBA promoter but in vivo does not prevent glnA expression from its own promoter, which is not nitrogen regulated . The glnB mutant strain does not grow on nitrate as a sole nitrogen source and it is Nod+, Fix+ . Two -24/-12 promoters, for the glnII and glnBA genes, are constitutively expressed in the glnB mutant, while two -35/-10-like promoters for glnA and ntrBC are unaffected . We propose that the glnB gene product, the PII protein, plays a negative role in the ability of NtrC to activate transcription from its target promoters and a positive role in the mechanism of nitrate utilization.

J Bacteriol, 1994 Jan, 176(2), 518 - 9
An insertional point mutation inactivates NolR repressor in Rhizobium meliloti 1021; Cren M et al.; In the majority of Rhizobium meliloti isolates, nod gene expression is controlled by NolR, but this is not the case in a few strains including the widely used laboratory strain 1021 . In 1021, the lack of NolR function was shown to be due to a single insertional mutation in the C-terminal coding sequence which abolished the DNA-binding ability, though the helix-turn-helix motif remained intact . This indicates that the C-terminal part of the protein is also essential for DNA binding . We conclude that in this species, control of nod gene expression involves NolR and strain 1021 represents an exception in which the NolR function was lost by a single event.

J Bacteriol, 1994 Jan, 176(1), 189 - 97
Molecular characterization, nucleotide sequence, and expression of the fliO, fliP, fliQ, and fliR genes of Escherichia coli; Malakooti J et al.; The fliL operon of Escherichia coli contains seven genes that are involved in the biosynthesis and functioning of the flagellar organelle . DNA sequences for the first three genes of this operon have been reported previously . A 2.2-kb PstI restriction fragment was shown to complement known mutant alleles of the fliO, fliP, fliQ, and fliR genes, the four remaining genes of the fliL operon . Four open reading frames were identified by DNA sequence analysis and correlated to their corresponding genes by complementation analysis . These genes were found to encode very hydrophobic polypeptides with molecular masses of 11.1, 26.9, 9.6, and 28.5 kDa for FliO, FliP, FliQ, and FliR, respectively . Analysis of recombinant plasmids in a T7 promoter-polymerase expression system enabled us to identify three of the four gene products . On the basis of DNA sequence analysis and in vivo protein expression, it appears that the fliP gene product is synthesized as a precursor protein with an N-terminal signal peptide of 21 amino acids . The FliP protein was homologous to proteins encoded by a DNA sequence upstream of the flaA gene of Rhizobium meliloti, to a gene involved in pathogenicity in Xanthomonas campestris pv . glycines, and to the spa24 gene of the Shigella flexneri . The latter two genes encode proteins that appear to be involved in protein translocation, suggesting that the FliP protein may have a similar function.

Mol Microbiol, 1994 Jan, 11(2), 315 - 21
Nodulating ability of Rhizobium tropici is conditioned by a plasmid-encoded citrate synthase; Pardo MA et al.; Rhizobium species elicit the formation of nitrogen-fixing root nodules through a complex interaction between bacteria and plants . Various bacterial genes involved in the nodulation and nitrogen-fixation processes have been described and most have been localized on the symbiotic plasmids (pSym) . We have found a gene encoding citrate synthase on the pSym plasmid of Rhizobium tropici, a species that forms nitrogen-fixing nodules on the roots of beans (Phaseolus vulgaris) and trees (Leucaena spp.) . Citrate synthase is a key metabolic enzyme that incorporates carbon into the tricarboxylic acid cycle by catalysing the condensation of acetyl-CoA and oxaloacetic acid to form citrate . R . tropici pcsA (the plasmid citrate synthase gene) is closely related to the corresponding genes of Proteobacteria . pcsA inactivation by a Tn5-mob insertion causes the bacteria to form fewer nodules (30-50% of the original strain) and to have a decreased citrate synthase activity in minimal medium with sucrose . A clone carrying the pcsA gene complemented all the phenotypic alterations of the pcsA mutant, and conferred Rhizobium leguminosarum bv . phaseoli (which naturally lacks a plasmid citrate synthase gene) a higher nodulation and growth capacity in correlation with a higher citrate synthase activity . We have also found that pcsA gene expression is sensitive to iron availability, suggesting a possible role of pcsA in iron uptake.

Mol Microbiol, 1994 Jan, 11(1), 165 - 73
Genetic characterization of a Rhizobium meliloti lactose utilization locus; Jelesko JG et al.; We identified several linked genes of a lactose regulon in Rhizobium meliloti . These were lacZ, the structural gene for beta-galactosidase; lacR, the lactose repressor gene; and two genes encoding proteins of unknown function, lacW and lacX . Insertion mutants in lacW and lacZ belonged to a single genetic complementation group, and lacW appeared to lie upstream of lacZ in an operon . Expression of lacZ, lacW and lacX was repressed by lacR, and expression of lacZ and lacW was derepressed by lactose . lacZ was not required for induction of lacW by lactose, suggesting that lactose itself, rather than a processed form of lactose, may be the actual inducer molecule . Expression of all three genes was repressed by succinate, and the lacR independence of this repression showed that inducer exclusion could not be the sole mechanism . This pattern of lac gene organization and regulation differs in several ways from that observed in enteric bacteria.

Biochimie, 1994, 76(2), 121 - 8
Purification of an alpha-L-fucoside-binding protein from Rhizobium lupini; Wisniewski JP et al.; Lectins associated with the bacterial cell surface of Rhizobium lupini strain LL13 were evidenced by erythrocyte agglutination, by aggregation of neoglycoprotein coated beads and by spectrofluorimetry using fluoresceinylated neoglycoproteins . At pH 5.0, a specific binding of the fluorescein-labelled neoglycoprotein bearing alpha-L-fucose was observed . The binding of this labelled neoglycoprotein is a saturable phenomenon and is inhibited by the same unlabelled neoglycoprotein . Extracts of R lupini obtained by disrupting a bacterial pellet through a French press were stabilized at pH 5.6 by gel filtration and purified to homogeneity by affinity chromatography on Agarose A4 substituted with alpha-L-fucose . A protein with a M(r) approximately 19,000 was specifically eluted from this affinity column with L-fucose . Isoelectric focusing of this sample yielded a single band with pI near 6.7 . This protein specifically aggregated L-Fuc-BSA-coated microspheres . The results obtained in the present study indicate that we have purified from Rhizobium lupini strain LL13, a L-fucose binding protein as a lectin.

Arch Microbiol, 1994, 161(5), 404 - 8
Characterization of the Rhizobium leguminosarum biovar phaseoli nifA gene, a positive regulator of nif gene expression; Michiels J et al.; We report the isolation, mutational analysis and the nucleotide sequence of the Rhizobium leguminosarum bv . phaseoli nifA gene . Comparison of the deduced amino acid sequence with other NifA sequences indicated the presence of the conserved central activator and the C-terminal DNA-binding domains . Nodules elicited by a R . leguminosarum bv . phaseoli nifA mutant were symbiotically ineffective . The expression of a nifA-gusA fusion was shown to be independent on the oxygen status of the cell . We cloned the three nifH copies of R . leguminosarum bv . phaseoli and determined the nucleotide sequence of their promoter regions . The expression of nifH-gusA fusions is induced under microaerobic conditions and is dependent on the presence of NifA.

Acta Biochim Pol, 1994, 41(1), 7 - 11
Siderophore activity in Rhizobium species isolated from different legumes; Derylo M et al.; Rhizobium strains isolated from nodules of the different legumes including wild-growing plants were examined for their siderophore activity . Fifteen of the 84 screened rhizobial strains were able to grow under conditions of limited iron supply . Nine of them gave orange halos in the assay with Chrom azurol S . Among these strains were Rhizobium sp . (Ononis) and Rhizobium (Genista), producing hydroxamates and phenolates . These compounds could promote the growth of siderophore-negative bacteria on iron-deficient media . The results imply that the hydroxamates from G1 and O1 strains may belong to the monohydroxamate class of siderophores.

Arch Microbiol, 1994, 161(4), 286 - 92
The adaptive acid tolerance response in root nodule bacteria and Escherichia coli; O'Hara GW et al.; Root nodule bacteria and Escherichia coli show an adaptive acid tolerance response when grown under mildly acidic conditions . This is defined in terms of the rate of cell death upon exposure to acid shock at pH 3.0 and expressed in terms of a decimal reduction time, D . The D values varied with the strain and the pH of the culture medium . Early exponential phase cells of three strains of Rhizobium leguminosarum (WU95, 3001 and WSM710) had D values of 1, 6 and 5 min respectively when grown at pH 7.0; and D values of 5, 20 and 12 min respectively when grown at pH 5.0 . Exponential phase cells of Rhizobium tropici UMR1899, Bradyrhizobium japonicum USDA110 and peanut Bradyhizobium sp . NC92 were more tolerant with D values of 31, 35 and 42 min when grown at pH 7.0; and 56, 86 and 68 min when grown at pH 5.0 . Cells of E . coli UB1301 in early exponential phase at pH 7.0 had a D value of 16 min, whereas at pH 5.0 it was 76 min . Stationary phase cells of R . leguminosarum and E . coli were more tolerant (D values usually 2 to 5-fold higher) than those in exponential phase . Cells of R . leguminosarum bv . trifolii 3001 or E . coli UB1301 transferred from cultures at pH 7.0 to medium at pH 5.0 grew immediately and induced the acid tolerance response within one generation . This was prevented by the addition of chloramphenicol . Acid-adapted cells of Rhizobium leguminosarum bv . trifolii WU95 and 3001; or E . coli UB1301, M3503 and M3504 were as sensitive to UV light as those grown at neutral pH.

Rev Argent Microbiol, 1994 Jan-Mar, 26(1), 1 - 8
{The Rhizobium-Prosopis symbiosis in the Argentinian Chaco Arido}; Abril A et al.; Low productivity soils can be improved by incorporation of adapted legumes species able to increase the nitrogen fixation when associated to Rhizobium strains . Two fast-growing Rhizobium strains were isolated from nodules of Prosopis alba . Infectivity and effectivity of the strains were assessed under controlled conditions, measuring the following parameters: nodule number, dry weight, nitrogen percentage and fixed nitrogen . P . flexuosa strains showed high infective capacity (144 nodules/plant) but low effectivity (21.4 mg fixed nitrogen in 75 days) . Although nodule number in P . alba was lower (23 nodules/plant) nitrogen fixation was higher (119.5 mg fixed nitrogen in 75 days) as compared with P . flexuosa . The isolated strains might be used as nitrogen donors in aride soils; they might be helpful to forestation of these ecosystems.

Crit Rev Microbiol, 1994, 20(2), 117 - 23
Molecular genetics of the glutamine synthetases in Rhizobium species; Espin G et al.; Soil bacteria of the genus Rhizobium and Bradyrhizobium establish symbiotic interactions with leguminous plants that result in the formation of specialized structures, the nodules, in which the bacteria differentiate into bacteroids and fix nitrogen . Rhizobial glutamine synthetase (GS) activity is very low in the nodule . The ammonia produced by the bacteroids is exported to the plant cell, where it is assimilated by the GS from the plant, whereas in the free-living state, Rhizobium and Bradyrhizobium species assimilate ammonia for growth . Another characteristic of these species is that they possess two glutamine synthetase isozymes, known as GSI and GSII . A third glutamine synthetase isozyme, called GSIII, has been found in R . meliloti and R . etli.

Microbiology, 1994 Jan, 140 ( Pt 1), 113 - 22
Rhizobium leguminosarum contains multiple chaperonin (cpn60) genes; Wallington EJ et al.; We have examined the heat shock response of Rhizobium leguminosarum . After normal growth at 28 degrees C, a 10 min heat shock at 37 degrees C induced the synthesis of proteins with approximate M(r) values of 90,000, 70,000, 60,000, 58,000, 19,000, 17,000 and 13,000 . A monoclonal antibody raised against the E . coli Cpn60 cross-reacted with proteins of M(r) 60,000 and 58,000 in R . leguminosarum, suggesting that both were Cpn60 homologues . Hybridization of an E . coli cpn60 probe to total DNA from Rhizobium leguminosarum also showed evidence for at least two cpn60 homologues . One of these was cloned and completely sequenced, and showed close homology to cpn60 sequences from other prokaryotes . The expression of this gene in E . coli failed to complement a cpn60 mutation, either for growth at high temperature or for growth of bacteriophage lambda . Hybridization of total R . leguminosarum DNA with a probe from this gene revealed the presence of a third putative cpn60 gene . Two further hybridizing clones were analysed and found to consist of two additional cpn60 sequences plus upstream regions containing putative cpn10 genes.

Antonie Van Leeuwenhoek, 1994, 66(1-3), 129 - 50
Oxygen control in Rhizobium; Batut J et al.; Rhizobia are gram-negative bacteria with two distinct habitats: the soil rhizosphere in which they have a saprophytic and, usually, aerobic life and a plant ecological niche, the legume nodule, which constitutes a microoxic environment compatible with the operation of the nitrogen reducing enzyme nitrogenase . The purpose of this review is to summarize the present knowledge of the changes induced in these bacteria when shifting to a microoxic environment . Oxygen concentration regulates the expression of two major metabolic pathways: energy conservation by respiratory chains and nitrogen fixation . After reviewing the genetic data on these metabolic pathways and their response to oxygen we will put special emphasis on the regulatory molecules which are involved in the control of gene expression . We will show that, although homologous regulatory molecules allow response to oxygen in different species, they are assembled in various combinations resulting in a variable regulatory coupling between genes for microaerobic respiration and nitrogen fixation genes . The significance of coordinated regulation of genes not essential for nitrogen fixation with nitrogen fixation genes will also be discussed.

Acta Microbiol Pol, 1994, 43(3-4), 381 - 8
The effect of L-tryptophane on yield of field bean and activity of soil microorganisms; Kucharski J et al.; The pot trial was performed to study the effect of L-tryptophane (as an auxin precursor) applied in the amount 0.3 and 3.0 mg per 1 kg of the soil on yield and the chemical composition of field bean . The effects of this compound on dehydrogenases activity in the cells Rhizobium leguminosarum isolated from root nodules, soil dehydrogenases activity and number of microorganisms from different systematic or physiological groups were also studied . The effects of L-tryptophane were compared to indole-3-acetic acid (IAA) after application to soil in the rate 0.2 mg per 1 kg of the soil of foliar spraying in the rate 20 mg Din 1 dm3 of distilled water . Studies were carried out in three experimental series: without microorganisms or with addition of Azotobacter sp . or Rhizobium leguminosarum biovar . viciae to the soil . It was found that L-tryptophane and IAA did not affect the yield of above ground part and roots of field bean and their effects on macronutrients concentration were not direct and dependent on the nutrient and experimental series . L-tryptophane and auxine increased the dehydrogenases activity in the cells of Rhizobium leguminosarum isolated from root nodules and the effect on the activity of soil dehydrogenases and urease was dependent on the rate of L-tryptophane . This chemical adversely affected the numbers of some microorganisms groups.

Antonie Van Leeuwenhoek, 1994, 65(2), 81 - 98
The molecular basis of the host specificity of the Rhizobium bacteria; Spaink HP; The interaction between soil bacteria belonging to the genera Rhizobium, Bradyrhizobium and Azorhizobium and leguminous plants results in the induction of a new plant organ, the root nodule . After invading these root nodules via infection threads the bacteria start to fix atmospheric nitrogen into ammonia which is beneficial for the host plant . This symbiotic interaction is highly host-specific in that each rhizobial strain is able to associate with only a limited number of host plant species . The subject of this presentation is the molecular mechanism by which the bacterium determines its host-specific characteristics . This mechanism appears to be based on at least two stages of molecular signaling between the bacterium and the plant host . In the first stage, flavonoids secreted by the plant root induce, in a host specific way, the transcription of bacterial genes which are involved in nodulation, the so-called nod genes . This leads to the second step of the signaling system: the production and secretion of lipo-oligosaccharide molecules by the Rhizobium bacteria . These signal molecules, which are acylated forms of small fragments of chitin, have various discernable effects on the roots of the host plants . One of these effects is the dedifferentiation of groups of cells located in the cortex which leads to the formation of nodule meristems . In their mitogenic activity the bacterial signals resemble several well-known plant hormones like auxins and cytokinins . However, there are two major differences: (i) the bacterial signals lead to the induction of a specific organ and (ii) they are host-specific in that only the signals produced by compatible bacteria are able to induce meristems . The nod genes determine this stage of host specificity by their essential role in the biosynthesis of the signal molecules . They appear to encode enzymes which are involved in the processes of fatty acid biosynthesis, fatty acid transfer, chitin synthesis and chitin modification . I will illustrate the statement that the nod gene products are ideal model enzymes for the study of these important processes because they are not needed in the free-living state of the bacteria.

Biochem Soc Symp, 1994, 60, 61 - 73
Bacterial and plant glycoconjugates at the Rhizobium-legume interface; Brewin NJ et al.; Many classes of bacterial and plant glycoconjugate have been shown to be involved in establishing the Rhizobium root nodule symbiosis with peas (Pisum sativum) . It was demonstrated, using techniques of molecular genetics, that a group of Rhizobium nodulation genes (nod genes) co-operate to synthesize a lipo-oligosaccharide signal molecule that specifically initiates nodule development on legume hosts . An additional gene function, encoded by nodX, has been found to extend the host range of Rhizobium leguminosarum bv . viciae to include nodulation of a pea mutant, cultivar Afghanistan; the nodX gene product specifies the addition of an acetyl group to the terminal N-acetylglucosamine residue at the reducing end of the pentasaccharide core of this signal molecule . Several other classes of bacterial glycoconjugate have also been shown by genetic analysis to be essential for normal nodule development and function: these include a capsular extracellular polysaccharide; lipopolysaccharide in the outer membrane; and cyclic glucans present in the periplasmic space . Potential functions for these glycoconjugates are discussed in the context of tissue and cell invasion by Rhizobium . Some plant components involved in symbiotic interactions have been identified by the analysis of nodule-specific gene expression (early nodulins) . Several of the cDNA clones encoding these early nodulins specify proline-rich proteins that presumably correspond to cell wall glycoproteins or membrane arabinogalactan proteins . Other plant glycoconjugates have been identified using monoclonal antibodies as probes . A plant glycoprotein present in intercellular spaces has been identified as a component of the luminal matrix of infection threads . Because it attaches to the surface of bacteria and is itself susceptible to oxidative cross-linking, this glycoprotein may be involved in limiting the progress of microbial infections . Endocytosis of bacteria into the plant cytoplasm is apparently driven by direct interactions between the bacterial surface and the plasma membrane that is exposed within an unwalled infection droplet; glycoprotein and glycolipid components of the plant membrane glycocalyx have been defined using monoclonal antibodies . Differentiation of endosymbiotic bacteroids is preceded by differentiation of the plant-derived peribacteroid membrane which encloses the symbiosome compartment . Using a monoclonal antibody that identifies a group of plant membrane-associated, inositol-containing glycolipids, we have identified a very early marker for the differentiation of peribacteroid membrane from plasma membrane.

Gene, 1993 Dec 8, 134(2), 145 - 52
Mutants of the two-component regulatory protein FixJ of Rhizobium meliloti that have increased activity at the nifA promoter; Weinstein M et al.; FixL and FixJ belong to a two-component regulatory system in Rhizobium meliloti that induces the expression of numerous nitrogen-fixation genes during symbiosis with alfalfa . FixJ is a positive activator required for transcription of the regulatory genes nifA and fixK, while FixL is an oxygen-binding hemoprotein capable of regulating the phosphorylation status of both itself and FixJ, in response to oxygen availability . In this study, we isolated four FixJ mutants that display increased activity at the nifA promoter (PnifA) in Escherichia coli . All four mutants possess amino acid changes in a domain of FixJ that is conserved in other response regulator proteins, and all exhibit increased activity at PnifA in R . meliloti that is dependent on the presence of FixL . One of the mutant proteins, while less efficient at accepting phosphate from a truncated derivative of FixL (FixL*), nevertheless has a phosphorylated form that is more stable than the phosphorylated form of wild-type (wt) FixJ and is more resistant to the phosphatase activity of FixL* . The wt FixJ-phosphate was found to have a half-life of approximately 4 h, which makes it an unusually long-lived response regulator protein . The exceptional stability of wt FixJ-phosphate and the altered phosphorylation properties observed for the mutant are discussed in relation to signal transduction in the FixLJ system.

J Appl Bacteriol, 1993 Dec, 75(6), 519 - 28
The survival of bacteria exposed to desiccation on surfaces associated with farm buildings; Bale MJ et al.; The survival of 11 species of Gram-negative and Gram-positive bacteria was examined on different surfaces exposed to desiccation . There were large variations between species; Pseudomonas spp . and Rhizobium leguminosarum biovars survived for less than 2 d, whilst Enterococcus spp . survived for more than 11 weeks . The type of surface on to which the bacteria were deposited affected survival, but with different effects between species . In addition the survival of spontaneous nalidixic acid-resistant (Nal-r) mutants of a natural Escherichia coli isolate were compared . Overall the differences were slight, but of seven resistant mutants, five survived better than the parent whilst one survived less well . Nine transposon insertion derivatives of one of the Nal-r mutants (ECO80) which survived better than the parent were compared; all survived similarly to the parent except ECO883 which survived less well . The growth characteristics of ECO883 and ECO80 were compared; at high osmotic pressures (> 0.4 mol 1-1 NaCl) ECO883 grew more slowly and showed a longer lag time than the parent . Of the osmoregulatory functions studied, ECO883 appeared to be altered with respect to K+ transport or accumulation, although the transposon insertion had occurred in a gene distant from known K+ transport genes.

FEMS Microbiol Lett, 1993 Dec 1, 114(2), 185 - 9
Role of the fixGHI region of Azorhizobium caulinodans in free-living and symbiotic nitrogen fixation; Mandon K et al.; A 19-kb DNA region containing genes sharing homology with Rhizobium meliloti fixNOQP and fixGHI was isolated from a genomic library of Azorhizobium caulinodans . Identity of fixG was confirmed by partial nucleotide sequencing . Mutant strains in the fixGHI region were constructed by deletion or Tn5 insertions . In contrast with the situation in R . meliloti, the mutants still displayed a significant nitrogenase activity in symbiosis.

FEMS Microbiol Lett, 1993 Dec 1, 114(2), 139 - 44
Identification and cloning of a cyclic beta-(1-->3), beta-(1-->6)-D-glucan synthesis locus from Bradyrhizobium japonicum; Bhagwat AA et al.; A cosmid clone complementing a cyclic beta-(1-->2)-glucan biosynthesis (ndvB) mutant of Rhizobium meliloti was isolated from a Bradyrhizobium japonicum gene library . This clone specified synthesis of beta-(1-->3), beta-(1-->6)-linked glucans in R . meliloti . The complemented strain was osmotically tolerant and symbiotically competent on alfalfa . Thus, beta-(1-->3), beta-(1-->6)-glucans can substitute functionally for beta-(1-->2)-glucans in R . meliloti.

Plant J, 1993 Dec, 4(6), 971 - 81
Molecular characterization and expression of alfalfa isoliquiritigenin 2'-O-methyltransferase, an enzyme specifically involved in the biosynthesis of an inducer of Rhizobium meliloti nodulation genes; Maxwell CA et al.; A cDNA clone encoding an O-methyltransferase (OMT) from alfalfa has been isolated, which methylates the 2'-hydroxyl of isoliquiritigenin (2',4,4'-trihydroxychalcone) to form 4,4'-dihydroxy-2'-methoxychalcone, the most potent of the nod-gene-inducing flavonoid derivatives released from alfalfa roots . The cDNA clone was identified on the basis of N-terminal sequence identity to purified S-adenosyl-L-methionine:isoliquiritigenin 2'-O-methyltransferase (chalcone OMT) and expression of enzymatically active chalcone OMT protein in Escherichia coli . The deduced amino acid sequence showed significant similarities to other OMTs . Chalcone OMT is encoded by a small gene family in alfalfa and related sequences are present in other legumes . The chalcone OMT gene is expressed primarily in alfalfa roots; transcript levels were highest during the first 2 weeks of development . The OMT transcript was also detected, to a much lesser extent, in root nodules . In contrast, chalcone isomerase (CHI), although expressed at high levels in roots, was found in all plant organs and had a somewhat different developmental expression pattern . Chalcone OMT transcripts were localized primarily to epidermal and cortical cells starting 1.5-2.0 mm behind the root tip, whereas CHI transcripts were present at approximately equal levels in epidermal, cortical and vascular tissues, both at the root tip and throughout the root . Chalcone OMT transcripts were elicitor-inducible in alfalfa cell suspension cultures, although only low levels of methoxychalcone accumulated . The implications of these results for plant-microorganism interactions are discussed.

Lipids, 1993 Nov, 28(11), 975 - 9
Phospholipid and fatty acid compositions of Rhizobium leguminosarum biovar trifolii ANU843 in relation to flavone-activated pSym nod gene expression; Orgambide GG et al.; The phospholipid and associated fatty acid compositions of the bacterial symbiont of clover, Rhizobium leguminosarum biovar trifolii wild-type ANU843, was analyzed by two-dimensional silica thin-layer chromatography, fast atom bombardment-mass spectrometry, flame-ionization detection gas-liquid chromatography and combined gas-liquid chromatography/mass spectrometry . The phospholipid composition included phosphatidylethanolamine (15%), N-methylphosphatidylethanolamine (47%), N,N-dimethylphosphatidylethanolamine (9%), phosphatidylglycerol (19%), cardiolipin (5%) and phosphatidylcholine (2%) . Fatty acid composition included predominantly cis-11-octadecenoic acid, lower levels of cis-9-hexadecenoic acid, hexadecanoic acid, 11-methyl-11-octadecenoic acid, octadecanoic acid, 11,12-methyleneoctadecanoic acid, eicosanoic acid and traces of branched, and di- and triunsaturated fatty acids . The influence of expression of the "nodulation" genes encoding symbiotic functions on the composition of these membrane lipids was examined in wild-type cells grown with or without the flavone inducer, 4',7-dihydroxyflavone and in mutated cells lacking the entire symbiotic plasmid where these genes reside, or containing single transposon insertions in selected nodulation genes . No significant changes in phospholipid or associated fatty acid compositions were detected by the above methods of analysis.

Mol Gen Genet, 1993 Nov, 241(3-4), 367 - 79
Identification and analysis of the Rhizobium meliloti exoAMONP genes involved in exopolysaccharide biosynthesis and mapping of promoters located on the exoHKLAMONP fragment; Becker A et al.; Sequence analysis of a 7.494 kb DNA fragment from megaplasmid 2 of Rhizobium meliloti 2011 involved in exopolysaccharide I (EPS I) biosynthesis revealed the presence of five exo genes designated exoA, exoM, exoN, exoO, and exoP . ExoN was found to show strong homology to a UDP-glucose pyrophosphorylase from Acetobacter xylinum, whereas ExoO displayed weak homologies to the NodC proteins from R . meliloti and R . loti . Surprisingly, different mutations in exoP resulted in divergent phenotypes . One exoP mutant was able to establish an effective symbiosis with alfalfa, although no EPS I polymer could be detected . In contrast, other exoP mutations prevented the formation of an effective symbiosis . The transcriptional organization of the exoA-exoP gene region has been analysed in conjunction with the exoH, exoK and exoL genes . Using exo-lacZ transcription fusions in association with plasmid integration mutagenesis a strong promoter was identified upstream of exoH, which is able to direct transcription of the whole exoHKLAMONP gene cluster . A much weaker promoter upstream of exoL was found to be involved in the transcription of the exoLAMONP genes . In addition, weak promoters were identified upstream of exoK, exoA, exoN and exoP.

J Bacteriol, 1993 Nov, 175(21), 7045 - 55
Genes needed for the modification, polymerization, export, and processing of succinoglycan by Rhizobium meliloti: a model for succinoglycan biosynthesis; Glucksmann MA et al.; The major acidic exopolysaccharide of Rhizobium meliloti, termed succinoglycan, is required for nodule invasion and possibly nodule development . Succinoglycan is a polymer of octasaccharide subunits composed of one galactose residue, seven glucose residues, and acetyl, succinyl, and pyruvyl modifications, which is synthesized on an isoprenoid lipid carrier . A cluster of exo genes in R . meliloti are required for succinoglycan production, and the biosynthetic roles of their gene products have recently been determined (T.L . Reuber and G . C . Walker, Cell 74:269-280, 1993) . Our sequencing of 16 kb of this cluster of exo genes and further genetic analysis of this region resulted in the discovery of several new exo genes and has allowed a correlation of the genetic map with the DNA sequence . In this paper we present the sequences of genes that are required for the addition of the succinyl and pyruvyl modifications to the lipid-linked intermediate and genes required for the polymerization of the octasaccharide subunits or the export of succinoglycan . In addition, on the basis of homologies to known proteins, we suggest that ExoN is a uridine diphosphoglucose pyrophosphorylase and that ExoK is a beta(1,3)-beta (1,4)-glucanase . We propose a model for succinoglycan biosynthesis and processing which assigns roles to the products of nineteen exo genes.

J Bacteriol, 1993 Nov, 175(21), 7033 - 44
Family of glycosyl transferases needed for the synthesis of succinoglycan by Rhizobium meliloti; Glucksmann MA et al.; Rhizobium meliloti produces an acidic exopolysaccharide, termed succinoglycan or EPS I, that is important for invasion of the nodules that it elicits on its host, Medicago sativa . Succinoglycan is a high-molecular-weight polymer composed of repeating octasaccharide subunits . These subunits are synthesized on membrane-bound isoprenoid lipid carriers, beginning with a galactose residue followed by seven glucose residues, and modified by the addition of acetate, succinate, and pyruvate . Biochemical characterizations of lipid-linked succinoglycan biosynthetic intermediates from previously identified exo mutant strains have been carried out in our laboratory (T . L . Reuber and G . C . Walker, Cell 74:269-280, 1993) to determine where each mutation blocks the biosynthetic pathway . We have carried out a fine structure genetic analysis of a portion of the cluster of exo genes present on the second symbiotic megaplasmid of R . meliloti and have identified several new genes . In addition, the DNA sequence of 16 kb of the exo cluster was determined and the genetic map was correlated with the DNA sequence . In this paper we present the sequence of a family of glycosyl transferases required for the synthesis of succinoglycan and discuss their functions.

J Bacteriol, 1993 Nov, 175(21), 6867 - 72
Oxygen-regulated in vitro transcription of Rhizobium meliloti nifA and fixK genes; Reyrat JM et al.; Oxygen concentration regulates the expression of nitrogen fixation genes in the symbiotic bacterium Rhizobium meliloti . We demonstrate that two proteins, FixL and FixJ, that belong to the two-component family of regulatory proteins are necessary and sufficient for oxygen-regulated in vitro transcription of the two key regulatory genes, nifA and fixK . We show directly that FixJ is a transcriptional activator, working in conjunction with the RNA polymerase sigma 70 holoenzyme . Addition of FixL122, a soluble form of the sensor FixL protein, to the transcription assay enhanced FixJ transcriptional activity in response to low oxygen concentration . This enhancement of FixJ activity was correlated with FixJ phosphorylation.

Mol Plant Microbe Interact, 1993 Nov-Dec, 6(6), 764 - 74
Biological activity of Rhizobium sp . NGR234 Nod-factors on Macroptilium atropurpureum; Relic B et al.; The broad host range of Rhizobium sp . NGR234 is based mainly on its ability to secrete a family of lipooligosaccharide Nod factors . To monitor Nod-factor purification, we used the small seeded legume Macroptilium atropurpureum, which responds evenly and consistently to Nod factors . At concentrations between approximately equal to 10(-11) M and 10(-9) M, this response takes the form of deformation of the root hairs . Higher concentrations (approximately equal to 10(-9) to 10(-7) M), provoked profound "shepherd's crook" type curling of the root hairs . Similar concentrations of Nod factors of Bradyrhizobium japonicum, Rhizobium leguminosarum, and R . meliloti also provoked marked curling of the root hairs, but the latter two species are unable to nodulate Macroptilium . On the other hand, plant hormones, hormone-like substances, inhibitors of hormone action, as well as substituents of Nod factors were without effect in this bioassay . We thus conclude that only Nod factors are capable of inducing shepherd's crook type curling of Macroptilium root hairs . Perturbations in the auxin-cytokinin balance induced "pseudo" nodulation on M . atropurpureum, as did NodNGR factors at concentrations between 10(-7) and 10(-6) M . Concomitant inoculation of Macroptilium with a NodABC- mutant of NGR234 and sulfated NodNGR factors (NodNGR{S}) gave rise to plants that slowly greened, showing that the NodNGR factors permitted entry of the Nod- mutant into the roots.

Mol Plant Microbe Interact, 1993 Nov-Dec, 6(6), 735 - 44
Analysis of the Rhizobium meliloti genes exoU, exoV, exoW, exoT, and exoI involved in exopolysaccharide biosynthesis and nodule invasion: exoU and exoW probably encode glucosyltransferases; Becker A et al.; Sequence analysis of a 5.780-kb DNA fragment originating from megaplasmid 2 of Rhizobium meliloti 2011 involved in biosynthesis of exopolysaccharide I (EPS I) and invasion of alfalfa nodules revealed the presence of five exo genes designated exoU, exoV, exoW, exoT, and exoI . ExoT resembled transmembrane proteins, whereas ExoI displayed a characteristic signal peptide . Sequence comparisons with several polysaccharide-polymerizing enzymes of both prokaryotic and eukaryotic origin indicated that exoW and exoU encode glucosyltransferases . Moreover, ExoV displayed weak homologies to the ExoO, ExoA, ExoL, and ExoM proteins of R . meliloti, which are also discussed as glucosyltransferases . Using exo-lacZ transcription fusions in connection with plasmid integration mutagenesis, promoters were identified in front of exoI, exoT, exoW, exoV, and exoU.R . meliloti 2011 strains with mutations in exoT, exoW, exoV, and exoU produced no detectable EPS I and were unable to infect alfalfa nodules, whereas exoI mutants synthesized a reduced amount of EPS I and did infect alfalfa nodules.

Mol Plant Microbe Interact, 1993 Nov-Dec, 6(6), 715 - 21
ENOD8, a novel early nodule-specific gene, is expressed in empty alfalfa nodules; Dickstein R et al.; The alfalfa ENOD8 nodule-specific gene is expressed in empty nodules elicited by exopolysaccharide-deficient Rhizobium meliloti, and it is expressed early in nodule development . An ENOD8 cDNA was sequenced and found to encode a novel product . Its deduced polypeptide sequence was found to be similar to the nonproline-rich domains of the putative polypeptides encoded by a class of anther-specific genes from Arabidopsis thaliana and Brassica napus . The role of the ENOD8 gene product is predicted to be in nodule organogenesis . ENOD8 is expressed in a developmental pathway triggered as a result of Rhizobium nodulation signal transduction.

J Mol Biol, 1993 Oct 5, 233(3), 336 - 48
Interactions of NodD at the nod Box: NodD binds to two distinct sites on the same face of the helix and induces a bend in the DNA; Fisher RF et al.; The Rhizobium meliloti nodD gene products are positive transcriptional activators of genes required for early stages of nodule morphogenesis in the R . meliloti-alfalfa symbiosis (nod genes) . The regulatory activity of NodD, a member of the LysR family of activator proteins, is mediated in part through its binding to conserved DNA sequences termed nod boxes which lie upstream of the inducible nod genes . Here we use interference footprinting to identify two NodD binding sites in the nodA, nodF and nodH nod boxes . These two binding sites are located on the same face of the DNA helix and can be separated by an additional 10 bp with retention of activity . By systematic alteration of the phasing of the two binding sites on the DNA helix, we showed that only constructs which contain both sites on the same side of the helix are recognized by NodD as determined by migration retardation assay and by in vivo activation of nod box-lacZ fusions . Moreover, NodD apparently induces a bend in the DNA upon binding at the nod box as shown by migration retardation behavior of circularly permuted nod box fragments.

Biochemistry, 1993 Oct 5, 32(39), 10430 - 5
Nodulation factors from Rhizobium tropici are sulfated or nonsulfated chitopentasaccharides containing an N-methyl-N-acylglucosaminyl terminus; Poupot R et al.; Phaseolus vulgaris (common bean) can be nodulated by several Rhizobium species . Among them, Rhizobium tropici has a relatively broad host range, as it is able to infect beans, Leucaena trees, and several other legumes . This work describes the isolation and the characterization of extracellular factors (Nod factors) whose production from R . tropici was triggered by the transcriptional activation of its nod genes . These factors consist of a chitopentaose backbone in which the N-acetyl group of the nonreducing end glucosaminyl residue is replaced by an N-methyl-N-vaccenoyl one . Some of these molecules are sulfated on position 6 of the terminal reducing glucosamine.

Plant J, 1993 Oct, 4(4), 727 - 33
Lipo-oligosaccharides of Rhizobium induce infection-related early nodulin gene expression in pea root hairs; Horvath B et al.; This paper shows that lipo-oligosaccharides (Nod factors) synthesized by Rhizobium bacteria elicit the induction of infection-related early nodulin genes (PsENOD5 and PsENOD12) in pea root hairs . R . leguminosarum bv . viciae secretes a mixture of Nod factors containing a C18 fatty acid chain with 4 (C18:4) or 1 double bond (C18:1) . Purified Nod factors harbouring either a C18:4 or a C18:1 acyl moiety induce the expression of the pea early nodulin genes, PsENOD5 and PsENOD12, but the kinetics of induction are different . The expression of both early nodulin genes is induced in a transient manner by the purified Nod factors while a mixture of the Nod factors extends the period during which these genes are expressed . In spite of the host-specific nature of the infection process, heterologous Nod factors of R . meliloti also induce the expression of PsENOD5 and PsENOD12 genes, though with a marked delay compared with the homologous compounds.

Int J Syst Bacteriol, 1993 Oct, 43(4), 761 - 7
Genomic heterogeneity among French Rhizobium strains isolated from Phaseolus vulgaris L; Laguerre G et al.; Levels of DNA relatedness between strains isolated from root nodules of Phaseolus vulgaris and reference strains of different Rhizobium species were determined by performing DNA-DNA hybridization experiments (S1 nuclease method) . The nine strains examined were members of three genomic groups previously delineated by a restriction fragment length polymorphism analysis among strains isolated from P . vulgaris at different sites in France . In agreement with the results of the restriction fragment length polymorphism analysis, three genomic species were found . We confirmed that one of these species corresponded to Rhizobium leguminosarum since the strain examined was 100% related to the type strain of this species . The other two species were new genomic species which were less than 21% related to reference strains belonging to other Rhizobium species, including Rhizobium etli and Rhizobium tropici, and were 18% related to each other . As determined by an analysis of partial 16S ribosomal DNA sequences, each of the genomic species was found to belong to a lineage independent from the lineages of previously described Rhizobium species . Nevertheless, they were included in the group formed by the fast-growing Rhizobium species . Both genomic species 1 and genomic species 2 contained a majority of strains which were capable of nodulating both P . vulgaris and Leucaena leucocephala, like R . tropici . However, they also contained strains with a nodulation phenotype restricted to P . vulgaris, like R . leguminosarum bv . phaseoli and R . etli bv . phaseoli . Our data are the first evidence that in Europe species other than R . leguminosarum nodulate P . vulgaris.

Mol Gen Genet, 1993 Oct, 241(1-2), 106 - 14
The membrane topology of the Rhizobium meliloti C4-dicarboxylate permease (DctA) as derived from protein fusions with Escherichia coli K12 alkaline phosphatase (PhoA) and beta-galactosidase (LacZ); Jording D et al.; The Rhizobium meliloti dctA gene encodes the C4-dicarboxylate permease which mediates uptake of C4-dicarboxylates, both in free-living and symbiotic cells . Based on the hydrophobicity of the DctA protein, 12 putative membrane spanning regions were predicted . The membrane topology was further analysed by isolating in vivo fusions of DctA to Escherichia coli alkaline phosphatase (PhoA) and E . coli beta-galactosidase (LacZ) . Of 10 different fusions 7 indicated a periplasmic and 3 a cytoplasmic location of the corresponding region of the DctA protein . From these data a two-dimensional model of DctA was constructed which comprised twelve transmembrane alpha-helices with the amino-terminus and the carboxy-terminus located in the cytoplasm . In addition, four conserved amino acid motifs present in many eukaryotic and prokaryotic transport proteins were observed.

Mol Microbiol, 1993 Oct, 10(2), 351 - 60
Resistance to nodulation of cv . Afghanistan peas is overcome by nodX, which mediates an O-acetylation of the Rhizobium leguminosarum lipo-oligosaccharide nodulation factor; Firmin JL et al.; Only some strains of Rhizobium leguminosarum biovar viciae can efficiently nodulate varieties of peas such as cv . Afghanistan, which carry a recessive allele that blocks efficient nodulation by most western isolates of R.I . viciae . One strain (TOM) which can nodulate cv . Afghanistan peas has a gene (nodX) that is required to overcome the nodulation resistance . Strain TOM makes significantly lower amounts of lipo-oligosaccharide nodulation factors than other strains of R.I . viciae and this effect appears to be due to lower levels of nod gene induction . These nodulation factors are similar to those from other R.I . viciae strains in that they consist of an oligomer of four or five beta 1-4-linked N-acetylglucosamine residues in which the terminal non-reducing glucosamine carries an O-acetyl group and a C18:4 or C18:1 N-acyl group . However, one of the nodulation factors made by strain TOM differs from the factors made by other strains of R.I . viciae in that it carries an O-acetyl group on the C-6 of the reducing N-acetylglucosamine residue . This acetylation is NodX-dependent and the pentameric nodulation factor is acetylated on the reducing N-acetylglucosamine residue whereas the tetrameric nodulation factor is not . Although the nodL gene product is also an O-acetyl transferase (it O-acetylates the C-6 of the terminal non-reducing glucosamine), there is very little similarity between the amino acid sequences of these two acetyl transferases.

Mol Microbiol, 1993 Oct, 10(2), 329 - 40
Homology between a genetic locus (mdoA) involved in the osmoregulated biosynthesis of periplasmic glucans in Escherichia coli and a genetic locus (hrpM) controlling pathogenicity of Pseudomonas syringae; Loubens I et al.; Membrane-derived oligosaccharides (MDO) of Escherichia coli are representative members of a family of glucans found in the periplasmic space of Gram-negative bacteria . The two genes forming the mdoGH operon are necessary for the synthesis of MDO . The nucleotide sequence (4759 bp) and the transcriptional start of this operon were determined . Both gene products were further characterized by gene fusion analysis . MdoG is a 56 kDa periplasmic protein whose function remains to be determined . MdoH, whose presence was shown to be necessary for normal glucosyl transferase activity, is a 97 kDa protein spanning the cytoplasmic membrane . To our surprise, these proteins are not homologous to the periplasmic glucan biosynthetic enzymes previously characterized in the Rhizobiaceae family . However, a considerable homology (69% identical nucleotides out of 2816) was discovered between mdoGH and the two genes present at the hrpM locus of the phytopathogenic bacterium Pseudomonas syringae pv . syringae . Functions of these genes remain mysterious but they are known to be required for both the expression of disease symptoms on host plants and the development of the hypersensitive reaction on non-host plants (Mills and Mukhopadhyay, 1990) . These results confirm the importance of periplasmic glucans for the physiological ecology of Gram-negative bacteria.

J Biol Chem, 1993 Sep 25, 268(27), 20134 - 42
Role of the Rhizobium meliloti nodF and nodE genes in the biosynthesis of lipo-oligosaccharidic nodulation factors; Demont N et al.; Rhizobia nodulation (nod) genes are involved in the synthesis of symbiotic signals, the Nod factors, which are mono-N-acylated chito-oligosaccharides . Nod factors elicit, in a specific manner, various plant responses on legume roots . In this report we address the question of the role of nodFEG genes in the synthesis of the acyl moiety of Rhizobium meliloti Nod factors . In a Nod factor-overproducing strain with the wild-type nod region, in addition to the delta 2,9-C16:2 and delta 2, 4,9-C16:3 acyl groups already described, delta 9-C16:1 was also found, together with a series of C18 to C26 (omega-1)-hydroxylated fatty acids . A deletion of nodE resulted in the absence of C16:2 and C16:3 fatty acids, which were replaced by vaccenic acid (delta 11-C18:1), but did not change the proportion of (omega-1)-hydroxylated fatty acids . A nodF deletion, non-polar with respect to nodE, resulted in the same alterations in the Nod factor N-acyl composition, showing that both nodF and nodE are required for the synthesis of the C16 polyunsaturated chains . In contrast, nodG mutations did not result in a detectable change in the Nod factor N-acyl moiety . When a plasmid carrying the nodFE genes of Rhizobium leguminosarum bv . viciae was introduced into R . meliloti nodFE- and nodFEG-deleted strains, Nod factors with polyunsaturated C18 fatty acids (C18:2, C18:3, and C18:4) could be detected . These results provide evidence that the molecular basis of allelic variation between the R . meliloti and R . leguminosarum bv . viciae host range nodFE genes lies in the fact that the two nodFE alleles specify the synthesis of unsaturated fatty acid substituents with a different carbon length.

Biochim Biophys Acta, 1993 Sep 13, 1144(2), 232 - 3
Cloning and sequence of the Rhizobium leguminosarum biovar phaseoli fixA gene; Michiels J et al.; We report the identification and cloning of Rhizobium leguminosarum biovar phaseoli fixABCX homologous genes and the complete nucleotide sequence of the fixA gene . The corresponding gene product is highly homologous to the Rhizobium meliloti and Azorhizobium caulinodans FixA proteins . Putative NtrA- and NifA-binding sites are identified in the fixA promoter region.

FEBS Lett, 1993 Sep 6, 330(1), 95 - 8
Uridylylation of the PII protein in Rhizobium leguminosarum; Colonna-Romano S et al.; Permeabilization with cetyl trimethyl ammonium bromide was used to study the post-translational modification of the PII protein in Rhizobium leguminosarum . Upon incubation with radioactive UTP a single band was obtained after SDS-PAGE and autoradiography . RNase resistance and snake venom phosphodiesterase sensitivity showed that radioactivity was bound through a phosphodiester bond to a protein which was absorbed by an antiserum specific for the PII protein . Uridylylation of the PII protein was shown to be dependent on the modifications of the glutamine/alpha-ketoglutarate ratio.

Mol Gen Genet, 1993 Sep, 240(3), 435 - 44
Genes at different regulatory levels are required for the ammonia control of nodulation in Rhizobium meliloti; Dusha I et al.; The expression of the nodulation genes nodABC of Rhizobium meliloti, which determine early response functions to plant host signals, is regulated by the level of ammonia, the primary product of symbiotic nitrogen fixation . We show that the pathway that links the ammonia-induced signal to the transcriptional control of the nodABC genes involves at least two regulatory levels . The fluctuating nitrogen level is sensed and the signal is mediated by the members of the general nitrogen regulatory (ntr) system, then transmitted to the syrM-nodD3 genes representing the nod-specific level of ammonia regulation . At low ammonia concentration, the activator protein NtrC exerts its effect via nodD3 . In conditions of nitrogen excess ntrR, involved in the repression of nod genes, may function in coordination with the syrM gene . Finally, the NodD3 protein may relay the nitrogen status signal to the transcriptional control of the nodABC genes.

Proc Natl Acad Sci U S A, 1993 Sep 1, 90(17), 8005 - 8
The primary structure of a fungal chitin deacetylase reveals the function for two bacterial gene products; Kafetzopoulos D et al.; Chitin deacetylase (EC 3.5.1.41) hydrolyzes the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin . A cDNA to the Mucor rouxii mRNA encoding chitin deacetylase was isolated, characterized, and sequenced . Protein sequence comparisons revealed significant similarities of the fungal chitin deacetylase to rhizobial nodB proteins and to an uncharacterized protein encoded by a Bacillus stearothermophilus open reading frame . These data suggest the functional homology of these evolutionarily distant proteins . NodB is a chitooligosaccharide deacetylase essential for the biosynthesis of the bacterial nodulation signals, termed Nod factors . The observed similarity of chitin deacetylase to the B . stearothermophilus gene product suggests that this gene encodes a polysaccharide deacetylase.

Plant Physiol, 1993 Sep, 103(1), 21 - 30
A gene that encodes a proline-rich nodulin with limited homology to PsENOD12 is expressed in the invasion zone of Rhizobium meliloti-induced alfalfa root nodules; Lobler M et al.; To define the early stages of the interaction between Rhizobium and host legumes, we have cloned and characterized three early nodulin-encoding sequences from an alfalfa (Medicago sativa L.) cDNA library by probing with a fragment of a cDNA clone for PsENOD12, an infection-related nodulin from pea (Pisum sativum L.) . Although the coding regions of the three clones are 95 to 98% homologous to each other, they are only 43% homologous to the pea clone . However, the putative signal peptide encoded by the alfalfa cDNA clones is 100% homologous to the PsENDO12 signal peptide . The spatial and temporal expression patterns of PsENOD12 and the alfalfa clones were compared . In situ hybridization experiments detected RNA transcripts in the invasion zone of mature nitrogen-fixing nodules, the same site where PsENOD12 mRNAs are found . Transcripts were also found by in situ hybridization in cells of Rhizobium meliloti exoH mutant-induced nodules penetrated by infection threads, but northern analysis did not detect transcripts in inf- (infection thread minus) nodules elicited by R . meliloti exoB nodules or in pseudonodules elicited by treatment with the auxin transport inhibitor N-1-(naphthyl)phthalamic acid . In addition, the alfalfa gene represented by these cDNA clones exhibited a temporal expression pattern that differed from that of PsENOD12, which is transiently expressed . These data, plus information derived from Southern blot analysis, indicate that we have isolated cDNA clones for a novel early nodulin, which we have designated MsENOD10 (Medicago sativa Early Nodulin 10).

Mol Microbiol, 1993 Sep, 9(6), 1223 - 7
The ORF1 of the gentamicin-resistance operon (aac) of Pseudomonas aeruginosa encodes adenosine 5'-phosphosulphate kinase; Satishchandran C et al.; The gentamicin-resistance operon of Pseudomonas aeruginosa (aac) contains two cistrons for which only the second gene product has an identified function . The 813bp second cistron (ORF2) encodes a protein that confers gentamicin resistance by catalysis of the transfer of an acetyl group from acetyl Coenzyme A to gentamicin . The first open reading frame (ORF1) encodes a 23.9 kDa protein that we have found, by enzyme activity and immunological reactivity, to be adenosine-5'-phosphosulphate (APS) kinase . APS kinase catalyses the transfer of the gamma phosphoryl group of ATP to the 3'-hydroxyl group of APS . The 70% sequence similarity between the Pseudomonas and Escherichia coli APS kinases suggests that the Pseudomonas enzyme may catalyse phosphoryl transfer to the 3'-hydroxyl group of other nucleotides such as dephosphocoenzyme A, as does the purified E . coli APS kinase . In extracts of pseudomonad cells we have also detected a higher molecular mass (70kDa) protein that cross-reacts with an anti-E . coli APS kinase antibody . This cross-reactive protein is also present in Pseudomonas strains lacking the gentamicin-resistance plasmid, and apparently reflects an APS kinase analogous to the nodQ-encoded high-molecular-weight APS kinase present in Rhizobium meliloti . Production of the Pseudomonas aac APS kinase was repressed by cysteine when expressed in E . coli, as is E . coli APS kinase . However, cysteine did not repress production of the Pseudomonas enzyme when the aac ORF1-encoded enzyme was expressed in a Pseudomonas strain, indicating differential regulation of gene expression in the two organisms.

Mol Microbiol, 1993 Sep, 9(6), 1157 - 68
A Rhizobium tropici DNA region carrying the amino-terminal half of a nodD gene and a nod-box-like sequence confers host-range extension; Sousa C et al.; Rhizobium tropici CIAT899 is a broad-host-range strain that, in addition to Phaseolus, nodulates other plant legumes such as Leucaena and Macroptilium . The narrow-host-range of Rhizobium leguminosarum biovars phaseoli (strain CE3) and trifolii (strain RS1051) can be extended to Leucaena esculenta and Phaseolus vulgaris plants, respectively, by the introduction of a DNA fragment 521 bp long, which carries 128 amino acids of the amino-terminal region of a nodD gene from R . tropici, as well as a putative nod-box-like sequence, divergently oriented . The 521 bp fragment, in the presence of L . esculenta or P . vulgaris root exudates, induced a R . leguminosarum bv . viciae nodA-lacZ fusion in either a CE3 or RS1051 background, respectively.

J Biol Chem, 1993 Aug 5, 268(22), 16293 - 7
Regulation of the kinase activity of heme protein FixL from the two-component system FixL/FixJ of Rhizobium meliloti; Gilles-Gonzalez MA et al.; The Rhizobium meliloti two-component system FixL/FixJ regulates nitrogen fixation in response to oxygen during symbiosis . FixJ is a transcriptional activator of critical nif and fix promoters; its in vivo activity is enhanced by FixL in diminished oxygen (David, M., Daveran, M.-L., Batut, J., Dedieu, A., Domergue, O., Ghai, J., Hertig, C., Boistard, P., and Kahn, D . (1988) Cell 54, 671-683; Virts, E . L., Stanfield, S . W., Helinski, D . R., and Ditta, G . S . (1988) Proc . Natl . Acad . Sci . U.S.A . 85, 3062-3065) . FixL* is a soluble truncated version of FixL that contains heme; it catalyzes its autophosphorylation and the phosphorylation of FixJ (Gilles-Gonzalez, M.A., Ditta, G . S., and Helinski, D . R . (1991) Nature 350, 170-172) . We examine the kinetics of phosphoryl transfer in this system . First, there is a slow autophosphorylation of FixL* in ATP that is accelerated in the absence of oxygen and in the presence of Mn2+ . This reaction is reversible, i.e . phospho-FixL* reacts with ADP to generate ATP . Since the reverse reaction is faster, most FixL* is not phosphorylated at equilibrium . Next, there is a rapid phosphoryl transfer directly from phospho-FixL* to FixJ that is unaffected by oxygen . Finally, phospho-FixJ is hydrolyzed; this reaction is very fast and not controlled by oxygen . We propose that in addition to the oxygen signal previously noted in vivo, energy charge and manganese concentration are also indicators of symbiosis that impact on the induction of nitrogen fixation genes.

Mol Microbiol, 1993 Aug, 9(3), 569 - 77
The ntrBC genes of Rhizobium leguminosarum are part of a complex operon subject to negative regulation; Patriarca EJ et al.; We report here that ntrB and ntrC genes of Rhizobium leguminosarum biovar phaseoli are cotranscribed with an open reading frame (called ORF1) of unknown function . The promoter region of the ORF1-ntrB-ntrC operon was mapped immediately upstream of ORF1 and two in vivo transcription initiation sites were identified, both preceded by -35/-10 promoter consensus sequences . Some major aspects differentiate R . leguminosarum from the enteric nitrogen regulatory system: the ntrBC genes are cotranscribed with ORF1 which is homologous to an ORF located upstream of ntrBC of R . capsulatus and to the ORF1 located upstream of the fis gene of Escherichia coli; ntrBC are not transcribed from a -24/-12 promoter and are only autogenously repressed . Moreover, the intracellular concentration of the NtrC protein increases when the bacterium is grown on ammonium salts, while under the same conditions the promoter of one of its target genes, glnII, is 12 times less active.

Genes Dev, 1993 Aug, 7(8), 1485 - 97
A Rhizobium meliloti homolog of the Escherichia coli peptide-antibiotic transport protein SbmA is essential for bacteroid development; Glazebrook J et al.; Alfalfa nodules induced by a Rhizobium meliloti strain carrying the bacA386::TnphoA mutation (formerly fix386::TnphoA) were examined by light and electron microscopy . These ineffective nodules were found to contain bacteria within infection threads, but no mature bacteroids were observed . A closer examination revealed that there were undeveloped senescent bacteroids in the plant cells of the nodule invasion zone, strongly suggesting that the symbiotic defect of the bacA386::TnphoA mutant is attributable to an early block in bacteroid development . The expression of the bacA gene in effective nodules was monitored with a bacA-phoA fusion and found to be strongest in the region where developing bacteroids are found . The bacA+ gene was cloned and sequenced . Sequence analysis indicated that BacA is probably an integral inner membrane protein with seven transmembrane domains and that it is extremely homologous to Escherichia coli SbmA, an inner membrane protein required for the uptake of microcin B17, a peptide antibiotic . Southern blotting experiments indicate that a gene closely related to bacA/sbmA is found in many bacteria, including some that invade eukaryotic cells . Possible roles for BacA in symbiosis are discussed.

J Bacteriol, 1993 Aug, 175(16), 5205 - 15
Characterization of genes for synthesis and catabolism of a new rhizopine induced in nodules by Rhizobium meliloti Rm220-3: extension of the rhizopine concept; Saint CP et al.; Rhizopines are selective growth substrates synthesized in nodules only by strains of rhizobia capable of their catabolism . We report the isolation and study of genes for the synthesis and catabolism of a new rhizopine, scyllo-inosamine (sIa), from alfalfa nodules induced by Rhizobium meliloti Rm220-3 . This compound is similar in structure to the previously described rhizopine 3-O-methyl-scyllo-inosamine from R . meliloti L5-30 (P.J . Murphy, N . Heycke, Z . Banfalvi, M.E . Tate, F.J . de Bruijn, A . Kondorosi, J . Tempe, and J . Schell, Proc . Natl . Acad . Sci . USA 84:493-497, 1987) . The synthesis (mos) and catabolism (moc) genes for the Rm220-3 rhizopine are closely linked and located on the nod-nif Sym plasmid . The mos genes are directly controlled by the NifA/NtrA regulatory system . A comparison of the sequence of the 5' regions of the two mos loci shows very extensive conservation of sequence as well as strong homology to the nifH coding region . Restriction mapping and hybridization to DNA from the four open reading frames (ORFs) of the L5-30 mos locus indicate the absence of mosA and presence of the other three ORFs (ORF1 and mosB and -C) in Rm220-3 . We suggest that the L5-30 mosA gene product is involved in the conversion of scyllo-inosamine to 3-O-methyl-scyllo-inosamine . Restriction fragment length polymorphism analysis of the moc regions of both strains shows that they are very similar . Regulation studies indicate that the moc region is not controlled by the common regulatory gene nifA, ntrA, and ntrC . We discuss the striking similarities in gene structure, location, and regulation between these two rhizopine loci in relation to the rhizopine concept.

J Bacteriol, 1993 Aug, 175(16), 5193 - 204
The Rhizobium meliloti rhizopine mos locus is a mosaic structure facilitating its symbiotic regulation; Murphy PJ et al.; The Rhizobium meliloti L5-30 mos locus, encoding biosynthesis of the rhizopine 3-O-methyl-scyllo-inosamine, is shown to be a mosaic structure . The mos locus consists of four open reading frames (ORFs) (ORF1 and mosABC) arranged in an operon structure . Within this locus, several domains of homology with other prokaryotic symbiotic genes (nifH, fixA, fixU, and nifT) are present, suggesting that this locus may represent a hot spot for rearrangement of symbiotic genes . Unusually, these domains are present in the coding as well as noncoding regions of the mos locus . Proteins corresponding to those encoded by mosABC, but not ORF1, have been detected in nodule extracts by using antibodies . As ORF1 shows extensive homology with the 5' region of the nifH gene (P.J . Murphy, N . Heycke, S.P . Trenz, P . Ratet, F.J . de Bruijn, and J . Schell, Proc . Natl . Acad . Sci . USA 85:9133-9137, 1988) and a frameshift mutation indicates that expression of this ORF is not required for mos activity, we propose that the mos locus has acquired a duplicated copy of nifH, including the promoter region, in order to become symbiotically regulated . Surprisingly, since the functions are likely different, MosA has an amino acid sequence similar to that of the DapA protein of Escherichia coli . The central domain of MosB has extensive homology with a range of diverse proteins involved with carbohydrate metabolism in either antibiotic or outer-cell-wall biosynthesis . This region is also common to the regulatory proteins DegT and DnrJ, suggesting a regulatory role for MosB . The structure of MosC is consistent with its being a membrane transport protein.

J Bacteriol, 1993 Aug, 175(15), 4922 - 6
Methoxylated fatty acids reported in Rhizobium isolates arise from chemical alterations of common fatty acids upon acid-catalyzed transesterification procedures; Orgambide GG et al.; We obtained from a phospholipid extract of wild-type Rhizobium leguminosarum bv . trifolii ANU843 methoxylated fatty acids that had been previously reported as constitutive unusual Rhizobium fatty acids . The use of deuterated reagents and subsequent gas-liquid chromatography-mass spectrometry analyses showed that these methoxylated fatty acid derivatives are the products of chemical alterations of common cyclopropane-containing and unsaturated fatty acids occurring during various acid-catalyzed transesterification treatments aimed at producing the methyl ester derivatives . Similar results were obtained from a phospholipid extract of Escherichia coli K-12 . In contrast, these chemical alterations were not induced by an alkaline methanolysis method of transesterification . If an acidic treatment is needed to release the fatty acids from the source molecule, the finding of unusual methoxylated fatty acids should be carefully confirmed with deuterated reagents.

Plant Physiol, 1993 Aug, 102(4), 1095 - 107
Interaction of a rhizobial DNA-binding protein with the promoter region of a plant leghemoglobin gene; Welters P et al.; A nucleotide sequence was identified approximately 650 bp upstream of the Sesbania rostrata leghemoglobin gene Srglb3 start codon, which interacts specifically with a proteinaceous DNA-binding factor found in nodule extracts but not in extracts from leaves or roots . The binding site for this factor was delimited using footprinting techniques . The DNA-binding activity of this factor was found to be heat stable, dependent on divalent cations, and derived from the (infecting) Azorhizobium caulinodans bacteria or bacteroids (A . caulinodans bacterial binding factor 1, AcBBF1) . A 9- to 10-kD protein was isolated from a free-living culture of A . caulinodans that co-purifies with the DNA-binding activity (A . caulinodans bacterial binding protein 1, AcBBP1) and interacts specifically with its target (S . rostrata bacterial binding site 1, SrBBS1) . The amino acid sequence of the N-terminal 27 residues of AcBBP1 was determined and was found to share significant similarity (46% identity; 68% similarity) with a domain of the herpes simplex virus major DNA-binding protein infected cell protein 8 (ICP8) . An insertion mutation in the SrBBS1 was found to result in a substantial reduction of the expression of a Srglb3-gus reporter gene fusion in nodules of transgenic Lotus corniculatus plants, suggesting a role for this element in Srglb3 promoter activity . Based on these results, we propose that (a) bacterial transacting factor(s) may play a role in infected cell-specific expression of the symbiotically induced plant lb genes.

Mol Microbiol, 1993 Aug, 9(4), 869 - 79
Sequence and molecular analysis of the nifL gene of Azotobacter vinelandii; Blanco G et al.; In both Klebsiella pneumoniae and Azotobacter vinelandii the nifL gene, which encodes a negative regulator of nitrogen fixation, lies immediately upstream of nifA . We have sequenced the A . vinelandii nifL gene and found that it is more homologous in its C-terminal domain to the histidine protein kinases (HPKs) than is K . pneumoniae NifL . In particular A . vinelandii NifL contains a conserved histidine at a position shown to be phosphorylated in other systems . Both NifL proteins are homologous in their N-termini to a part of the Halobacterium halobium bat gene product; Bat is involved in regulation of bacterio-opsin, the expression of which is oxygen sensitive . The same region showed homology to the haem-binding N-terminal domain of the Rhizobium meliloti fixL gene product, an oxygen-sensing protein . Like K . pneumoniae NifL, A . vinelandii NifL is shown here to prevent expression of nif genes in the presence of NH+4 or oxygen . The sequences found homologous in the C-terminal regions of NifL, FixL and Bat might therefore be involved in oxygen binding or sensing . An in-frame deletion mutation in the nifL coding region resulted in loss of repression by NH+4 and the mutant excreted high amounts of ammonia during nitrogen fixation, thus confirming a phenotype reported earlier for an insertion mutation . In addition, nifLA are cotranscribed in A . vinelandii as in K . pneumoniae, but expression from the A . vinelandii promoter requires neither RpoN nor NtrC.

Mol Microbiol, 1993 Aug, 9(4), 751 - 60
The transcriptional activator HlyU of Vibrio cholerae: nucleotide sequence and role in virulence gene expression; Williams SG et al.; HlyU upregulates expression of the haemolysin, HlyA, of Vibrio cholerae . DNA sequence analysis indicates that HlyU is an 11.9 kDa protein containing a putative helix-turn-helix motif and belonging to a family of small regulatory proteins, including NoIR (Rhizobium meliloti), SmtB (Synechococcus PCC 7942) and ArsR (plasmids R773, Escherichia coli; pI258, Staphylococcus aureus; and pSX267, Staphylococcus xylosus) . An hlyU mutant was constructed by insertional inactivation, and found to be deficient in the production of both the haemolysin and a 28 kDa secreted protein . The mutant was assessed for virulence in the infant mouse cholera model, revealing a 100-fold increase in the LD50 . This suggests that HlyU promotes expression of virulence determinant(s) in vivo.

Mol Gen Genet, 1993 Aug, 240(2), 258 - 64
The bacterial attachment site of the temperate Rhizobium phage 16-3 overlaps the 3' end of a putative proline tRNA gene; Papp I et al.; Bacteriophage 16-3 inserts its genome into the chromosome of Rhizobium meliloti strain 41 (Rm41) by site-specific recombination . The DNA regions around the bacterial attachment site (attB) and one of the hybrid attachment sites bordering the integrated prophage (attL) were cloned and their nucleotide sequences determined . We demonstrated that the 51 bp region, where the phage and bacterial DNA sequences are identical, is active as a target site for phage integration . Furthermore it overlaps the 3' end of a putative proline tRNA gene . This gene shows 79% similarity to the corresponding proline tRNA-like genomic target sequence of certain integrative plasmids in Actinomycetes.

Mol Gen Genet, 1993 Aug, 240(2), 213 - 20
Cloning and characterization of an ftsZ homologue from a bacterial symbiont of Drosophila melanogaster; Holden PR et al.; A 1194 bp open reading frame that codes for a 398 amino acid peptide was cloned from a lambda gt11 library of Drosophila melanogaster genomic DNA . The predicted peptide sequence is very similar to three previously characterized protein sequences that are encoded by the ftsZ genes in Escherichia coli, Bacillus subtilis and Rhizobium meliloti . The FtsZ protein has a major role in the initiation of cell division in prokaryotic cells . Using a tetracycline treatment that eradicates bacterial parasites from insects, the ftsZ homologue has been found to be derived from a bacterium that lives within the D . melanogaster strain . However, polymerase chain reaction (PCR) amplification of the gene from treated embryos suggests that it is not derived from a gut bacterium . Nevertheless, by amplifying and characterizing part of the 16S rRNA from this bacterium we have been able to demonstrate that it is a member of the genus Wolbachia, a parasitic organism that infects, and disturbs the sexual cycle of various strains of Drosophila simulans . We suggest that this ftsZ homologue is implicated in the cell division of Wolbachia, an organism that fails to grow outside the host organism . Sequence and alignment analysis of this ftsZ homologue show the presence of a potential GTP-binding motif indicating that it may function as a GTPase . The consequences of this function particularly with respect to its role in cell division are discussed.

Cell, 1993 Jul 30, 74(2), 269 - 80
Biosynthesis of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti; Reuber TL et al.; The exo genes of Rhizobium meliloti are needed for the synthesis of an acidic exopolysaccharide, succinoglycan . We have assigned biosynthetic roles to the products of the exo genes by characterizing succinoglycan biosynthetic intermediates from exo mutant strains . We propose a model of succinoglycan biosynthesis in which the products of the exoY and exoF genes function in the addition of the first sugar, galactose, to the lipid carrier; the products of the exoA, exoL, exoM, exoO, exoU, and exoW genes function in subsequent sugar additions; and the product of the exoV gene functions in the addition of pyruvate . The products of the exoP, exoQ, and exoT genes are required for polymerization of the octasaccharide subunits or transport of the completed polymer.

Mol Microbiol, 1993 Jul, 9(1), 17 - 29
Molecular cloning and characterization of a sym plasmid locus that regulates cultivar-specific nodulation of soybean by Rhizobium fredii USDA257; Meinhardt LW et al.; Rhizobium fredii strain USDA257 produces nitrogen-fixing nodules on primitive soybean cultivars such as Peking but fails to nodulate agronomically improved cultivars such as McCall . Transposon-mutant 257DH4 has two new phenotypes: it nodulates McCall, and its ability to do so is sensitive to the presence of parental strain USDA257, i.e . it is subject to competitive nodulation blocking . We have isolated a cosmid containing DNA that corresponds to the site of transposon insertion in 257DH4 and have localized Tn5 on an 8.0 kb EcoRI fragment . The 5596 bp DNA sequence that surrounds the insertion site contains seven open reading frames . Five of these, designated nolBTU, ORF4, and nolV, are closely spaced and of the same polarity . nolW and nolX are of the opposite polarity . The initiation codon for nolW lies 155 bp upstream from that of nolB, and its is separated from nolX by 281 bp . The predicted NolT and NolW proteins have putative membrane-spanning regions . The N-terminus of the hypothetical NolW protein also has limited homology to NodH of Rhizobium meliloti, but none of the deduced protein sequences has significant homology to known nodulation gene products . Site-directed mutagenesis with mudII1734 confirms that inactivation of nolB, nolT, nolU, nolV, nolW, or nolX extends host range for nodulation to McCall soybean . This phenotype could not be genetically dissected from sensitivity to competitive nodulation blocking . Expression of nolBTU and nolX is induced as much as 30-fold by flavonoid signal molecules, even though these genes lack nod-box promoters . Histochemical staining of McCall roots inoculated with nolB-, nolU-, or nolX-lacZ fusions verifies that these genes are expressed continuously from preinfection to the stage of the functional nodule . Although a nolU-ORF4-nolV clone hybridizes to a single 8.0 kb EcoRI fragment from 10 strains of R . fredii and broad-host-range Rhizobium sp . NGR234, hybridizing sequences are not detectable in other rhizobia.

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