J Bacteriol, 1997 Sep, 179(17), 5264 - 70 FnrN controls symbiotic nitrogen fixation and hydrogenase activities in Rhizobium leguminosarum biovar viciae UPM791; Gutierrez D et al.; Rhizobium leguminosarum bv . viciae UPM791 contains a second copy of the fnrN gene, which encodes a redox-sensitive transcriptional activator functionally homologous to Escherichia coli Fnr . This second copy (fnrN2) is located in the symbiotic plasmid, while fnrN1 is in the chromosome . Isolation and sequencing of the fnrN2 gene revealed that the deduced amino acid sequence of FnrN2 is 87.5% identical to the sequence of FnrN1, including a conserved cysteine-rich motif characteristic of Fnr-like proteins . Individual R . leguminosarum fnrN1 and fnrN2 mutants exhibited a Fix+ phenotype and near wild-type levels of nitrogenase and hydrogenase activities in pea (Pisum sativum L.) nodules . In contrast, an fnrN1 fnrN2 double mutant formed ineffective nodules lacking both nitrogenase and hydrogenase activities . Unlike the wild-type strain and single fnrN1 or fnrN2 mutants, the fnrN1 fnrN2 double mutant was unable to induce micro-oxic or bacteroid activation of the hypBFCDEX operon, which encodes proteins essential for hydrogenase synthesis . In the search for symbiotic genes that could be controlled by FnrN, a fixNOQP operon, putatively encoding a micro-oxically induced, bacteroid-specific cbb3-type terminal cytochrome oxidase, was isolated from strain UPM791 and partially sequenced . The fixNOQP operon was present in a single copy located in the symbiotic plasmid, and an anaerobox was identified in the fixN promoter region . Consistent with this, a fixNOQP'-lacZ fusion was shown to be highly induced in micro-oxic cells of the wild-type strain . A high level of micro-oxic induction was also observed in single fnrN1 and fnrN2 mutants, but no detectable induction was observed in the fnrN1 fnrN2 double mutant . The lack of expression of fixNOQP in the fnrN1 fnrN2 double mutant is likely to cause the observed Fix- phenotype . These data demonstrate that, contrary to the situation in other rhizobia, FnrN controls both hydrogenase and nitrogenase activities of R . leguminosarum bv . viciae UPM791 in the nodule and suggest that this strain lacks a functional fixK gene. FEBS Lett, 1997 Aug 4, 412(3), 485 - 9 Mechanism of gluconate synthesis in Rhizobium meliloti by using in vivo NMR; Portais JC et al.; The dehydrogenation of {1-(13)C}- and {2-(13)C}glucose into gluconate was monitored by NMR spectroscopy in living cell suspensions of two Rhizobium meliloti strains . The synthesis of gluconate was accompanied, in the cellular environment, by the formation of two gluconolactones, a gamma-lactone being detected in addition to the expected delta-lactone . These lactones--as well as the gluconate--could be further metabolized by the cells . The delta-lactone was utilized faster than the gamma-lactone . The presence--in significant amounts--and the relative stability of the lactones raise the question of their possible physiological significance. Int J Biol Macromol, 1997 Aug, 21(1-2), 3 - 9 Identification of glucuronan lyase from a mutant strain of Rhizobium meliloti; Michaud P et al.; The Rhizobium meliloti M5N1CS (NCIMB 40472) mutant strain wich induces nodule formation on alfalfa roots, produces a (1 --> 4)-beta-D-glucuronan partially acetylated . During fermentation under specific conditions, the molecular weight of the polymer decrease, the presence of polysaccharide degrading enzyme was suspected . A glucuronan lyase was identified, this new bacterial lyase produces d.p . 4 oligoglucuronans, substituents (acetates) present on the substrate reduced the enzyme activity. Microbiology, 1997 Aug, 143 ( Pt 8), 2817 - 24 A new nos gene downstream from nosDFY is essential for dissimilatory reduction of nitrous oxide by Rhizobium (Sinorhizobium) meliloti; Chan YK et al.; Rhizobium (Sinorhizobium) meliloti strains capable of dissimilatory nitrous oxide reduction (Nos+) carry a nosRZDFY gene cluster on a 10.1 kb EcoRI fragment of the nod megaplasmid near the fixGHIS genes . These nos genes are arranged in three complementation groups and the 10.1 kb EcoRI fragment is sufficient to confer Nos activity to R . meliloti strains lacking such activity . An overlapping HindIII fragment containing the nosRZDFY genes but missing a 0-6 kb HindIII-EcoRI downstream segment was found incapable of imparting Nos activity to strains unable to reduce nitrous oxide, suggesting the presence of other nos gene(s) in this region . Tn5 introduced near the HindIII site resulted in mutants with a Nos- phenotype . Complete sequence analysis of nosY showed that it was well-conserved with respect to that of Pseudomonas stutzeri . Two previously unreported genes downstream of nosY in R . meliloti were also revealed . Contiguous with nosY was a sequence showing 63% identity with the ORFL protein of P . stutzeri . It appeared to be in the same operon as nosDFY and was predicted to encode a membrane lipoprotein similar to the putative NosL of P . stutzeri . Unlike the latter protein, however, amino acid sequences typical of metal-binding sites and cysteine residues indicative of the active site of protein disulphide isomerase were absent in the predicted NosL of R . mellioti . The Tn5 mutations resulting in a Nos- phenotype were localized within a 966 nucleotide gene 31 nucleotides downstream of nosDFYL with the same orientation . The new gene, nosX, was determined to be in a separate complementation group . It encoded a periplasmic protein with homology in the C-terminal domain with Rnff of Rhodobacter capsulatus and with a hypothetical Escherichia coli protein, YOJK . It was concluded that there are seven genes constituting the nos cluster in R . meliloti . They are organized in four complementation groups and in the same orientation, spanning a distance of about 9 kb on the nod megaplasmid. J Bacteriol, 1997 Aug, 179(16), 5087 - 93 Rhizobium sp . strain NGR234 NodZ protein is a fucosyltransferase; Quesada-Vincens D et al.; Rhizobium sp . strain NGR234 produces a large family of lipochitooligosaccharide Nod factors carrying specific substituents . Among them are 3-O- (or 4-O-) and 6-O-carbamoyl groups, an N-methyl group, and a 2-O-methylfucose residue which may bear either 3-O-sulfate or 4-O-acetyl substitutions . Investigations on the genetic control of host specificity revealed a number of loci which directly affect Nod factor structure . Here we show that insertion and frameshift mutations in the nodZ gene abolish fucosylation of Nod factors . In vitro assays using GDP-L-fucose as the fucose donor show that fucosyltransferase activity is associated with the nodZ gene product (NodZ) . NodZ is located in the soluble protein fraction of NGR234 cells . Together with extra copies of the nodD1 gene, the nodZ gene and its associated nod box were introduced into ANU265, which is NGR234 cured of the symbiotic plasmid . Crude extracts of this transconjugant possess fucosyltransferase activity . Fusion of a His6 tag to the NodZ protein expressed in Escherichia coli yielded a protein able to fucosylate both nonfucosylated NodNGR factors and oligomers of chitin . NodZ is inactive on monomeric N-acetyl-D-glucosamine and on desulfated Rhizobium meliloti Nod factors . Kinetic analyses showed that the NodZ protein is more active on oligomers of chitin than on nonfucosylated NodNGR factors . Pentameric chitin is the preferred substrate . These data suggest that fucosylation occurs before acylation of the Nod factors. Genetics, 1997 Aug, 146(4), 1211 - 20 Megaplasmid and chromosomal loci for the PHB degradation pathway in Rhizobium (Sinorhizobium) meliloti; Charles TC et al.; Chromosomal and megaplasmid loci that affect the poly-3-hydroxybutyrate (PHB) degradation pathway in Rhizobium meliloti were identified . A clone that restores the ability of certain R . meliloti mutants with defined deletions in megaplasmid pRmeSU47b to use 3-hydroxybutyrate or acetoacetate as the sole carbon source was isolated from a cosmid library of R . meliloti genomic DNA . Tn5 insertion mutagenesis, followed by merodiploid complementation analysis, demonstrated that the locus consists of at least four transcriptional units, bhbA-D . We also identified loci involved in 3-hydroxybutyrate and/or acetoacetate utilization by screening for mutants that had lost the ability to use 3-hydroxybutyrate as the sole carbon source while retaining the ability to use acetate (thus ensuring an intact glyoxylate cycle and gluconeogenic pathway) . These mutants fell into four classes, as determined by replicon mobilization experiments and genetic linkage in phage transduction; one class corresponded to the bhb locus on pRmeSU47b, two classes mapped to different regions on the chromosome and the fourth, bdhA, represented by a single mutant, mapped to another pRmeSU47b locus, near bacA . The bdhA mutant is deficient in 3-hydroxybutrate dehydrogenase activity. FEMS Microbiol Lett, 1997 Aug 1, 153(1), 43 - 9 A helicase gene (helO) in Rhizobium meliloti WSM419; Reeve WG et al.; A 2.8 kb BamHI DNA fragment adjacent to a BamHI fragment containing actR-actS (a sensor/regulator pair required for low pH tolerance in Rhizobium meliloti WSM419) was cloned and sequenced . A computer predicted protein of 821 amino acids, designated HelO, showed extensive similarity with 'DEAH' motif helicases . Expression of helO was higher at pH 7.0 than pH 5.8 and it did not require the product of the actR gene . Inactivation of helO by insertion of a omega interposon at codon 40 did not affect nodulation, growth or tolerance to low pH, high temperature, osmolarity or elevated levels of copper or zinc. Gene, 1997 Jul 18, 194(1), 1 - 8 Deletion of Escherichia coli groEL is complemented by a Rhizobium leguminosarum groEL homologue at 37 degrees C but not at 43 degrees C; Ivic A et al.; Bacterial Cpn60 proteins (homologues to the Escherichia coli GroEL protein) are often examined for function by testing their ability to complement a temperature sensitive mutation in the E . coli groEL gene . Such tests suffer from two drawbacks: the Cpn600 protein may come from a strain with a lower optimum growth temperature than E . coli, and the requirements for successful complementation in E . coli are likely to be more stringent at 43 degrees C than at lower temperatures . Here we describe the construction of a strain of E . coli where the chromosomal gene for the essential molecular chaperone GroEL has been deleted, with GroEL being expressed from a tightly regulated plasmid borne copy of the gene . The deletion can be transduced into strains expressing heterologous Cpn60 proteins, to test for complementation at any temperature . We show that a Cpn60 protein from the bacterium Rhizobium leguminosarum can function to allow E . coli growth at 37 degrees C but not at 43 degrees C . By switching off the plasmid borne groEL gene, the effects of progressive depletion of GroEL protein from E . coli cells can also be monitored at any temperature. Plant Mol Biol, 1997 Jul, 34(5), 771 - 80 Cloning of a WD-repeat-containing gene from alfalfa (Medicago sativa): a role in hormone-mediated cell division? McKhann HI, Frugier F, Petrovics G, de la Pena TC, Jurkevitch E, Brown S, Kondorosi E, Kondorosi A, Crespi M. Rhizobium meliloti can interact symbiotically with Medicago plants thereby inducing the formation of root nodules . Screening of a young nodule cDNA library led to the isolation of a cDNA from Medicago sativa, Msgbl, that comprises a new member of the RACK1 (Receptor of Activated C Kinase) subfamily of WD-repeat proteins . This subfamily shows homology to the beta-subunit of heterotrimeric G proteins . Besides RACK1, this subfamily contains several plant genes including the well characterized auxin-inducible ArcA of tobacco . The Msgbl gene is strongly expressed in young embryos and in leaves, and is induced upon cytokinin treatment of roots . Whereas northern analysis failed to reveal differences in expression between total RNA from roots and nodules, in situ hybridization demonstrated that the transcript was most abundant in dividing cells of nodule primordia and in the nodule meristem . Msgbl may be related to the signal transduction acting in response to hormone-mediated cell division. Microbiology, 1997 Jul, 143 ( Pt 7), 2209 - 21 Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum; Walshaw DL et al.; Mutants of Rhizobium leguminosarum were selected that were altered in the uptake activity of the general amino acid permease (Aap) . The main class of mutant maps to sucA and sucD, which are part of a gene cluster mdh-sucCDAB, which codes for malate dehydrogenase (mdh), succinyl-CoA synthetase (sucCD) and components of the 2-oxoglutarate dehydrogenase complex (sucAB) . Mutation of either sucC or sucD prevents expression of 2-oxoglutarate dehydrogenase (sucAB) . Conversely, mutation of sucA or sucB results in much higher levels of succinyl-CoA synthetase and malate dehydrogenase activity . These results suggest that the genes mdh-sucCDAB may constitute an operon . suc mutants, unlike the wild-type, excrete large quantities of glutamate and 2-oxoglutarate . Concomitant with mutation of sucA or sucD, the intracellular concentration of glutamate but no 2-oxoglutarate was highly elevated, suggesting that 2-oxoglutarate normally feeds into the glutamate pool . Elevation of the intracellular glutamate pool appeared to be coupled to glutamate excretion as part of an overflow pathway for regulation of the TCA cycle . Amino acid uptake via the Aap of R . leguminosarum was strongly inhibited in the suc mutants, even though the transcription level of the aap operon was the same as the wild-type . This is consistent with previous observations that the Aap, which influences glutamate excretion in R . leguminosarum, has uptake inhibited when excretion occurs . Another class of mutant impaired in uptake by the Aap is mutated in polyhydroxybutyrate synthase (phaC) . Mutants of succinyl-CoA synthetase (sucD) or 2-oxoglutarate dehydrogenase (sucA) form ineffective nodules . However, mutants of aap, which are unable to grow on glutamate as a carbon source in laboratory culture, show wild-type levels of nitrogen fixation . This indicates that glutamate is not an important carbon and energy source in the bacteroid . Instead glutamate synthesis, like polyhydroxybutyrate synthesis, appears to be a sink for carbon and reductant, formed when the 2-oxoglutarate dehydrogenase complex is blocked . This is in accord with previous observations that bacteroids synthesize high concentrations of glutamate . Overall the data show that the TCA cycle in R . leguminosarum is regulated by amino acid excretion and polyhydroxybutyrate biosynthesis which act as overflow pathways for excess carbon and reductant. FEMS Microbiol Lett, 1997 Jul 1, 152(1), 57 - 64 The general amino acid permease of Rhizobium leguminosarum strain 3841 is negatively regulated by the Ntr system; Walshaw DL et al.; Cosmid-borne and chromosomal lacZ fusions to aapJ . aapQ and aapM were used to examine the nitrogen regulation of the general amino acid permease (Aap) of Rhizobium leguminosarum strain 3841 . Transcription of the first gene of the operon (aapJ), which encodes the periplasmic binding protein, was 2-4-fold higher than aapQ and aapM, which encode the integral membrane proteins, under various growth conditions . This may be due to the presence of a putative stem loop in the intergenic region between aapJ and aapQ . All aap fusions were derepressed 3-5-fold after growth on glutamate as a nitrogen source, which effectively causes nitrogen limitation . An ntrC mutant was derepressed for transcription of the aap operon and had high rates of amino acid transport when grown on ammonia as the nitrogen source . Thus NtrC negatively regulates the aap operon, contrary to its usual role in positive gene activation . These results confirm that the aap-operon is subject to complex regulation involving both transcriptional and post-transcriptional factors. Int J Syst Bacteriol, 1997 Jul, 47(3), 874 - 9 Phylogenetic and genetic relationships of Mesorhizobium tianshanense and related rhizobia; Tan ZY et al.; The genetic and phylogenetic relationships for strains of Mesorhizobium tianshanense and its relatives were compared by an analysis of the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins, DNA-DNA hybridization, and full 16S rRNA gene sequencing . The strains of M . tianshanense formed a cluster which was distinct from those of other rhizobium species in the clustering analysis of SDS-PAGE . DNA-DNA relatedness between A-1BS (type strain of M . tianshanense) and the type or reference strains for Mesorhizobium loti, M . huakuii, M . ciceri, M . mediterraneum, and cluster U, an unnamed rhizobial group, ranged from 4.4 to 43.8% . The phylogenetic analysis based on the 16S rRNA gene sequences showed that M . tianshanense was closely related to the Mesorhizobium phylogenetic branch and could be distinguished from the other four species in this branch . These results further confirmed that these bacteria constitute a distinct rhizobial species. Int J Syst Bacteriol, 1997 Jul, 47(3), 870 - 3 Rhizobium hainanense sp . nov., isolated from tropical legumes; Chen WX et al.; A fast-growing rhizobial group isolated from leguminous plants in Hainan Province, a tropical region of China, is proposed as a new Rhizobium species on the basis of 16S rRNA gene sequencing . DNA-DNA hybridization, and phenotypic characterization . This new species belongs to the phylogenetic branch which includes Rhizobium leguminosarum . We propose the name Rhizobium hainanense sp . nov . for this species . The strain CCBAU 57015 (166) is the type strain; it has been deposited in the culture collection of Beijing Agricultural University, People's Republic of China. J Bacteriol, 1997 Jul, 179(14), 4545 - 52 The 75-kilodalton antigen of Bartonella bacilliformis is a structural homolog of the cell division protein FtsZ; Padmalayam I et al.; A genomic library of Bartonella bacilliformis was constructed and screened with human anti-Bartonella serum from a patient with the chronic, verruga peruana phase of bartonellosis . An immunoreactive clone isolated from this library was found to code for a 591-amino-acid protein with a high degree of sequence similarity to the FtsZ family of proteins . The degree of amino acid identity between the B . bacilliformis protein (FtsZ{Bb}) and the other FtsZ proteins is especially pronounced over the N-terminal 321 amino acids (N-terminal domain) of the sequence, with values ranging from 45% identity for the homolog from Micrococcus luteus (FtsZ{Ml}) to 91% identity for the homolog from Rhizobium melliloti, (FtsZ{Rm1}) . All of the functional domains required for FtsZ activity are conserved in FtsZ(Bb) and are located within the N-terminal domain of the protein . FtsZ(Bb) is approximately twice as large as most of the other FtsZ proteins previously reported, a property it shares with FtsZ(Rm1) . Like the Rhizobium homolog, FtsZ(Bb) has a C-terminal region of approximately 256 amino acids that is absent in the other FtsZ proteins . Evidence is presented that implicates this region in the protein's antigenicity and suggests that, unlike most other FtsZ homologs, FtsZ(Bb) is at least partly exposed at the cell surface . PCR analysis revealed that an ftsZ gene similar in size to the B . bacilliformis gene is present in Bartonella henselae, a bacterium that is closely related to B . bacilliformis. J Bacteriol, 1997 Jul, 179(13), 4172 - 8 Divergence in nitrogenases of Azoarcus spp., Proteobacteria of the beta subclass; Hurek T et al.; Nitrogenase is a functionally constant protein catalyzing N2 reduction, which is found in many phylogenetic lineages of Archaea and Bacteria . A phylogenetic analysis of nif genes may provide insights into the evolution of the bacterial genomes . Moreover, it may be used to study diazotrophic communities, when classical isolation techniques may fail to detect all contributing populations . Among six species of the genus Azoarcus, diazotrophic Proteobacteria of the beta subclass, the deduced amino acid sequences of nifH genes of two species were unusually divergent from each other . Nitrogenases of the "authentic" Azoarcus branch formed a monophyletic unit with those of gamma Proteobacteria, thus being in accordance with 16S ribosomal DNA phylogeny . The nitrogenase proteins of the two aberrant strains clustered within the alpha proteobacterial clade with rhizobial nitrogenases . This relationship was supported by bootstrap values of 87 to 98% obtained by various distance and maximum parsimony methods . Phylogenetic distances of NifH proteins indicate a possible lateral gene transfer of nif genes to Azoarcus from a common donor of the alpha subclass at the time of species diversification or several more recent, independent transfers . Application of the phylogenetic analysis to DNA isolated from environmental samples demonstrated novel habitats for Azoarcus: in guts of termites and rice grown in Japan, nifH genes belonging to the authentic Azoarcus branch were detected . This is the first evidence suggesting the occurrence of Azoarcus spp . in a plant other than its originally described host, Kallar grass . Moreover, evidence for expression of nif genes inside grass roots was obtained by in situ hybridization studies with antisense nifH probes. Mol Plant Microbe Interact, 1997 Jul, 10(5), 665 - 76 Evolutionary diversity of symbiotically induced nodule MADS box genes: characterization of nmhC5, a member of a novel subfamily; Heard J et al.; Unique organs called nodules form on legume roots in response to intracellular infection by soil bacteria in the genus Rhizobium . This study describes a new MADS box gene, nmhC5, which along with nmh7 (J . Heard and K . Dunn, Proc . Natl . Acad . Sci . USA 92:5273-5277, 1995), is expressed in alfalfa (Medicago sativa) root nodules . Together, these genes represent the first putative transcription factors identified in nodules . Expression in a root-derived structure supports the growing sentiment that MADS box proteins have diverse roles in plant development . Comparison of the putative translation product of nmhC5 with those of other reported members of the MADS box family suggests that the overall structure of nmhC5 is conserved . Evolutionary analysis among the MADS box family showed that nmhC5 is orthologous to a root-expressed clone in Arabidopsis thaliana, agl17, and that nmh7 is similar to the floral subfamily with AP3 (DefA)/PI (Glo) . Consistent with a prediction of homodimer formation, NMHC5 was shown to bind a CArG consensus sequence in vitro . In contrast, NMH7, which shows structural similarity to MADS box proteins that form heterodimers, did not bind the CArG element in an in vitro DNA-binding assay, suggesting the existence of an unknown dimer partner . The root-derived MADS box genes constitute a novel subfamily of vegetatively expressed MADS box genes . The evolutionary diversity between nmh7 and nmhC5 could represent an overall mechanistic conservation between plant developmental processes or could mean that nmh7 and nmhC5 make fundamentally different contributions to the development of the nodule. Mol Plant Microbe Interact, 1997 Jul, 10(5), 605 - 16 Functional and regulatory analysis of the two copies of the fixNOQP operon of Rhizobium leguminosarum strain VF39; Schluter A et al.; DNA corresponding to two copies of the Rhizobium leguminosarum bv . viciae strain VF39 fixNOQP operon coding for a putative symbiotic terminal oxidase of the heme-copper oxidase superfamily was cloned, sequenced, and genetically analyzed . The first copy is located upstream of the fixK-fixL region on plasmid pRleVF39c, whereas the second copy resides on the nodulation plasmid pRleVF39d . Insertional mutagenesis with antibiotic resistance cassettes confirmed that both copies were functional, and that the presence of at least one functional copy was required for nitrogen fixation . The deduced amino acid sequences of both fixN genes are highly similar (95% identity) and contain 15 putative transmembrane helices, suggesting that the fixN gene products are integral membrane proteins . Furthermore, six histidine residues predicted to be the ligands for a heme-copper binuclear center and a low-spin heme b are conserved in both R . leguminosarum fixN proteins . The deduced fixO and fixP gene products show characteristics of membrane-bound monoheme and diheme cytochrome c, respectively . Upstream of both fixN copies putative Fnr-consensus binding sites (anaeroboxes) were found that differ in certain base pairs . As R . leguminosarum VF39 possesses two members of the Fnr/FixK regulator family, FnrN and FixK, the possible differential regulation of both fixN copies was analyzed with fixN-gusA reporter gene fusions . Both fixN fusions were induced under free-living microaerobic conditions and in the symbiotic zone of the root nodule . Induction of the expression of fixNc and fixNd was highly reduced in a fnrN mutant background and in a fixL mutant background, whereas fixK was only marginally involved in fixN regulation . Residual expression of fixN was observed in an fnrN/fixK double mutant. Proc Natl Acad Sci U S A, 1997 Jun 10, 94(12), 6019 - 24 Hydrogenase genes from Rhizobium leguminosarum bv . viciae are controlled by the nitrogen fixation regulatory protein nifA; Brito B et al.; Rhizobium leguminosarum bv . viciae expresses an uptake hydrogenase in symbiosis with peas (Pisum sativum) but, unlike all other characterized hydrogen-oxidizing bacteria, cannot express it in free-living conditions . The hydrogenase-specific transcriptional activator gene hoxA described in other species was shown to have been inactivated in R . leguminosarum by accumulation of frameshift and deletion mutations . Symbiotic transcription of hydrogenase structural genes hupSL originates from a -24/-12 type promoter (hupSp) . A regulatory region located in the -173 to -88 region was essential for promoter activity in R . leguminosarum . Activation of hupSp was observed in Klebsiella pneumoniae and Escherichia coli cells expressing the K . pneumoniae nitrogen fixation regulator NifA, and in E . coli cells expressing R . meliloti NifA . This activation required direct interaction of NifA with the essential -173 to -88 regulatory region . However, no sequences resembling known NifA-binding sites were found in or around this region . NifA-dependent activation was also observed in R . etli bean bacteroids . NifA-dependent hupSp activity in heterologous hosts was also absolutely dependent on the RpoN sigma-factor and on integration host factor . Proteins immunologically related to integration host factor were identified in R . leguminosarum . The data suggest that hupSp is structurally and functionally similar to nitrogen fixation promoters . The requirement to coordinate nitrogenase-dependent H2 production and H2 oxidation in nodules might be the reason for the loss of HoxA in R . leguminosarum and the concomitant NifA control of hup gene expression . This evolutionary acquired control would ensure regulated synthesis of uptake hydrogenase in the most common H2-rich environment for rhizobia, the legume nodule. Mol Gen Genet, 1997 Jun, 255(2), 131 - 40 Structure and role in symbiosis of the exoB gene of Rhizobium leguminosarum bv trifolii; Sanchez-Andujar B et al.; The Rhizobium leguminosarum bv trifolii exoB gene has been isolated by heterologous complementation of an exoB mutant of R . meliloti . We have cloned a chromosomal DNA fragment from the R . leguminosarum bv trifolii genome that contains an open reading frame of 981 bp showing 80% identity at the amino acid level to the UDP-glucose 4-epimerase of R . meliloti . This enzyme produces UDP-galactose, the donor of galactosyl residues for the lipid-linked oligosaccharide repeat units of various heteropolysaccharides of rhizobia . An R . leguminosarum bv trifolii exoB disruption mutant differed from the wild type in the structure of both the acidic exopolysaccharide and the lipopolysaccharide . The acidic exopolysaccharide made by our wild-type strain is similar to the Type 2 exopolysaccharide made by other R . leguminosarum bv trifolii wild types . The exopolysaccharide made by the exoB mutant lacked the galactose residue and the substitutions attached to it . The exoB mutant induced the development of abnormal root nodules and was almost completely unable to invade plant cells . Our results stress the importance of exoB in the Rhizobium-plant interaction. Mol Microbiol, 1997 Jun, 24(6), 1119 - 29 Sulphation of Rhizobium sp . NGR234 Nod factors is dependent on noeE, a new host-specificity gene; Hanin M et al.; Rhizobia secrete specific lipo-chitooligosaccharide signals (LCOs) called Nod factors that are required for infection and nodulation of legumes . In Rhizobium sp . NGR234, the reducing N-acetyl-D-glucosamine of LCOs is substituted at C6 with 2-O-methyl-L-fucose which can be acetylated or sulphated . We identified a flavonoid-inducible locus on the symbiotic plasmid pNGR234a that contains a new nodulation gene, noeE, which is required for the sulphation of NGR234 Nod factors (NodNGR) . noeE was identified by conjugation into the closely related Rhizobium fredii strain USDA257, which produces fucosylated but non-sulphated Nod factors (NodUSDA) . R . fredii transconjugants producing sulphated LCOs acquire the capacity to nodulate Calopogonium caeruleum . Furthermore, mutation of noeE (NGRdelta noeE) abolishes the production of sulphated LCOs and prevents nodulation of Pachyrhizus tuberosus . The sulphotransferase activity linked to NoeE is specific for fucose . In contrast, the sulphotransferase NodH of Rhizobium meliloti seems to be less specific than NoeE, because its introduction into NGRdelta noeE leads to the production of a mixture of LCOs that are sulphated on C6 of the reducing terminus and sulphated on the 2-O-methylfucose residue . Together, these findings show that noeE is a host-specificity gene which probably encodes a fucose-specific sulphotransferase. J Lipid Res, 1997 Jun, 38(6), 1229 - 41 Structural requirements of Rhizobium chitolipooligosaccharides for uptake and bioactivity in legume roots as revealed by synthetic analogs and fluorescent probes; Philip-Hollingsworth S et al.; Rhizobium chitolipooligosaccharides (CLOSs) are heterogeneous fatty acylated N-acetyl glucosamine oligomers with variations in both the polar (hydrophilic) oligosaccharide head group and the non-polar (hydrophobic) fatty acyl chain . They trigger root hair deformation and cortical cell divisions in legume roots during development of the nitrogen-fixing root-nodule symbiosis . It has been proposed that only certain unique molecular species of CLOSs made by a particular rhizobia can elicit these responses on the corresponding legume host, suggesting that receptor-mediated perception of CLOSs serves as a basis of symbiotic specificity . We evaluated the relative symbiotic importance of the hydrophilic and hydrophobic structural domains of CLOSs by comparing the biological activities of CLOSs from wild type R . leguminosarum bv . trifolii ANU843 with that of various synthetic analogs . These tests were performed in axenic bioassays on the compatible symbiotic host, white clover (Trifolium repens) and the incompatible non-host legume, alfalfa (Medicago sativa) . Fluorochrome-tagged derivatives of the native CLOSs and the analogs were also prepared in order to evaluate the uptake and localization patterns of these molecules within host root cells . The results indicate a direct link between uptake and biological activities of Rhizobium CLOSs on legume roots . The smallest CLOS analog taken up and biologically active on white clover and alfalfa was a N-fatty acylglucosamine, without an essential requirement of oligomerization, fatty N-acyl unsaturation, or acetate/sulfate functionalization . This suggests that N-fattyacylglucosamine is the common minimum structure required and sufficient for uptake and biological activity of CLOS glycolipids in these legumes, and that the various specific modifications of its polar head group and hydrophobic tail modulate its inherent ability to further express these activities, thus influencing which legumes are capable of responding to CLOSs rather than dictating their biological activities per se. FASEB J, 1997 Jun, 11(7), 517 - 25 Sulfation and sulfotransferases 6: Biochemistry and molecular biology of plant sulfotransferases; Varin L et al.; It is now well established that, in mammals, sulfate conjugation constitutes an important reaction in the transformation of xenobiotics and in the modulation of the biological activity of steroid hormones and neurotransmitter . The presence of a sulfate group on some molecules can also be a prerequisite for their biological function . For example, it is well known that the sulfate groups are directly involved in the molecular interaction between heparin and antithrombin III . In plants, sulfation also seems to play an important role in the intermolecular recognition and signaling processes, as indicated by the requirement of a sulfate moiety for the biological activity of gallic acid glucoside sulfate in the seismonastic and gravitropic movements of plants, and of Nod RM1 in the cortical cell division during early nodule initiation in Rhizobium meliloti-alfalfa interaction . In addition, recent studies indicate that flavonoid conjugates, including the sulfate esters, may play a role in the regulation of plant growth by strongly binding the naphthylphthalamic acid receptor, thus blocking the quercetin-stimulated accumulation of the auxin phytohormone . Although several sulfated metabolites are known to accumulate in a variety of plant species, the study of enzymes that catalyze the sulfation reaction in plants lagged considerably compared to those conducted with their mammalian homologs . This apparent lack of interest may have been because the function of plant-sulfated metabolites is difficult to predict, since their accumulation is often restricted to a limited number of species . Despite this limitation, several plant sulfotransferases (STs) have been characterized at the biochemical level, and the cDNA clones encoding six plant STs have been isolated . Based on sequence homology, the plant ST coding sequences are grouped under the SULT3 family, also known as the flavonol ST family . This review summarizes our current knowledge of the plant STs and focuses on the functional significance of the sulfate conjugation in plant growth, development, and adaptation to stress. Microbiology, 1997 Jun, 143 ( Pt 6), 1951 - 8 Regulation of exopolysaccharide production in Rhizobium leguminosarum biovar viciae WSM710 involves exoR; Reeve WG et al.; A mildly acid-sensitive mutant of Rhizobium leguminosarum bv . viciae WSM710 (WR6-35) produced colonies which were more mucoid in phenotype than the wild-type . Strain WR6-35 contained a single copy of Tn5 and the observed mucoid phenotype, acid sensitivity and Tn5-induced kanamycin resistance were 100% co-transducible using phage RL38 . WR6-35 produced threefold more exopolysaccharide (EPS) than the wild-type in minimal medium devoid of a nitrogen source . EPS produced by the mutant and the wild-type was identical as determined by proton NMR spectra . An EcoRI rhizobial fragment containing Tn5 and flanking rhizobial sequences was cloned from the mutant, restriction mapped and sequenced . There was extensive similarity between the ORF disrupted by Tn5 in R . leguminosarum bv . viciae WR6-35 and the exoR gene of Rhizobium (Sinorhizobium) meliloti Rm1021 (71.3% identity over 892 bp) . At the protein level there was 70% identity and 93.3% similarity over 267 amino acids with the ExoR protein of R . meliloti Rm1021 . Hydrophilicity profiles of the two proteins from these two rhizobia are superimposable . This gene in R . leguminosarum bv . viciae was thus designated exoR . The data suggest that Tn5 has disrupted a regulatory gene encoding a protein that negatively modulates EPS biosynthesis in R . leguminosarum bv . viciae WSM710 . Despite earlier suggestions that EPS production and acid tolerance might be positively correlated, disruption of exoR in either R . leguminosarum bv . viciae or R . meliloti and its associated overproduction of EPS does not result in a more acid-tolerant phenotype than the wild-type when cultures are screened on conventional laboratory agar. J Bacteriol, 1997 Jun, 179(12), 4053 - 5 Acyl-acyl carrier protein is a donor of fatty acids in the NodA-dependent step in biosynthesis of lipochitin oligosaccharides by rhizobia; Ritsema T et al.; NodA controls transfer of a fatty acid in the biosynthesis of lipochitin oligosaccharides by rhizobia . In an in vitro assay, we used de-N-acetylated chitin oligosaccharides substituted with an O-acetyl moiety as acyl acceptor substrates . We show that acyl-acyl carrier protein is used as a donor in NodA-directed fatty acid transfer. Nat Biotechnol, 1997 Jun, 15(6), 564 - 9 Generation of Rhizobium strains with improved symbiotic properties by random DNA amplification (RDA) Mavingui P, Flores M, Romero D, Martinez-Romero E, Palacios R. To select for bacterial strains with enhanced phenotypes, random fragments of a whole genome, or a defined region of the genome, are cloned in a nonreplicating vector . The resulting plasmids are integrated by recombination into the homologous DNA region of the original strain . Integration gives rise to a nontandem direct duplication of the corresponding DNA region separated by the vector moiety of the plasmid . Recombination between the direct repeats leads to tandem duplication and further amplification of the entire integrated DNA, including the vector . Bacteria harboring the amplified DNA are selected by increasing the dosage of an antibiotic corresponding to a resistance marker of the integrated vector . Pooled strains carrying amplifications are then challenged with a selective pressure for the desired phenotype . After repeated selection cycles, the most fit strains are isolated . We used this process, which we called random DNA amplification, to select Rhizobium strains with increased competitiveness for nodule formation . Derivatives containing randomly amplified DNA regions of the symbiotic plasmid of Rhizobium tropici CFN299 strain were generated . Pools of amplified strains were inoculated onto various tropical legumes . After several cycles of selection through plants, amplified derivatives showing an increased competitiveness for nodule formation with the leguminous plant Macroptilium atropurpureum were obtained. J Bacteriol, 1997 Jun, 179(11), 3706 - 10 Transcriptional regulation of delta-aminolevulinic acid dehydratase synthesis by oxygen in Bradyrhizobium japonicum and evidence for developmental control of the hemB gene; Chauhan S et al.; An increased demand for cytochromes is associated with symbiotic development and microaerobic metabolism in the bacterium Bradyrhizobium japonicum, and evidence suggests that hemB, rather than hemA, is the first essential bacterial heme synthesis gene in symbiosis with soybean . Steady-state levels of mRNA and protein encoded by hemB were strongly and rapidly induced by O2 deprivation as determined by RNase protection and immunoblot analyses, but hemH message was not induced . Oxygen limitation resulted in a greater-than-10-fold increase in the rate of hemB mRNA synthesis as determined by transcriptional runoff experiments, whereas hemH transcription was unaffected by the O2 status . Thus, hemB is a regulated gene in B . japonicum and is transcriptionally controlled by O2 . Unlike the expression in parent strain I110, hemB expression was not affected by O2 in the fixJ strain 7360, and O2-limited cultures of the mutant contained quantities of hemB mRNA and protein that were comparable to uninduced levels found in aerobic cells . In addition, spectroscopic analysis of cell extracts showed that increases in b- and c-type cytochromes and the disappearance of cytochrome aa3 in response to microaerobic growth in wild-type cells were not observed in the fixJ mutant . FixJ is a key transcriptional regulator that mediates O2-dependent differentiation in rhizobia, and therefore hemB expression is under developmental control . Furthermore, the data suggest a global control of cytochrome expression and heme biosynthesis in response to the cellular O2 status. Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 5483 - 8 Rhizobium gone native: unexpected plasmid stability of indigenous Rhizobium leguminosarum; Wernegreen JJ et al.; Lateral transfer of bacterial plasmids is thought to play an important role in microbial evolution and population dynamics . However, this assumption is based primarily on investigations of medically or agriculturally important bacterial species . To explore the role of lateral transfer in the evolution of bacterial systems not under intensive, human-mediated selection, we examined the association of genotypes at plasmid-encoded and chromosomal loci of native Rhizobium, the nitrogen-fixing symbiont of legumes . To this end, Rhizobium leguminosarum strains nodulating sympatric species of native Trifolium were characterized genetically at plasmid-encoded symbiotic (sym) regions (nodulation AB and nodulation CIJT loci) and a repeated chromosomal locus not involved in the symbiosis with legumes . Restriction fragment length polymorphism analysis was used to distinguish genetic groups at plasmid and chromosomal loci . The correlation between major sym and chromosomal genotypes and the distribution of genotypes across host plant species and sampling location were determined using chi2 analysis . In contrast to findings of previous studies, a strict association existed between major sym plasmid and chromosomal genetic groups, suggesting a lack of successful sym plasmid transfer between major Rhizobium chromosomal types . These data indicate that previous observations of sym plasmid transfer in agricultural settings may seriously overestimate the rates of successful conjugation in systems not impacted by human activities . In addition, a nonrandom distribution of Rhizobium genotypes across host plant species and sampling site demonstrates the importance of both factors in shaping Rhizobium population dynamics. Mol Gen Genet, 1997 May, 254(6), 665 - 73 Genetic evidence for 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a negative effector of cytochrome terminal oxidase cbb3 production in Rhizobium etli; Soberon M et al.; A Rhizobium etli Tn5mob-induced mutant (CFN035) exhibits an enhanced capacity to oxidize N,N,N',N', tetramethyl-p -phenylenediamine (TMPD), a presumptive indicator of elevated cytochrome c terminal oxidase activity . Sequencing of the mutated gene in CFN035 revealed that it codes for the amidophosphoribosyl transferase enzyme (PurF) that catalyzes the first step in the purine biosynthetic pathway . Two c-type cytochromes with molecular weights of 32 and 27 kDa were produced in strain CFN035, which also produced a novel CO-reactive cytochrome (absorbance trough at 553 nm), in contrast to strain CE3 which produced a single 32 kDa c-type protein and did not produce the 553 nm CO-reactive cytochrome . A wild-type R . etli strain that expresses the Bradyrhizobium japonicum fixNOQP genes, which code for the symbiotic cytochrome terminal oxidase cbb3, produced similar absorbance spectra (a trough at 553 nm in CO-difference spectra) and two c-type proteins similar in size to those of strain CFN035, suggesting that CFN035 also produces the cbb3 terminal oxidase . The expression of a R . etli fixN-lacZ gene fusion was measured in several R . etli mutants affected in different steps of the purine biosynthetic pathway . Our analysis showed that purF, purD, purQ, purL, purY, purK and purE mutants expressed three-fold higher levels of the fixNOQP operon than the wild-type strain . The derepressed expression of fixN was not observed in a purH mutant . The purH gene product catalyzes the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-form-aminoimidazole-4-carboxamide ribonucleotide (FAICAR) and inosine . Supplementation with AICA riboside lowered the levels of fixN expression in the purF mutants . These data are consistent with the possibility that AICAR, or a closely related metabolite, is a negative effector of the production of the symbiotic terminal oxidase cbb3 in R . etli. Microbiology, 1997 May, 143 ( Pt 5), 1639 - 48 Increased pyruvate orthophosphate dikinase activity results in an alternative gluconeogenic pathway in Rhizobium (Sinorhizobium) meliloti; Osteras M et al.; The formation of phosphoenolpyruvate (PEP) is a major step in the gluconeogenic pathway in which tricarboxylic acid (TCA) cycle intermediates are converted to hexose sugars . In Rhizobium (now Sinorhizobium) meliloti this step is catalysed by the enzyme PEP carboxykinase (PCK) which converts oxaloacetate to PEP . R . meliloti Pck- mutants grow very poorly with TCA cycle intermediates as the sole source of carbon . Here, the isolation and mapping of suppressor mutations which allow Pck- mutants to grow on succinate and other TCA cycle intermediates is reported . Tn5 insertions which abolished the suppressor phenotype and mapped to the suppressor locus were located within the pod gene encoding pyruvate orthophosphate dikinase (PPDK) . Strains carrying suppressor mutations had increased PPDK activity compared to the wild-type . The suppressor phenotype was dependent on the combined activities of malic enzyme and PPDK, which thus represent an alternative route for the formation of PEP in R . meliloti . PPDK activity was not required for symbiotic N2 fixation. Microbiology, 1997 May, 143 ( Pt 5), 1557 - 65 Escherichia coli flavohaemoglobin (Hmp) reduces cytochrome c and Fe(III)-hydroxamate K by electron transfer from NADH via FAD: sensitivity of oxidoreductase activity to haem-bound dioxygen; Poole RK et al.; Escherichia coli flavohaemoglobin (Hmp) reduced purified mitochondrial cytochrome c aerobically in a reaction that was not substantially inhibited by superoxide dismutase, demonstrating that superoxide anion, the product of O2 reduction by Hmp, did not contribute markedly to cytochrome c reduction . Cytochrome c was reduced by Hmp even in the presence of 0.5 mM CO, when the haem B was locked in the ferrous, low-spin state, demonstrating that electron transfer to cytochrome c from NADH was via FAD, not haem . Hmp also reduced the ferrisiderophore complex Fe(III)-hydroxamate K from Rhizobium leguminosarum bv . viciae anaerobically in a CO-insensitive manner, but at low rates and with low affinity for this substrate . The NADH-cytochrome c oxidoreductase activity of Hmp was slightly sensitive to the binding and reduction of O2 at the haem . The Vmax of cytochrome c reduction fell from 7.1 s-1 in the presence of 0.5 mM CO to 5.0 s-1 in the presence of 100 microM O2, with no significant change in K(m) for cytochrome c (6.8 to 7.3 microM, respectively) . O2 at near-micromolar concentrations diminished cytochrome c reduction to a similar extent as did 100 microM O2 . Thus, Hmp acts as a reductase of broad specificity, apparently without involvement of electron transfer via the globin-like haem . These data are consistent with the hypothesis that Hmp could act as an intracellular sensor of O2 since, in the absence of O2, electron flux from FAD to other electron acceptors increases . However, the nature of such acceptors in vivo is not known and alternative models for O2 sensing are also considered. Development, 1997 May, 124(9), 1781 - 7 Ethylene provides positional information on cortical cell division but is not involved in Nod factor-induced root hair tip growth in Rhizobium-legume interaction; Heidstra R et al.; Nod factors secreted by Rhizobium leguminosarum bv . viciae induce root hair deformation, involving a reinitiation of tip growth, and the formation of nodule primordia in Vicia sativa (vetch) . Ethylene is a potent inhibitor of cortical cell division, an effect that can be counteracted by applying silver ions (Ag+) or aminoethoxy-vinylglycine (AVG) . In contrast to the inhibitory effect on cortical cell division, ethylene promotes the formation of root hairs (which involves tip growth) in the root epidermis of Arabidopsis . We investigate the possible paradox concerning the action of ethylene, putatively promoting Nod factor induced tip growth whilst, at the same time, inhibiting cortical cell division . We show, by using the ethylene inhibitors AVG and Ag+, that ethylene has no role in the reinitiation of root hair tip growth induced by Nod factors (root hair deformation) in vetch . However, root hair formation is controlled, at least in part, by ethylene . Furthermore, we show that ACC oxidase, which catalizes the last step in ethylene biosynthesis, is expressed in the cell layers opposite the phloem in that part of the root where nodule primordia are induced upon inoculation with Rhizobium . Therefore, we test whether endogenously produced ethylene provides positional information controlling the site where nodule primordia are formed by determining the position of nodules formed on pea roots grown in the presence of AVG or Ag+. Genes Dev, 1997 May 1, 11(9), 1194 - 206 The Rhizobium meliloti PII protein, which controls bacterial nitrogen metabolism, affects alfalfa nodule development; Arcondeguy T et al.; Symbiotic nitrogen fixation involves the development of specialized organs called nodules within which plant photosynthates are exchanged for combined nitrogen of bacterial origin . To determine the importance of bacterial nitrogen metabolism in symbiosis, we have characterized a key regulator of this metabolism in Rhizobium meliloti, the uridylylatable P(II) protein encoded by glnB . We have constructed both a glnB null mutant and a point mutant making nonuridylylatable P(II) . In free-living conditions, P(II) is required for expression of the ntrC-dependent gene glnII and for adenylylation of glutamine synthetase I . P(II) is also required for efficient infection of alfalfa but not for expression of nitrogenase . However alfalfa plants inoculated with either glnB mutant are nitrogen-starved in the absence of added combined nitrogen . We hypothesize that P(II) controls expression or activity of a bacteroid ammonium transporter required for a functional nitrogen-fixing symbiosis . Therefore, the P(II) protein affects both Rhizobium nitrogen metabolism and alfalfa nodule development. Mol Plant Microbe Interact, 1997 May, 10(4), 506 - 16 New Rhizobium leguminosarum flavonoid-induced proteins revealed by proteome analysis of differentially displayed proteins; Guerreiro N et al.; Proteome analysis was used to establish the first two-dimensional protein map of Rhizobium . R . leguminosarum bv . trifolii strain ANU843 was grown in defined medium in the presence and absence of the flavonoid 7,4'-dihydroxyflavone . Over 1,700 constitutive proteins were resolved, representing about 30% of the estimated genomic output . Proteome analysis of flavonoid-treated cells was done to reveal differentially displayed proteins . The results showed that while the global expression pattern of proteins was largely unaltered by the treatment, four inducible proteins were observed . The four inducible proteins and 20 constitutively expressed proteins were subjected to sequence analysis to provide internal standards for the construction of a two-dimensional Rhizobium protein data base . The identity of 12 proteins, including NodE and NodB, was established . NodE was present throughout the growth of the cells but was diminished in amount in stationary phase cells whereas NodB was not detected in the later stages of growth . Two of the induced proteins sequenced did not match any known nodulation gene product, with one of these being present in mid-late log and stationary phase cells and possessing four consecutive His residues at the N-terminal sequencing was successful with 100 to 200 fmol of protein . Proteome analysis provides a sensitive new tool to examine plant-microbe interactions. Mol Plant Microbe Interact, 1997 May, 10(4), 488 - 98 hrpf of Xanthomonas campestris pv . vesicatoria encodes an 87-kDa protein with homology to NoIX of Rhizobium fredii; Huguet E et al.; The gram-negative bacterium Xanthomonas campestris pv . vesicatoria is the causal agent of bacterial spot disease on pepper and tomato plants . The main hrp (hypersensitive reaction and pathogenicity) gene cluster in X . campestris pv . vesicatoria spans a 23-kb chromosomal region, comprising six complementation groups designated hrpA to hrpF . Analysis of the hrpF locus revealed a single open reading frame encoding HrpF (86.4 kDa) . HrpF is predominantly hydrophilic, and contains two hydrophobic domains in the C terminus . An interesting feature is the presence of two imperfect direct repeats in the N-terminal region . Deletion studies showed that one repeat is sufficient for function . Epitope tagging of HrpF allowed detection of the protein in X . campestris pv . vesicatoria . Subcellular localization studies suggest that HrpF is both in the soluble fraction and in the inner membrane . Interestingly, HrpF is 48% identical (67% similar) to the Rhizobium fredii NoIX protein that is part of the host specificity locus . Since several Hrp proteins are believed to be components of the types of III hrp protein secretion apparatus, allowing export of proteins essential for the interaction with the plant, the possible role of hrpF and NoIX in secretion is discussed. Appl Environ Microbiol, 1997 May, 63(5), 2038 - 46 Root colonization of different plants by plant-growth-promoting Rhizobium leguminosarum bv . trifolii R39 studied with monospecific polyclonal antisera; Schloter M et al.; Monospecific polyclonal antisera raised against Rhizobium leguminosarum bv . trifolii R39, a bacterium which was isolated originally from red clover nodules, were used to study the colonization of roots of leguminous and nonleguminous plants (Pisum sativum, Lupinus albus, Triticum aestivum, and Zea mays) after inoculation . Eight weeks after inoculation of soil-grown plants, between 0.1 and 1% of the total bacterial population in the rhizospheres of all inoculated plants were identified as R . leguminosarum bv . trifolii R39 . To characterize the associative colonization of the nonleguminous plants by R.leguminosarum bv . trifolii R39 in more detail, a time course study was performed with inoculated roots of Z . mays . R . leguminosarum bv . trifolii R39 was found almost exclusively in the rhizosphere soil and on the rhizoplane 4 weeks after inoculation . Colonization of inner root tissues was detected only occasionally at this time . During the process of attachment of R . leguminosarum bv . trifolii R39 to the rhizoplane, bacterial lipopolysaccharides were overexpressed, and this may be important for plant-microbe interaction . Fourteen weeks after inoculation, microcolonies of R . leguminosarum bv . trifolii R39 were detected in lysed cells of the root cortex as well as in intracellular space of central root cylinder cells . At the beginning of flowering (18 weeks after inoculation), the number of R . leguminosarum bv . trifolii R39 organisms decreased in the rhizosphere soil, rhizoplane, and inner root tissue. J Bacteriol, 1997 May, 179(9), 3079 - 83 Sinorhizobium teranga bv . acaciae ORS1073 and Rhizobium sp . strain ORS1001, two distantly related Acacia-nodulating strains, produce similar Nod factors that are O carbamoylated, N methylated, and mainly sulfated; Lorquin J et al.; We have determined the structures of Nod factors produced by strains representative of Sinorhizobium teranga bv . acaciae and the so-called cluster U from the Rhizobium loti branch, two genetically different symbionts of particular Acacia species . Compounds from both strains were found to be similar, i.e., mainly sulfated, O carbamoylated, and N methylated, indicating a close relationship between host specificity and Nod factor structure, regardless of the taxonomy of the bacterial symbiont. J Bacteriol, 1997 May, 179(9), 3076 - 8 Heme compounds as iron sources for nonpathogenic Rhizobium bacteria; Noya F et al.; Many animal-pathogenic bacteria can use heme compounds as iron sources . Like these microorganisms, rhizobium strains interact with host organisms where heme compounds are available . Results presented in this paper indicate that the use of hemoglobin as an iron source is not restricted to animal-pathogenic microorganisms . We also demonstrate that heme, hemoglobin, and leghemoglobin can act as iron sources under iron-depleted conditions for Rhizobium meliloti 242 . Analysis of iron acquisition mutant strains indicates that siderophore-, heme-, hemoglobin-, and leghemoglobin-mediated iron transport systems expressed by R . meliloti 242 share at least one component. Proc Natl Acad Sci U S A, 1997 Apr 29, 94(9), 4336 - 41 Bacterial nodulation protein NodZ is a chitin oligosaccharide fucosyltransferase which can also recognize related substrates of animal origin; Quinto C et al.; The nodZ gene, which is present in various soil bacteria such as Bradyrhizobium japonicum, Azorhizobium caulinodans, and Rhizobium loti, is involved in the addition of a fucosyl residue to the reducing N-acetylglucosamine residue of lipochitin oligosaccharide (LCO) signal molecules . Using an Escherichia coli strain that produces large quantities of the NodZ protein of B . japonicum, we have purified the NodZ protein to homogeneity . The purified NodZ protein appears to be active in an in vitro transfucosylation assay in which GDP-beta-fucose and LCOs or chitin oligosaccharides are used as substrates . The products of the in vitro reaction using chitin oligosaccharides as substrate were studied by using mass spectrometry, linkage analysis, and composition analysis . The data show that one fucose residue is added to C6 of the reducing-terminal N-acetylglucosamine residue . The substrate specificity of NodZ protein was analyzed in further detail, using radiolabeled GDP-beta-fucose as the donor . The results show that chitin oligosaccharides are much better substrates than LCOs, suggesting that in Rhizobium NodZ fucosylates chitin oligosaccharides prior to their acylation . The free glycan core pentasaccharides of N-linked glycoproteins are also substrates for NodZ . Therefore, the NodZ enzyme seems to have an activity equivalent to that of the enzyme involved in the addition of the C6-linked fucosyl substituent in the glycan core of N-linked glycoproteins in eukaryotes . Oligosaccharides that contain only one N-acetylglucosamine at the reducing terminus are also substrates for NodZ, although in this case very high concentrations of such oligosaccharides are needed . An example is the leukocyte antigen Lewis-X, which can be converted by NodZ to a novel fucosylated derivative that could be used for binding studies with E-selectin. FEMS Microbiol Lett, 1997 Apr 15, 149(2), 165 - 72 Cloning and transcriptional analysis of the lipA (lipoic acid synthetase) gene from Rhizobium etli; Tate R et al.; We report here the isolation of a Rhizobium etli gene involved in lipoic acid metabolism, the lipA gene, which complements a lipA mutant strain of Escherichia coli . A promoter region (lipAp) was mapped immediately upstream of lipA and two in vivo transcription initiation sites were identified, preceded by sequences showing some homology to the -10/-35 promoter consensus sequences . The activity of the lipAp was found not to be regulated either by the carbon source or by the addition of lipoic acid . Moreover, quantitative analysis of the lipA transcript by RNase protection assays indicated its down-regulation during entry into stationary phase. Biochemistry, 1997 Apr 8, 36(14), 4178 - 87 Characterization of ferrous FixL-nitric oxide adducts by resonance Raman spectroscopy; Lukat-Rodgers GS et al.; Resonance Raman spectra of the nitric oxide adducts of the ferrous forms of two soluble truncations of Rhizobium meliloti FixL, FixL* and FixLN, are reported . At room temperature, four isotope sensitive vibrations are observed for both ferrous FixL*-NO and ferrous FixLN-NO . For FixL*-NO, they are observed at 558, 525, 450, and 1675 cm(-1) and are assigned to v(Fe-NO) of a six-coordinate nitrosyl adduct, v(Fe-NO) of a five-coordinate nitrosyl adduct, delta(Fe-NO) of a six-coordinate nitrosyl adduct, and v(N-O) of a five-coordinate nitrosyl adduct, respectively . Similar frequencies are observed for the FixLN-NO isotope sensitive bands . On the basis of the frequencies and spectral separation of the v(Fe-NO) and delta(Fe-NO) modes, the Fe-N-O unit is concluded to have a bent geometry similar to those observed for the nitrosyl adducts of ferrous hemoglobin and myoglobin . Both proteins can be converted to predominantly five-coordinate nitrosyl adducts by lowering the temperature . In low-temperature resonance Raman spectra of FixL*-NO and FixLN-NO, the 558 cm(-1) bands are significantly decreased in intensity and v(Fe-NO)5-c (the Fe-NO stretching vibration for the five-coordinate nitrosyl adduct) is observed at 529 and 526 cm(-1), respectively . Analysis of the v3 and v8 vibrations for these nitrosyl adducts also supports the presence of both five- and six-coordinate nitrosyl adducts of FixL* and FixLN at room temperature and the conversion to predominantly five-coordinate nitrosyl adducts at low temperatures . This temperature-dependent interconversion is reversible . The possible physiological relevance of the thermally accessible five-coordinate state is discussed . The width of v(Fe-NO)6-c at half-height is 1.3 times broader in FixLN-NO than in FixL*-NO, suggesting that the Fe-N-O geometry is more homogeneous in FixL*-NO . In low-temperature spectra of FixLN-NO, a second v(N-O)5-c band is observed, indicating that more than one conformation is attainable in the five-coordinate FixLN-NO . This second v(N-O)5-c is not observed for five-coordinate FixL*-NO, further suggesting a more conformationally restricted nitrosyl heme in FixL* . These variations in the vibrations involving the Fe-NO unit indicate that the kinase domain influences the heme structure . The spectral differences are discussed in terms of the interdomain interactions that result in ligation-dependent mediation of the kinase activity. Rev Argent Microbiol, 1997 Apr-Jun, 29(2), 98 - 102 Growth of a leguminous tree (Centrolobium tomentosum Guill . ex Benth.) inoculated with Rhizobium and mycorrhizal fungi; Marques MS et al.; Leguminous trees are being suggested for revegetation programs due to their ability to develop associations with rhizobia and mycorrhizal fungi . The growth of a native species of the Tropical Atlantic Forest, Centrolobium tomentosum, was evaluated in a native forest soil and in a Eucalyptus forest soil under different treatments of inoculation . C . tomentosum produced more biomass under nursery conditions after inoculation with Rhizobium BHICB-Ab1 associated with arbuscular mycorrhizal (AM) . This treatment improved shoot and root growth and nodule weight under forest soil condition, while in eucalyptus soil only shoot biomass and nodule weight were significantly modified . In another experiment, using forest soil, height and stem diameter were also increased by dual inoculation procedures . The height and diameter growth promoting effect was observed when BHICB-Ab1 was used as inoculant associated with AM, but not with BHICB-Ab1 alone . In contrast, plants inoculated with BHICB-Ab3 alone were similar in height and diameter growth, to those which were inoculated with BHICB-Ab3 associated with AM . These results suggest that benefits of dual inoculation depend on triparty symbiosis and especially on the choice of Rhizobium strain. Curr Biol, 1997 Apr 1, 7(4), R223 - 6 Nodulation: finding the lost common denominator; Peters NK; The products of the 'common' nodulation genes of Rhizobium catalyze the synthesis of signal molecules and were once thought to have similar functions in all Rhizobium species; subtle differences in the activities of these gene products have now been discovered that influence the host range of Rhizobium species. Microbiology, 1997 Apr, 143 ( Pt 4), 1381 - 8 The soybean cultivar specificity gene nolX is present, expressed in a nodD-dependent manner, and of symbiotic significance in cultivar-nonspecific strains of Rhizobium (Sinorhizobium) fredii; Bellato C et al.; Rhizobium (now Sinorhizobium) fredii is a symbiotic nitrogen-fixing bacterium that can nodulate soybean in a cultivar-specific manner . This process is governed by a set of negatively acting nodulation genes termed nolXWBTUV . These genes prevent R . fredii strain USDA257 from infecting soybean cultivars such as McCall, but they do not block nodulation of cultivar Peking . R . fredii strain USDA191 contains DNA sequences that hybridize to nolXWBTUV, yet it forms normal nitrogen-fixing nodules on both McCall and Peking soybean . These sequences were isolated and their structure and function examined in comparison to nolXWBTUV of strain USDA257 . Restriction maps of the two loci are identical, as is a 2-4 kb DNA sequence that corresponds to nolX and its promoter region . Expression of nolX by strain USDA191 is flavonoid-dependent in culture and readily detectable in nodules . The gene is not inducible in a mutant of strain USDA191 that lacks the regulatory nodD1 gene, and its expression is greatly attenuated in a nodD2 mutant . nolX is also present and flavonoid-inducible in HH103, a second R . fredii strain that nodulates McCall soybean normally . Inactivation of nolX in strain HH103, USDA191 or USDA257 leads to retardation of initial nodulation rates on soybean cultivars such as Peking and to acquisition of the capacity to form nitrogen-fixing nodules on two species of Erythrina . nolX is thus of symbiotic significance in all three strains, even though it regulates soybean cultivar specificity only in strain USDA257. Lett Appl Microbiol, 1997 Apr, 24(4), 296 - 300 Development of an acute and chronic ecotoxicity assay using lux-marked Rhizobium leguminosarum biovar trifolii; Paton GI et al.; A soil isolate of Rhizobium leguminosarum bv . trifolii was marked with a lux CDABE gene cassette to enable the expression of bioluminescence . The suitability of the bacterium as a soil pollution biosensor was assessed using acute and chronic assays . Bacterial bioluminescence responded sensitively to the metals studied . The order of sensitivity was found to be Cd > Ni = Zn > Cu for the acute test and Cd > Ni = Zn = Cu for the chronic test . The sensitive response of the biosensor highlighted its potential for use as an indicator of soil pollution. J Appl Microbiol, 1997 Apr, 82(4), 477 - 84 Evaluation of the resolving power of three different DNA fingerprinting methods to discriminate among isolates of a natural Rhizobium meliloti population; Niemann S et al.; In a comparative study, the PCR-based RAPD and ERIC fingerprint methods were evaluated for their resolving power to discriminate among 21 isolates of a natural Rhizobium meliloti population . PCR fingerprint patterns were analysed by using an automated laser fluorescent (ALF) DNA sequencer, thus allowing the automated on-line storage of data . Results obtained were compared to a classification system using insertion sequence (IS) fingerprinting . Both PCR fingerprint methods were comparable in their ability to resolve differences amongst Rh . meliloti isolates . Grouping of strains on the basis of their RAPD as well as their ERIC fingerprints correlated with grouping of strains according to their IS fingerprints . Moreover, strains displaying identical PCR patterns could be further differentiated according to their IS fingerprints, thus allowing a detailed insight into phylogenetic relationship among strains . The automated evaluation of strain-specific fingerprint patterns has the potential to become a valuable tool for studies of bacterial population genetics . Moreover, the rapid identification of single strains, e.g . pathogens in epidemiological studies seems feasible. Mol Plant Microbe Interact, 1997 Apr, 10(3), 401 - 6 Sequence and mutational analysis of the 6.7-kb region containing nodAFEG genes of Rhizobium sp . strain N33: evidence of DNA rearrangements; Cloutier J et al.; A 6.7-kb region upstream of nodBC genes in Rhizobium sp . strain N33 was shown to contain the nodAFEG genes and an open reading frame designated orfZ . The open reading frames for these genes contain 591, 282, 1209, 738, and 1,338 nucleotides respectively . Homologues of these genes were found in other rhizobia with the exception of orfZ, for which there was no counterpart found in the Genbank/EMBL database . Tn5 mutagenesis in nodEG and in the intergenic nodG-B region has shown a Nod+ phenotype on their temperate hosts Onobrychis viciifolia and Astragalus cicer . The nodules formed on O . viciifolia plants by these mutants were altered in shape and size . However, on A . cicer there was only a reduction in the number of nodules formed, compared with the wild-type strain . Sequence analysis of the orfZ-nodA and nodG-B intergenic regions indicates the presence of truncated nodD genes. Mol Plant Microbe Interact, 1997 Apr, 10(3), 388 - 93 Induction of chalcone synthase expression by rhizobia and nod factors in root hairs and roots; Krause A et al.; Chalcone synthase (CHS) of Vigna unguiculata is encoded by a gene family that is abundantly transcribed in leaves and nodules . Inoculation with Rhizobium sp . NGR234, which nodulates V . unguiculata, or with NGR delta nodABC, a mutant deficient in Nod factor production, induced rapid accumulation of CHS mRNAs in roots and root hairs . As both Nod+ and Nod- bacteria provoke responses, induction of CHS gene expression may involve symbiotic or defense responses . Four days after inoculation with the wild-type Rhizobium sp., the transcript levels increased in roots but decreased in root hairs . Use of a region unique to the 5' end of a specific CHS gene (VuCHS1) showed that increases of transcript levels in root hairs 24 h after inoculation with both rhizobia were specific to this gene . Transcripts of this gene in roots were only detectable 4 days after treatment with NGR234 . It is possible therefore that accumulation of VuCHS1 follows the infection pathway of rhizobia entering legume roots . Purified Nod factors induced accumulation of transcripts, showing that they might be part of the signal transduction pathway leading to CHS expression. Nat Biotechnol, 1997 Apr, 15(4), 363 - 8 Modification of rhizobacterial populations by engineering bacterium utilization of a novel plant-produced resource; Savka MA et al.; The ability to catabolize distinct nutrients produced by a plant may be a factor in the successful colonization of that host by a bacterium when in competition with other rhizosphere microorganisms . We tested this hypothesis by examining the influence of a novel substrate produced by a transgenic plant on root colonization by near-isogenic bacteria, differing only in their ability to use the resource . When inoculated alone, both bacteria colonized the roots of the normal and transgenic plants with equal kinetics and to indistinguishable levels . When the two bacteria were coinoculated, the catabolizer reached a population density significantly higher than that of the noncatabolizer on the roots of the resource-producing plant . No such advantage was observed on the roots of normal plants . These results support the theory that resources produced and exuded by a plant host can confer a selective advantage to microorganisms that use the substrate. J Bacteriol, 1997 Apr, 179(7), 2356 - 62 Genetic analysis of the Rhizobium meliloti nifH promoter, using the P22 challenge phage system; Ashraf SI et al.; In several genera of bacteria, the sigma54-RNA polymerase holoenzyme (E sigma54) is a minor form of RNA polymerase that is responsible for transcribing genes whose products are involved in diverse metabolic processes . E sigma54 binds to the promoters of these genes to form a closed promoter complex . An activator protein is required for the transition of this closed promoter complex to an open complex that is transcriptionally competent . In this study, the P22-based challenge phage system was used to investigate interactions between E sigma54 and the Rhizobium meliloti nifH promoter . Challenge phages were constructed in which the R . meliloti nifH promoter replaced the binding site for the Mnt protein, a repressor of the phage P22 ant gene . When a Salmonella typhimurium strain that overexpressed sigma54 was infected with these challenge phages, E sigma54 bound to the nifH promoter and repressed transcription of the ant gene as seen by the increased frequency of lysogeny . Following mutagenesis of challenge phages that carried the R . meliloti nifH promoter, mutant phages that could form plaques on an S . typhimurium strain that overexpressed sigma54 were isolated . These phages had mutations within the nifH promoter that decreased the affinity of the promoter for E sigma54 . The mutations were clustered in seven highly conserved residues within the -12 and -24 regions of the nifH promoter. J Bacteriol, 1997 Apr, 179(7), 2132 - 40 The rkpGHI and -J genes are involved in capsular polysaccharide production by Rhizobium meliloti; Kiss E et al.; The first complementation unit of the fix-23 region of Rhizobium meliloti, which comprises six genes (rkpAB-CDEF) exhibiting similarity to fatty acid synthase genes, is required for the production of a novel type of capsular polysaccharide that is involved in root nodule development and structurally analogous to group II K antigens found in Escherichia coli (G . Petrovics, P . Putnoky, R . Reuhs, J . Kim, T . A . Thorp, K . D . Noel, R . W . Carlson, and A . Kondorosi, Mol . Microbiol . 8:1083-1094, 1993; B . L . Reuhs, R . W . Carlson, and J . S . Kim, J . Bacteriol . 175:3570-3580, 1993) . Here we present the nucleotide sequence for the other three complementation units of the fix-23 locus, revealing the presence of four additional open reading frames assigned to genes rkpGHI and -J . The putative RkpG protein shares similarity with acyltransferases, RkpH is homologous to short-chain alcohol dehydrogenases, and RkpJ shows significant sequence identity with bacterial polysaccharide transport proteins, such as KpsS of E . coli . No significant homology was found for RkpI . Biochemical and immunological analysis of Tn5 derivatives for each gene demonstrated partial or complete loss of capsular polysaccharides from the cell surface; on this basis, we suggest that all genes in the fix-23 region are required for K-antigen synthesis or transport. J Bacteriol, 1997 Apr, 179(7), 2103 - 8 Rhizobium nodulation protein NodC is an important determinant of chitin oligosaccharide chain length in Nod factor biosynthesis; Kamst E et al.; Synthesis of chitin oligosaccharides by NodC is the first committed step in the biosynthesis of rhizobial lipochitin oligosaccharides (LCOs) . The distribution of oligosaccharide chain lengths in LCOs differs between various Rhizobium species . We expressed the cloned nodC genes of Rhizobium meliloti, R . leguminosarum bv . viciae, and R . loti in Escherichia coli . The in vivo activities of the various NodC proteins differed with respect to the length of the major chitin oligosaccharide produced . The clearest difference was observed between strains with R . meliloti and R . loti NodC, producing chitintetraose and chitinpentaose, respectively . In vitro experiments, using UDP-{14C}GlcNAc as a precursor, show that this difference reflects intrinsic properties of these NodC proteins and that it is not influenced by the UDP-GlcNAc concentration . Analysis of oligosaccharide chain lengths in LCOs produced by a R . leguminosarum bv . viciae nodC mutant, expressing the three cloned nodC genes mentioned above, shows that the difference in oligosaccharide chain length in LCOs of R . meliloti and R . leguminosarum bv . viciae is due only to nodC . The exclusive production of LCOs which contain a chitinpentaose backbone by R . loti strains is not due to NodC but to end product selection by Nod proteins involved in further modification of the chitin oligosaccharide . These results indicate that nodC contributes to the host specificity of R . meliloti, a conclusion consistent with the results of several studies which have shown that the lengths of the oligosaccharide backbones of LCOs can strongly influence their activities on host plants. FEMS Microbiol Lett, 1997 Mar 15, 148(2), 145 - 51 Molecular studies on a new genetic locus linked to the common nodulation genes in Bradyrhizobium japonicum; Luka S et al.; ORFA, an actively transcribed genetic locus linked to the common nodulation genes in Bradyrhizobium japonicum USDA110, was sequenced and analysed . The expression of ORFA is neither dependent on the regulatory proteins NifA, NtrC, NtrB and NodD1 nor on either copy of sigma 54, RpoN1 and RpoN2 . The transcriptional start site of ORFA was determined and found to overlap the oppositely transcribed nodZ gene by 224 nucleotides . An appropriately located -10 sequence identical to the consensus proposed for rhizobia and a homologous -35 region were identified upstream of the transcriptional start site . ORFA showed no significant homologies to known sequences in gene databases, and its mutational inactivation had no effect on the nodulation of five legume species . Nevertheless, ORFA seems to be conserved among bradyrhizobia, since an ORFA probe hybridised to total DNA extracted from other Bradyrhizobium strains. Arch Microbiol, 1997 Mar 7, 167(2/3), 182 - 6 The requirement for exopolysaccharide precedes the requirement for flavolan-binding polysaccharide in nodulation of Leucaena leucocephala by Rhizobium loti Hotter GS, Scott DB. Rhizobium loti strain PN4115 (NZP2213 str-1) ineffectively nodulates Leucaena leucocephala, i.e., strain PN4115 induces nodulation (Nod+) and is able to invade these nodules (Inv+), but fails to fix nitrogen (Fix-) . Strain PN4115 does not synthesize a flavolan-binding polysaccharide (FBP), which is synthesized by the fully effective (Nod+Inv+Fix+) R . loti strain PN184 (NZP2037 str-1) . The FBP may offer protection from prodelphinidin-rich flavolans synthesized by Lc . leucocephala . In this work, we show that exopolysaccharide (EPS)-negative mutants derived from strain PN4115 have a more severe ineffective phenotype (Nod+Inv-Fix-) on Lc . leucocephala than strain PN4115 . This suggests that EPS from strain PN4115 is functional during invasion of Lc . leucocephala and that the requirement for EPS precedes the requirement for FBP. Plant J, 1997 Mar, 11(3), 407 - 20 A carbonic anhydrase gene is induced in the nodule primordium and its cell-specific expression is controlled by the presence of Rhizobium during development; Coba de la Pena T et al.; Under nitrogen starvation, Rhizobium meliloti is able to induce nitrogen-fixing nodules on alfalfa roots . Certain alfalfa cultivars spontaneously develop pseudonodules in the absence of bacteria . A transcript, Msca1, expressed in spontaneous and R . meliloti-induced nodules, that codes for a carbonic anhydrase (CA), an enzyme catalyzing the hydration of CO2 has been identified . This is the first CA gene cloned from a non-photosynthetic tissue in plants . Msca1 was activated initially in all cells of the bacterium-induced nodule primordium and was also induced by cytokinin treatment of alfalfa roots . The presence of CA enzymatic activity in different nodule types was demonstrated . Thus, Msca1 is a new early nodulin gene with a function possibly related to the increased amyloplast deposition of the dividing cortical cells . Msca1 transcripts were subsequently found mainly in a peripheral envelope of cells in developing and mature nodules . This novel pattern of gene expression is controlled by the presence of the bacterium inside the nodule . Sucrose synthase and phosphoenol pyruvate carboxylase (PEPC), other genes of the carbon fixation metabolism, were expressed in the same peripheral cells and even more strongly in the nitrogen-fixing region . Analysis of expression patterns of these genes indicated that early CA function may not be related to carbon fixation through PEPC . CA might be acting in pH regulation and/or CO2/HCO3-transport during nodule initiation . Thus, carbonic anhydrase may play different roles at several stages of nodule development and function. Microbiology, 1997 Mar, 143 ( Pt 3), 813 - 22 Analysis of a Rhizobium leguminosarum gene encoding a protein homologous to glutathione S-transferases; Tawfiq Alkafaf NK et al.; A novel Rhizobium leguminosarum gene, gstA, the sequence of which indicated that it was a member of the gene family of glutathione S-transferases (GSTs), was identified . The homology was greatest to the GST enzymes of higher plants . The Rhizobium gstA gene was normally expressed at a very low level . The product of gstA was over-expressed and purified from Escherichia coli . It was shown to bind to the affinity matrix glutathione-Sepharose, but no enzymic GST activity with 1-chloro-2,4-dinitrobenzene as substrate was detected . gstA encoded a polypeptide of 203 amino acid residues with a calculated molecular mass of 21990 Da . Transcribed divergently from gstA is another gene, gstR, which was similar in sequence to the LysR family of bacterial transcriptional regulators . A mutation in gstR had no effect on the transcription of itself or gstA under the growth conditions used here . Mutations in gstA and gstR caused no obvious phenotypic defect and the biological functions of these genes remain to be determined. J Bacteriol, 1997 Mar, 179(6), 2068 - 72 Isolation and characterization of Rhizobium etli mutants altered in degradation of asparagine; Huerta-Zepeda A et al.; Rhizobium etli mutants unable to grow on asparagine as the nitrogen and carbon source were isolated . Two kinds of mutants were obtained: AHZ1, with very low levels of aspartase activity, and AHZ7, with low levels of asparaginase and very low levels of aspartase compared to the wild-type strain . R . etli had two asparaginases differentiated by their thermostabilities, electrophoretic mobilities, and modes of regulation . The AHZ mutants nodulated as did the wild-type strain and had nitrogenase levels similar to that of the wild-type strain. Mol Plant Microbe Interact, 1997 Mar, 10(2), 290 - 301 Cloning and characterization of four genes of Rhizobium leguminosarum bv . trifolii involved in exopolysaccharide production and nodulation; van Workum WA et al.; Four different genes of Rhizobium leguminosarum bv . trifolii strain RBL5599 involved in exopolysaccharide (EPS) production were identified by complementation of Tn5-induced EPS-deficient mutants (Exo mutants) with a cosmid bank . On one cosmid pssA was located, which was found to be almost identical to the pss4 gene from R . leguminosarum bv . viciae VF39 and highly homologous to a family of glycosyl transferases . Two pssA mutants, exo2 and exo4, were characterized and found to produce 19 and 1% of the wild-type amount of EPS, respectively . The three other genes were found to be closely linked on a different complementing cosmid . pssC revealed similarity to exoM and exoW of R . meliloti, both encoding glucosyl transferases involved in the synthesis of succinoglycan . A mutation in this gene (mutant exo50) did reduce EPS synthesis to 27% of the wild-type amount . We found an operon closely linked to pssC, consisting of two overlapping genes, pssD and pssE, that is essential for EPS production . Homology of pssD and pssE was found with cps14F and cps14G of Streptococcus pneumoniae, respectively: two genes responsible for the second step in capsule polysaccharide synthesis . Furthermore, pssD and pssE were homologous to the 5' and 3' parts, respectively, of spsK of Sphingomonas S88, which encodes a putative glycosyl transferase . Structural analysis of EPS produced by Exo mutants exo2, exo4, and exo50 showed it to be identical to that of the parental strain RBL5599, with the exception of acetyl groups esterified to one of the glucose residues being absent. Mol Plant Microbe Interact, 1997 Mar, 10(2), 215 - 20 Nod factor-induced expression of leghemoglobin to study the mechanism of NH4NO3 inhibition on root hair deformation; Heidstra R et al.; Nod factors secreted by Rhizobium leguminosarum by, viciae induce root hair deformation, the formation of nodule primordia, and the expression of early nodulin genes in Vicia sativa (vetch) . Root hair deformation is induced within 3 h in a small, susceptible zone (+/-2 mm) of the root . NH4NO3, known to be a potent blocker of nodule formation, inhibits root hair deformation, initial cortical cell divisions, and infection thread formation . To test whether NH4NO3 affects the formation of a component of the Nod factor perception-transduction system, we studied Nod factor-induced gene expression . The differential display technique was used to search for marker genes, which are induced within 1 to 3 h after Nod factor application . Surprisingly, one of the isolated cDNA clones was identified as a leghemoglobin gene (VsLb1), which is induced in vetch roots within 1 h after Nod factor application . By using the drug brefeldin A, it was then shown that VsLb1 activation does not require root hair deformation . The pVsLb1 clone was used as a marker to show that in vetch plants grown in the presence of NH4NO3, Nod factor perception and transduction leading to gene expression are unaffected. Phytochemistry, 1997 Mar, 44(6), 991 - 5 Enzymic O-methylation of isoliquiritigenin and licodione in alfalfa and licorice cultures; Ichimura M et al.; S-Adenosyl-L-methionine (SAM): isoliquiritigenin (2',4,4'-trihydroxychalcone) 2'-O-methyltransferase (CHMT) of alfalfa (Medicago sativa) catalyses the formation of 4,4'-dihydroxy-2'-methoxychalcone, which is the most potent inducer of nodulation-genes of Rhizobium meliloti, the symbiont of alfalfa which forms nitrogen-fixing nodules . SAM: licodione 2'-O-methyltransferase (LMT) is involved in the biosynthesis of a retrochalcone in cultured licorice (Glycyrrhiza echinata) cells and has been shown to be induced as a defence response of the cells . Because licodione exists in an equilibrium mixture of tautomeric 2',4,4',beta-tetrahydroxychalcone (major) and 1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)-1,3-propanedione (minor), the apparent mode of action of both enzymes is very similar . In this study, cultured alfalfa cells were shown to exhibit rapid and transient increases in the extractable activities of both CHMT and LMT after treatment with yeast extract (YE) . Treatment of solution-cultured alfalfa seedlings with YE also resulted in a similar induction of both CHMT and LMT activities in the roots, but no activity was detected in the shoots . These activities were attributed to a single gene product, the CHMT protein, as extracts of Escherichia coli transformed with the CHMT cDNA exhibited both CHMT and LMT activities . In contrast, in G . echinata cells, LMT was induced after YE treatment, but no CHMT activity was observed . It is concluded that alfalfa CHMT and licorice LMT are distinct enzymes, the former displaying the wider substrate specificity. Curr Microbiol, 1997 Mar, 34(3), 167 - 72 Choline and glycine betaine uptake in various strains of Rhizobia isolated from nodules of Vicia faba var . major and Cicer arietinum l.: modulation by salt, choline, and glycine betaine; Brhada F et al.; Two strains of Rhizobia isolated from nodules of Vicia faba var . major and one strain isolated from nodules of Cicer arietinum L . were characterized for salt resistance . The presence of 1 mM glycine betaine or choline in a minimal medium with added NaCl had a beneficial role on the growth of the three strains . Both molecules were found to be taken up by cells obtained at low osmolarity, and whereas glycine betaine uptake activity was stimulated significantly in cells grown in the presence of 0.15 M NaCl, choline uptake activity was strongly inhibited by salt in all tested strains . However, in cells grown with exogenous choline,the uptake inhibition exerted by salt was relieved, mainly in the strain isolated from nodules of C . arietinum L . On the basis of kinetics determinations, in control cells as well as in salt-stressed cells, only high-affinity activities were observed for glycine betaine and choline(apparent Kms between 3 and 18 micro;M) . Periplasmic proteins that bound glycine betaine or choline were identified . In nondenaturing conditions, these proteins extracted from the various strains showed different electrophoretic mobility with always a less negative entire charge than the analogous proteins from Rhizobium meliloti. Proc Natl Acad Sci U S A, 1997 Feb 18, 94(4), 1270 - 5 Evidence that eukaryotic triosephosphate isomerase is of alpha-proteobacterial origin; Keeling PJ et al.; We have cloned and sequenced genes for triosephosphate isomerase (TPI) from the gamma-proteobacterium Francisella tularensis, the green non-sulfur bacterium Chloroflexus aurantiacus, and the alpha-proteobacterium Rhizobium etli and used these in phylogenetic analysis with TPI sequences from other members of the Bacteria, Archaea, and Eukarya . These analyses show that eukaryotic TPI genes are most closely related to the homologue from the alpha-proteobacterium and most distantly related to archaebacterial homologues . This relationship suggests that the TPI genes present in modern eukaryotic genomes were derived from an alpha-proteobacterial genome (possibly that of the protomitochondrial endosymbiont) after the divergence of Archaea and Eukarya . Among these eukaryotic genes are some from deeply branching, amitochondrial eukaryotes (namely Giardia), which further suggests that this event took place quite early in eukaryotic evolution. Arch Microbiol, 1997 Feb-Mar, 167(2-3), 182 - 6 The requirement for exopolysaccharide precedes the requirement for flavolan-binding polysaccharide in nodulation of Leucaena leucocephala by Rhizobium loti; Hotter GS et al.; Rhizobium loti strain PN4115 (NZP2213 str-1) ineffectively nodulates Leucaena leucocephala, i.e., strain PN4115 induces nodulation (Nod+) and is able to invade these nodules (Inv+), but fails to fix nitrogen (Fix-) . Strain PN4115 does not synthesize a flavolan-binding polysaccharide (FBP), which is synthesized by the fully effective (Nod+Inv+Fix+) R . loti strain PN184 (NZP2037 str-1) . The FBP may offer protection from prodelphinidin-rich flavolans synthesized by Lc . leucocephala . In this work, we show that exopolysaccharide (EPS)-negative mutants derived from strain PN4115 have a more severe ineffective phenotype (Nod+Inv-Fix-) on Lc . leucocephala than strain PN4115 . This suggests that EPS from strain PN4115 is functional during invasion of Lc . leucocephala and that the requirement for EPS precedes the requirement for FBP. Int J Biol Macromol, 1997 Feb, 20(1), 1 - 7 Effect of o-acyl substituents on the functional behaviour of Rhizobium meliloti succinoglycan; Ridout MJ et al.; The effects of selective removal of acetyl or succinyl substituents on the functionality of succinoglycan polysaccharide have been studied by comparing the behaviour of the polysaccharides isolated from native Rhizobium meliloti strain Rm1021, and genetically modified R . meliloti species . Removal of the succinyl groups was found to dramatically improve pseudoplasticity of the aqueous succinoglycan samples and also increase the cooperativity of the order-disorder transition exhibited by the polysaccharide . Removal of the acetyl substituent led to a decrease in the order-disorder transition temperature, whereas the removal of the succinyl groups led to an increase. Can J Microbiol, 1997 Feb, 43(2), 164 - 77 Identification of soil bacteria expressing a symbiotic plasmid from Rhizobium leguminosarum bv . trofolii; Sivakumaran S et al.; A hundred strains of non-nodulating, Gram-negative, rod-shaped bacteria were isolated from clover-ryegrass pastures on three different soil types and from a sandy loam under lupins . When crossed with Escherichia coli PN200 containing the cointegrate plasmid pPN1, 11 transconjugants gained the ability to form nodules on the roots of white clover (Trifolium repens cv . Grasslands Huia) . A nodA probe indicated that they had gained nodulation genes . The identities of these 11 strains and 4 others derived from earlier work on non-nodulating root nodule bacteria, were determined by ribotyping, DNA-DNA hybridization, and partial 16S rRNA sequencing . Good agreement was obtained between the three methods, and 11 of the strains were identified as Rhizobium leguminosarum (6), Rhizobium loti (2), Rhizobium etli (1), Rhizobium tropici (1), and Sinorhizobium meliloti (1) . DNA-DNA hybridization indicated that the remaining four strains were related to the Rhizobium leguminosarum reference strains . The existence of several species of non-nodulating rhizobia in pasture soil, including species for which the normal host plant was absent, is discussed in relation to the fate of symbiotic plasmids from Rhizobium seed inoculants . It is also suggested that new species should be named for the geographical region from which they are first isolated rather than the host plant. Microbiology, 1997 Feb, 143 ( Pt 2), 489 - 98 Properties of NAD(+)- and NADP(+)-dependent malic enzymes of Rhizobium (Sinorhizobium) meliloti and differential expression of their genes in nitrogen-fixing bacteroids; Driscoll BT et al.; The wild-type NAD(+)-dependent malic enzyme (dme) gene of Rhizobium (now Sinorhizobium) meliloti was cloned and localized to a 3.1 kb region isolated on the cosmid pTH69 . This cosmid complemented the symbiotic nitrogen fixation (Fix-) phenotype of R . meliloti dme mutants . The dme gene was mapped by conjugation to between the cys-11 and leu-53 markers on the R . meliloti chromosome . beta-Galactosidase activities measured in bacterial strains carrying either dme-lacZ or tme-lacZ gene fusions (the tme gene encodes NADP(+)-dependent malic enzyme) indicated that the dme gene was expressed constitutively in free-living cells and in N2-fixing bacteroids whereas expression of the tme gene was repressed in bacteroids . The R . meliloti dme gene product (DME) was overexpressed in and partially purified from Escherichia coli . The properties of this enzyme, together with those of the NADP(+)-dependent malic enzyme (TME) partially purified from R . meliloti dme mutants, were determined . Acetyl-CoA inhibited DME but not TME activity . This result supports the hypothesis that DME, together with pyruvate dehydrogenase, forms a pathway in which malate is converted to acetyl-CoA. Appl Environ Microbiol, 1997 Feb, 63(2), 661 - 4 Cloning and sequence analysis of genes encoding xylanases and acetyl xylan esterase from Streptomyces thermoviolaceus OPC-520; Tsujibo H et al.; Three genes encoding two types of xylanases (STX-I and STX-II) and an acetyl xylan esterase (STX-III) from Streptomyces thermoviolaceus OPC-520 were cloned, and their DNA sequences were determined . The nucleotide sequences showed that genes stx-II and stx-III were clustered on the genome . The stx-I, stx-II, and stx-III genes encoded deduced proteins of 51, 35.2, and 34.3 kDa, respectively . STX-I and STX-II bound to both insoluble xylan and crystalline cellulose (Avicel) . Alignment of the deduced amino acid sequences encoded by stx-I, stx-II, and stx-III demonstrated that the three enzymes contain two functional domains, a catalytic domain and a substrate-binding domain . The catalytic domains of STX-I and STX-II showed high sequence homology to several xylanases which belong to families F and G, respectively, and that of STX-III showed striking homology with an acetyl xylan esterase from S . lividans, nodulation proteins of Rhizobium sp., and chitin deacetylase of Mucor rouxii . In the C-terminal region of STX-I, there were three reiterated amino acid sequences starting from C-L-D, and the repeats were homologous to those found in xylanase A from S . lividans, coagulation factor G subunit alpha from the horseshoe crab, Rarobacter faecitabidus protease I, beta-1,3-glucanase from Oerskovia xanthineolytica, and the ricin B chain . However, the repeats did not show sequence similarity to any of the nine known families of cellulose-binding domains (CBDs) . On the other hand, STX-II and STX-III contained identical family II CBDs in their C-terminal regions. J Bacteriol, 1997 Feb, 179(4), 1375 - 84 The 32-kilobase exp gene cluster of Rhizobium meliloti directing the biosynthesis of galactoglucan: genetic organization and properties of the encoded gene products; Becker A et al.; Proteins directing the biosynthesis of galactoglucan (exopolysaccharide II) in Rhizobium meliloti Rm2011 are encoded by the exp genes . Sequence analysis of a 32-kb DNA fragment of megaplasmid 2 containing the exp gene cluster identified previously (J . Glazebrook and G . C . Walker, Cell 56:661-672, 1989) revealed the presence of 25 open reading frames . Homologies of the deduced exp gene products to proteins of known function suggested that the exp genes encoded four proteins involved in the biosynthesis of dTDP-glucose and dTDP-rhamnose, six glycosyltransferases, an ABC transporter complex homologous to the subfamily of peptide and protein export complexes, and a protein homologous to Rhizobium NodO proteins . In addition, homologies of three Exp proteins to transcriptional regulators, methyltransferases, and periplasmic binding proteins were found . The positions of 26 Tn5 insertions in the exp gene cluster were determined, thus allowing the previously described genetic map to be correlated with the sequence . Operon analysis revealed that the exp gene cluster consists of five complementation groups . In comparison to the wild-type background, all exp complementation groups were transcribed at a substantially elevated level in the regulatory mucR mutant. Science, 1997 Jan 24, 275(5299), 527 - 30 A Legume Ethylene-Insensitive Mutant Hyperinfected by Its Rhizobial Symbiont Penmetsa RV, Cook DR. Development of the Rhizobium-legume symbiosis is controlled by the host plant, although the underlying mechanisms have remained obscure . A mutant in the annual legume Medicago truncatula exhibits an increase of more than an order of magnitude in the number of persistent rhizobial infections . Physiological and genetic analyses indicate that this same mutation confers insensitivity to the plant hormone ethylene for multiple aspects of plant development, including nodulation . These data support the hypothesis that ethylene is a component of the signaling pathway controlling rhizobial infection of legumes. Microbios, 1997, 89(360-361), 187 - 96 Alteration of the symbiotic properties of Rhizobium meliloti by plasmid manipulations; Radeva G et al.; Mutants with symbiotic activity were obtained by random Tn5-Mob mutagenesis of Rhizobium meliloti 41 . The place of the Tn5 insertion was localized on a cryptic middle-sized plasmid pRme41a . Mobilization of the labelled plasmid pRme41a::Tn5-Mob into R . meliloti 114, which did not contain such a plasmid led to a complete loss of the nodulation ability of the recipient strain . The Nod-phenotype was a result of a large deletion in the symbiotic (pSym) plasmid of R . meliloti 114. Acta Biochim Pol, 1997, 44(1), 1 - 12 Nodulation genes in the Rhizobium--plant signal exchange; Lorkiewicz Z; The process of the host-plant recognition by rhizobia is complex and multi-step . The interaction between legumes and microorganisms results in the induction of the root nodule . This symbiotic interaction is highly host-specific . Bacteria within nodules fix atmospheric nitrogen . This process is of immense ecological and economic significance . The subject of this presentation is the molecular mechanism by which the bacterium determines its host-specific characteristics . First flavonoids secreted by the plant roots induce the transcription of bacterial genes involved in nodulation, the so-called nod genes . This leads to the next step of the signalling system, i.e . the production and secretion of lipo-oligosaccharide molecules by rhizobia . These signal molecules have various discernible effects on the roots of the host leguminous plants . The bacterial nodulation factors were isolated and structurally identified as substituted and N-acylated chitin oligosaccharides . These prokaryotic 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 . More recent studies implicate involvement of root cell membrane depolarization and ion selective channels in the communication processes that initiate nodule formation. Rev Argent Microbiol, 1997 Jan-Mar, 29(1), 24 - 31 {Inoculation of chickpeas with Rhizobium sp . native to the province of Córdoba, Argentina}; Abril A et al.; Native strains of Rhizobium were selected from a chickpea growing area with the purpose of producing inoculants tolerant to environmental conditions of the province of Cordoba . The strains were selected for nitrogen fixing capacity and specificity with four plant genotypes using the following parameters: number of nodules, plant biomass and nitrogen content . Native population of Rhizobium spp in the province of Cordoba showed better competition ability than foreign strains with similar response to different plant genotypes. Microbiology, 1997 Jan, 143 ( Pt 1), 127 - 34 High affinity iron acquisition in Rhizobium leguminosarum requires the cycHJKL operon and the feuPQ gene products, which belong to the family of two-component transcriptional regulators; Yeoman KH et al.; The cycHJKL operon of Rhizobium leguminosarum has previously been shown to be involved in the maturation of cytochrome c, possibly by its involvement in the covalent attachment of haem to the apoprotein . Mutations in the cycHJKL genes abolish symbiotic nitrogen fixation . Here, we show that cyc mutants are pleiotropically defective . They have lost a high affinity iron acquisition system due to their failure to make or to export siderophores . They also accumulate protoporphyrin IX, the immediate precursor of haem . A model to account for these phenotypes is presented . Immediately upstream of cycH is a gene, lipA, which is predicted to encode an outer-membrane lipoprotein . Further upstream of lipA, there are two other genes, whose products are similar in sequence to the widespread family of two-component transcriptional regulators . These two genes, feuP and feuQ, did not affect the transcription of lipA, or of the cycHJKL operon . However, a mutation in feuQ also led to the loss of the high affinity iron uptake system, although siderophores were still produced. Plant Physiol, 1997 Jan, 113(1), 45 - 57 Cell-specific expression of the promoters of two nonlegume hemoglobin genes in a transgenic legume, Lotus corniculatus; Andersson CR et al.; The promoters of the hemoglobin genes from the nitrogen-fixing tree Parasponia andersonii and the related nonnitrogen-fixing Trema tomentosa both confer beta-glucuronidase reporter gene expression to the central zone of the nodules of a transgenic legume, Lotus corniculatus . beta-Glucuronidase expression was high in the uninfected interstitial cells and parenchyma of the surrounding boundary layer and was low in the Rhizobium-infected cells . This contrasts with the expression of both the P . andersonii hemoglobin protein in P . andersonii nodules and the endogenous Lotus leghemoglobins that are expressed in the infected cells at very high levels . The expression pattern of the P . andersonii and T . tomentosa hemoglobin promoters in L . corniculatus resembles that of a nonsymbiotic hemoglobin gene from Casuarina glauca, which was introduced into this legume, and suggests that only the nonsymbiotic functions of the P . andersonii promoter are being recognized . Deletion of the distal segments of both the P . andersonii and T . tomentosa promoters identified regions important for the control of their tissue-specific and temporal activity in Lotus . Potential regulatory elements, which enhance nodule expression and suppress nonnodule expression, were also identified and localized to a distal promoter segment . A proximal AAGAG motif is present in the P . andersonii, T . tomentosa, and nonsymbiotic Casuarina hemoglobin genes . Mutation of this motif in the P . andersonii promoter resulted in a significant reduction in both the nodule and root expression levels in L . corniculatus . Some of the regulatory motifs characterized are similar to, but different from, the nodulin motifs of the leghemoglobins. Mol Microbiol, 1997 Jan, 23(1), 85 - 93 The Rhizobium meliloti putA gene: its role in the establishment of the symbiotic interaction with alfalfa; Jimenez-Zurdo JI et al.; Little is known about the energy sources used by rhizobia during colonization, invasion and root nodule formation on leguminous plants . We have recently reported that an impaired proline metabolism in rhizobium meliloti leads to a reduced nodulation efficiency and competitiveness on alfalfa roots . In the present study we have characterized the R . meliloti proline dehydrogenase gene (putA) and addressed the question of its role in symbiosis . This rhizobial gene encodes a 1224-amino-acid-long polypeptide which is homologous to enteric bacteria, Rhodobacter capsulatus and Bradyrhizobium japonicum PutA proteins . Like the situation in these bacteria, sequence analysis identified the proline dehydrogenase (PDH) and pyrroline-5-carboxylate dehydrogenase (P5CDH) domains in the R . meliloti putA-encoded protein . Beta-galactosidase assays performed with free-living cells carrying a putA-lacZ transcriptional fusion revealed that R . meliloti putA gene expression is induced by proline, autoregulated by its encoded product, and independent of the general nitrogen regulatory system (Ntr) . In addition, analysis of putA expression during the different steps of the symbiotic interaction with alfalfa showed that expression of this gene is turned on by the root exudates (RE), during root invasion and nodule formation, but not in differentiated nitrogen-fixing bacteroids . Furthermore, we show that the PutA- phenotype leads to a significant reduction of alfalfa root colonization by R . meliloti. Mol Plant Microbe Interact, 1997 Jan, 10(1), 124 - 31 The Vicia faba leghemoglobin gene VfLb29 is induced in root nodules and in roots colonized by the arbuscular mycorrhizal fungus Glomus fasciculatum; Fruhling M et al.; To investigate similarities between symbiotic interactions of broad bean (Vicia faba) with rhizobia and mycorrhizal fungi, plant gene expression induced by both microsymbionts was compared . We demonstrated the exclusive expression of 19 broad bean genes, including VfENOD2, VfENOD5, VfENOD12 and three different leghemoglobin genes, in root nodules . In contrast, the leghemoglobin gene VfLb29 was found to be induced not only in root nodules, but also in broad bean roots colonized by the mycorrhizal fungus Glomus fasciculatum . In uninfected roots, none of the 20 nodulin transcripts investigated was detectable . VfLb29 has an unusually low sequence homology with all other broad bean leghemoglobins as well as with leghemoglobins from other legumes . It can be regarded as a novel kind of leghemoglobin gene not described until now and the induction of which is common to symbiotic interactions of broad bean with both Rhizobium and a mycorrhizal fungus. Mol Plant Microbe Interact, 1997 Jan, 10(1), 95 - 101 Rhizobia modulate root-hair-specific expression of extensin genes; Arsenijevic-Maksimovic I et al.; Three cDNAs (ext3, ext127, and ext26), originally isolated by differential screening from a root-hair cDNA library of Vigna unguiculata, were found to encode extensin-like cell wall proteins . Transcripts homologous to these cDNAs were only detected in root hairs where mRNA levels decreased 1 day after inoculation with rhizobia . This coincided with the onset of root-hair deformation, the first morphological step in the Rhizobium-legume interaction . Decreases in transcript levels following inoculation with wild-type Rhizobium sp . NGR234 were more pronounced than with NGR delta nodABC, a mutant deficient in Nod-factor production . Inoculation with a rhizobial strain carrying a mutation in a gene encoding a transcriptional activator for nod genes (NGR delta nodD1) did not repress mRNA levels, indicating that a second nodulation signal may be present that is nodD dependent . Application of purified NodNGR factors only affected transcript levels of ext3 . The genomic locus of the gene homologous to ext26 (Ext26G) was cloned . In the 5' flanking region, several potential TATA boxes and CAP signals were identified . Part of the promoter region shares homology with the Pisum sativum seed lectin promoter and the Nicotiana tabacum nitrate reductase promoter region . Nonetheless, the function of these homologous regions in gene regulation is unknown. J Bacteriol, 1997 Jan, 179(2), 364 - 9 Dissection of the transcription machinery for housekeeping genes of Bradyrhizobium japonicum; Beck C et al.; By using a PCR approach, the Bradyrhizobium japonicum sigA gene, which encodes the primary RNA polymerase sigma factor, sigma80, was cloned and its nucleotide sequence was established . The deduced protein is highly homologous to the SigA protein of Rhizobium meliloti (72% amino acid sequence identity) but less so to RpoD of Escherichia coli (51% identity) . Well conserved is the C-terminal end of the protein, which is probably involved in promoter recognition and binding of the RNA polymerase core enzyme . A remarkable feature of the primary sequence is an alanine- and proline-rich segment of 24 amino acids between conserved regions 1 and 2, which might function as an interdomain linker . We purified the B . japonicum RNA polymerase holoenzyme . One of the subunits had an apparent molecular mass of 90 kDa and corresponded to the sigA gene product, as judged by N-terminal amino acid sequencing . The purified RNA polymerase was used in an in vitro transcription system to determine the transcription start sites of the rrn and groESL4 operons . They were identical to those previously identified in vivo . The rrn promoter was cloned upstream of a rho-independent terminator, yielding a transcript of about 240 bases . This served as a suitable template to analyze promoter activity . Then mutant derivatives of the rrn promoter were constructed and tested in in vitro transcription experiments . Several base pairs essential for promoter activity were thus identified . The results suggest that the well-characterized -35/-10 promoter class is predominantly used in B . japonicum for the expression of "housekeeping" genes. J Bacteriol, 1997 Jan, 179(1), 209 - 16 Genetic analysis of the Rhizobium meliloti bacA gene: functional interchangeability with the Escherichia coli sbmA gene and phenotypes of mutants; Ichige A et al.; The Rhizobium meliloti bacA gene encodes a function that is essential for bacterial differentiation into bacteroids within plant cells in the symbiosis between R . meliloti and alfalfa . An Escherichia coli homolog of BacA, SbmA, is implicated in the uptake of microcin B17, microcin J25 (formerly microcin 25), and bleomycin . When expressed in E . coli with the lacZ promoter, the R . meliloti bacA gene was found to suppress all the known defects of E . coli sbmA mutants, namely, increased resistance to microcin B17, microcin J25, and bleomycin, demonstrating the functional similarity between the two proteins . The R . meliloti bacA386::Tn(pho)A mutant, as well as a newly constructed bacA deletion mutant, was found to show increased resistance to bleomycin . However, it also showed increased resistance to certain aminoglycosides and increased sensitivity to ethanol and detergents, suggesting that the loss of bacA function causes some defect in membrane integrity . The E . coli sbmA gene suppressed all these bacA mutant phenotypes as well as the Fix- phenotype when placed under control of the bacA promoter . Taken together, these results strongly suggest that the BacA and SbmA proteins are functionally similar and thus provide support for our previous hypothesis that BacA may be required for uptake of some compound that plays an important role in bacteroid development . However, the additional phenotypes of bacA mutants identified in this study suggest the alternative possibility that BacA may be needed for membrane integrity, which is likely to be critically important during the early stages of bacterial differentiation within plant cells. Carbohydr Res, 1996 Dec 24, 296, 23 - 37 Cyclolaminarinose . A new biologically active beta-(1-->3) cyclic glucan; Pfeffer PE et al.; A unique glucan has been isolated from a recombinant strain of a Rhizobium meliloti TY7, a cyclic beta-(1-->2) glucan mutant carrying a locus specifying beta-(1-->3; 1-->6) glucan synthesis from Bradyrhizobium japonicum USDA110 . This compound, which appears to have considerable hydrophobic affinity, was separated from a perchloric acid cell extract by adsorption to a C-18 silica column . Unlike those cyclic glucans previously isolated from Rhizobium meliloti or Bradyrhizobium japonicum, this molecule contains neither phosphoglycerol nor phosphocholine substituents, respectively . 2D NMR, FAB mass spectrometric analysis and high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) confirmed that this glucan is a single, cyclic decasaccharide (cyclolaminarinose) in which one of the residues is substituted in its 6-position with beta-laminarabiose . This structural assignment was confirmed by mass spectral and NMR analyses of the product obtained from two consecutive Smith degradations . Unlike the complex 13C spectrum of the unoxidized material, the spectrum of this product consisted of only six resonances due to rapid time averaging of its symmetrical structure on the relatively slow NMR timescale . Synthesis of this newly described cyclic beta-glucan in the R . meliloti ndvB mutant restored the symbiotic and hypoosmotic adaptation characteristics of the R . meliloti wild type strain. Proc Natl Acad Sci U S A, 1996 Dec 24, 93(26), 15305 - 10 The common nodABC genes of Rhizobium meliloti are host-range determinants; Roche P et al.; Symbiotic bacteria of the genus Rhizobium synthesize lipo-chitooligosaccharides, called Nod factors (NFs), which act as morphogenic signal molecules on legume hosts . The common nodABC genes, present in all Rhizobium species, are required for the synthesis of the core structure of NFs . NodC is an N-acetylglucosaminyltransferase, and NodB is a chitooligosaccharide deacetylase; NodA is involved in N-acylation of the aminosugar backbone . Specific nod genes are involved in diverse NF substitutions that confer plant specificity . We transferred to R . tropici, a broad host-range tropical symbiont, the ability to nodulate alfalfa, by introducing nod genes of R . meliloti . In addition to the specific nodL and nodFE genes, the common nodABC genes of R . meliloti were required for infection and nodulation of alfalfa . Purified NFs of the R . tropici hybrid strain, which contained chitin tetramers and