Rhizobacter

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 were partly N-acylated with unsaturated C16 fatty acids, were able to elicit nodule formation on alfalfa . Inactivation of the R . meliloti nodABC genes suppressed the ability of the NFs to nodulate alfalfa . Studies of NFs from nodA, nodB, nodC, and nodI mutants indicate that (i) NodA of R . meliloti, in contrast to NodA of R . tropici, is able to transfer unsaturated C16 fatty acids onto the chitin backbone and (ii) NodC of R . meliloti specifies the synthesis of chitin tetramers . These results show that allelic variation of the common nodABC genes is a genetic mechanism that plays an important role in signaling variation and in the control of host range.

J Biol Chem, 1996 Dec 13, 271(50), 32126 - 36
A special acyl carrier protein for transferring long hydroxylated fatty acids to lipid A in Rhizobium; Brozek KA et al.; Lipid A, the hydrophobic anchor of lipopolysaccharides in the outer membranes of Gram-negative bacteria, varies in structure among different Rhizobiaceae . The Rhizobium meliloti lipid A backbone, like that of Escherichia coli, is a beta1'-6-linked glucosamine disaccharide that is phosphorylated at positions 1 and 4' . Rhizobium leguminosarum lipid A lacks both phosphates, but contains aminogluconate in place of the proximal glucosamine 1-phosphate, and galacturonic acid instead of the 4'-phosphate . A peculiar feature of the lipid As of all Rhizobiaceae is acylation with 27-hydroxyoctacosanoic acid, a long hydroxylated fatty acid not found in E . coli . We now describe an in vitro system, consisting of a membrane enzyme and a cytosolic acyl donor from R . leguminosarum, that transfers 27-hydroxyoctacosanoic acid to (Kdo)2-lipid IVA, a key lipid A precursor common to both E . coli and R . leguminosarum . The 27-hydroxyoctacosanoic acid moiety was detected in the lipid product by mass spectrometry . The membrane enzyme required the presence of Kdo residues in the acceptor substrate for activity . The cytosolic acyl donor was purified from wild-type R . leguminosarum using the acylation of (Kdo)2-{4'-32P}-lipid IVA as the assay . Amino-terminal sequencing of the purified acyl donor revealed an exact 19-amino acid match with a partially sequenced gene (orf*) of R . leguminosarum . Orf* contains the consensus sequence, DSLD, for attachment of 4'-phosphopantetheine . When the entire orf* gene was sequenced, it was found to encode a protein of 92 amino acids . Orf* is a new kind of acyl carrier protein because it is only approximately 25% identical both to the constitutive acyl carrier protein (AcpP) and to the inducible acyl carrier protein (NodF) of R . leguminosarum . Mass spectrometry of purified active Orf* confirmed the presence of 4'-phosphopantetheine and 27-hydroxyoctacosanoic acid in the major species . Smaller mass peaks indicative of Orf* acylation with hydroxylated 20, 22, 24, and 26 carbon fatty acids were also observed . Given the specialized function of Orf* in lipid A acylation, we suggest the new designation AcpXL.

J Biol Chem, 1996 Dec 13, 271(50), 32119 - 25
Lipopolysaccharide core glycosylation in Rhizobium leguminosarum . An unusual mannosyl transferase resembling the heptosyl transferase I of Escherichia coli; Kadrmas JL et al.; The lipopolysaccharide structure of the nitrogen-fixing bacterium Rhizobium leguminosarum differs from that of Escherichia coli in several ways, one of which is the sugar composition of the core . The E . coli inner core consists of 3-deoxy-D-manno-octulosonic acid (Kdo) and L-glycero-D-manno-heptose (heptose), while the inner core of R . leguminosarum contains 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo), mannose, galactose, and galacturonic acid . The two Kdo residues and their linkages appear to be identical in both species . The linkages of heptose in E . coli and of mannose in R . leguminosarum to Kdo are both alpha1-5 . We now characterize a membrane-associated glycosyl transferase in R . leguminosarum extracts that incorporates mannose into nascent lipopolysaccharide, using Kdo2-lipid IVA as the acceptor and GDP-mannose (or synthetic ADP-mannose) as the donor . The mannosyl transferase is associated with the inner membrane . The apparent Km values for GDP-mannose and Kdo2-lipid IVA are 4.3 microM and 7.1 microM, respectively, in the presence of excess co-substrate . Extracts of E . coli do not catalyze GDP-mannose-dependent glycosylation of Kdo2-lipid IVA, but they are active when ADP-mannose is substituted for GDP-mannose . Given the structural similarity of ADP-mannose to ADP-heptose, we examined the possibility that heptosyl transferase I of E . coli (the product of the rfaC gene) catalyzes mannose transfer from ADP-mannose to Kdo2-lipid IVA . Extracts of E . coli mutants defective in the rfaC gene are unable carry out ADP-mannose-dependent glycosylation of Kdo2-lipid IVA . Plasmids bearing rfaC+ not only restore the missing activity but also direct its overexpression . Our assay using ADP-mannose as a substitute for ADP-heptose (which is not readily available) should facilitate the purification and characterization of heptosyl transferase I of E . coli . The GDP-mannose-dependent enzyme of R . leguminosarum may represent a functional equivalent of E . coli RfaC.

J Biol Chem, 1996 Dec 13, 271(50), 32112 - 8
Lipopolysaccharide biosynthesis in Rhizobium leguminosarum . Novel enzymes that process precursors containing 3-deoxy-D-manno-octulosonic acid; Brozek KA et al.; The lipopolysaccharide of Rhizobium leguminosarum differs from that of other Gram-negative organisms . R . leguminosarum lipid A lacks phosphate groups, but it contains a galacturonic acid residue at the 4'-position and an aminogluconate moiety in place of the usual glucosamine 1-phosphate unit . R . leguminosarum lipid A is esterified with a peculiar long chain fatty acid, 27-hydroxyoctacosanoate, not found in enteric Gram-negative bacteria, and the inner core of R . leguminosarum contains mannose and galactose in place of heptose . Despite these differences, the biosynthesis of R . leguminosarum lipid A is initiated by the same seven enzyme pathway as in Escherichia coli (Raetz, C . R . H . (1993) J . Bacteriol . 175, 5745-5753) to form the phosphorylated precursor, (Kdo)2-lipid IVA, which is then processed differently . We now describe several novel Rhizobium-specific enzymes that recognize and modify (Kdo)2-lipid IVA . The 1- and 4'-phosphatases were detected using (Kdo)2-{1-32P}-lipid IVA and (Kdo)2-{4'-32P}-lipid IVA, respectively, as shown by release of 32Pi . In the presence of GDP-mannose and/or UDP-galactose, membranes of R . leguminosarum first transferred mannose and then galactose to (Kdo)2-{4'-32P}-lipid IVA . In addition, at least two hydrophobic metabolites were generated from (Kdo)2-{4'-32P}-lipid IVA in a manner that was dependent upon both membranes and a cytosolic factor from R . leguminosarum . These compounds are attributed to novel acylations of (Kdo)2-{4'-32P}-lipid IVA . E . coli membranes and cytosol did not catalyze any of the unique reactions detected in R . leguminosarum extracts . Our findings establish the conservation and versatility of (Kdo)2-lipid IVA as a lipid A precursor in bacteria.

Gene, 1996 Dec 5, 182(1-2), 129 - 35
Cloning and sequence analysis of the Rhizobium etli ccmA and ccmB genes involved in c-type cytochrome biogenesis; Aguilar GR et al.; In this paper we describe the sequence analysis of two Rhizobium etli genes (ccmA and ccmB) which are believed to participate in the transport of the haem moiety to the periplasm in other bacterial species . The characterized DNA region was isolated by complementation of a R . etli Tn5mob induced mutant (CFN4201) which was affected in the production of c-type cytochromes . Sequence analysis of this region identified three open reading frames, two were identified as the ccmA and ccmB genes . The predicted protein sequence of ccmA showed significant homology with ATP binding proteins of the ABC-type transporter systems, while ccmB encodes for a hydrophobic protein probably associated with the ccmA gene product . The Tn5mob insertion in CFN4201 strain was located in the carboxy terminus of CcmB . Restriction mapping of the EcoRI fragment containing the Tn5mob insertion showed that it involved a deletion of approximately 1.5 kb . Mutagenesis of the wild-type region with a miniMu transposon and complementation analysis showed that the mutation in ccmB, and not the deletion, was responsible for the phenotype of CFN4201 strain and that ccmA and ccmB are independent transcription units . We found that a region located downstream of ccmB is reiterated twice, one near the chromosomal ccmA-ccmB locus while the second in plasmid e . Finally, CFN4201 membranes had detectable levels of C1 apoprotein which did not contain bound haem . This data could suggest that haem binding to the apoprotein occurs after the translocation of the apo form of c1 to the membrane.

Arch Latinoam Nutr, 1996 Dec, 44(4 Suppl 1), 44S - 47S
{Agricultural and nutritional importance of legumes}; Montilla JJ; The main ecophysiologic, agronomic and economic feature of legume plants is the development of tubercles and nodules in their apical system . Nodule formation occurs in most legume species provided a compatible type of Rhizobium bacteria is present in the soil . Nitrogen fixation in nodules renders these plants independent of nitrogen fertilizers, the most expensive of all goods in modern cereal agriculture . Considering that soils may get enriched in nitrogen through fixation in nodules and the decomposition of foliage when the aerial parts of legume plants are used as green fertilizers, only through the inclusion of legume crops within planned harvest schemes, it would be possible to achieve success in large scale production strategies . Legume crops are extensively produced in temperate climates areas in which, in addition to their use in animal nutrition, yields of 18 kg per person per year are obtained . In contrast, in the Third World countries located in tropical areas, legume production is scarce, with annual yields of 9 kg per person per year . Currently, it is proposed that the energy and protein intake should match that of the developed countries 40 years ago (i.e . 3000 Kcal and 70 g protein per day); for this, it would be necessary to have an average availability of 60 g of legume seeds per person per day . Therefore, the production of legume seeds should be increased . In addition, research aimed to study and exploit the agronomic potential of this rich botanical family should be strengthened through the formation of interdisciplinary groups.

Biochem Genet, 1996 Dec, 34(11-12), 453 - 65
Identification of two glutaminases in Rhizobium etli; Duran S et al.; We present evidence that Rhizobium etli has two glutaminases differentiated by their thermostability and electrophoretic mobility . The thermostable glutaminase (B) is constitutive, in contrast with the thermolabile glutaminase (A), which is positively regulated by glutamine and negatively regulated by ammonium and by the carbon source . In distinction to glutaminase A, glutaminase B plays a minor role in the utilization of glutamine as a carbon source, but it may play a role in maintaining the balance of glutamine and glutamate . By complementation of the Rhizobium etli LM16 mutant that lacks glutaminase A, we have cloned the gene that codes for this enzyme.

Microbiol Res, 1996 Dec, 151(4), 433 - 9
Strains of the genus Serratia as beneficial rhizobacteria of oilseed rape with antifungal properties; Kalbe C et al.; Isolates of Serratia have been isolated from the rhizosphere of oilseed rape . The percentage of Serratia in this microenvironment was determined as 12.4% of the total antifungal bacteria . Serratia liquefaciens, S . plymuthica and S . rubidaea were found . All of the isolates showed an antifungal activity against different phytopathogenic fungi in vitro but the efficiency of strains was different . The antifungal mechanisms of 18 selected strains were investigated . Direct antifungal effect may be based on antibiosis (production of prodigiosin and pyrrolnitrin) and production of lytic enzymes (chitinases and beta-1,3-glucanases) . Potent siderophores were secreted by the strains to improve the availability of iron . No strain was able to produce cyanide . Most of the strains secrete the plant growth hormone indole-acetic-acid which can directly promote the growth of roots . The mechanisms were specific for each isolate.

Microbiology, 1996 Dec, 142 ( Pt 12), 3381 - 8
The cytochrome bc1 complex but not CycM is necessary for symbiotic nitrogen fixation by Rhizobium leguminosarum; Wu G et al.; Following Tn5 mutagenesis of Rhizobium leguminosarum biovar viciae, two mutants in one complementation group were identified as being unable to fix nitrogen in pea nodules . Spectroscopic analysis revealed that the mutants had lowered levels of c-type cytochromes and cytochromes aa3, but increased levels of cytochrome d . Cells of the mutants were greatly reduced in their ability to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine but membranes prepared from them had increased levels of succinate- and NADH-dependent respiration . NADH oxidation by the mutants was insensitive to the respiratory inhibitor antimycin A, that targets the cytochrome bc1 complex . Molecular analysis of the mutants revealed that they were affected in the cytochrome bc1 complex . One of the mutants contained Tn5 in a gene homologous to that encoding cytochrome c1, and in the other the Tn5 was in DNA homologous to that encoding the cytochrome b component of the cytochrome bc1 complex . Haem staining revealed that haem proteins of M(r)31,000 and M(r)23,000 were absent from membranes from the mutants whereas an additional soluble c-type cytochrome protein of M(r)23,000 was present . We conclude that the larger of these two haem proteins corresponds to cytochrome c1 and, in its absence, the protein of M(r)23,000 does not remain associated with the membrane . Formation of this M(r)23,000 component was specifically blocked in a third respiratory-defective mutant which contained Tn5 in a region of DNA showing homology to a Bradyrhizobium Japonicum gene previously shown to encode the membrane-bound c-type cytochrome CycM . Although the cytochrome bc1 complex is essential for symbiotic nitrogen fixation, the other membrane-bound c-type cytochrome (CycM) is not.

Plant Mol Biol, 1996 Dec, 32(6), 1043 - 53
Localization of plasma membrane H+-ATPase in nodules of Phaseolus vulgaris L; Campos F et al.; Legume nodules have specialized transport functions for the exchange of carbon and nitrogen compounds between bacteroids and root cells . Plasma membrane-type (vanadate-sensitive) H+-ATPase energizes secondary active transporters in plant cells and it could drive exchanges across peribacteroidal and plasmatic membranes . A nodule cDNA corresponding to a major isoform of Phaseolus vulgaris H+-ATPase (designated BHA1) has been cloned . BHA1 is a functional proton pump because after removal of its inhibitory domain and can complement a yeast mutant unable to synthesize a H+-ATPase . BHA1 is not nodule-specific, since it is also expressed in roots of uninfected plants . It belongs to the subfamily of plasma membrane H+-ATPases defined by the Arabidopsis AHA1, AHA2 and AHA3 genes and the tobacco PMA4 and corn MHA2 genes . In situ hybridization in nodule sections indicates high expression of BHA1 limited to uninfected cells . These results were confirmed by immunocytochemistry . The relatively low expression of plasma membrane-type H+-ATPase in Rhizobium-infected cells put a note of caution on the origin of the vanadate-sensitive ATPase described in preparations of peribacteroidal membranes . Also, our results indicate that active transport in symbiotic nodules is most intense at the plasma membrane of uninfected cells and support a specialized role of uninfected tissue for nitrogen transport.

J Nat Prod, 1996 Dec, 59(12), 1137 - 42
Biological activities of the nortropane alkaloid, calystegine B2, and analogs: structure-function relationships; Goldmann A et al.; Calystegines, polyhydroxy nortropane alkaloids, are a recently discovered group of plant secondary metabolites believed to influence rhizosphere ecology as nutritional sources for soil microorganisms and as glycosidase inhibitors . Evidence is presented that calystegines mediate nutritional relationships under natural conditions and that their biological activities are closely correlated with their chemical structures and stereochemistry . Assays using synthetic (+)- and (-)-enantiomers of calystegine B2 established that catabolism by Rhizobium meliloti, glycosidase inhibition, and allelopathic activities were uniquely associated with the natural, (+)-enantiomer . Furthermore, the N-methyl derivative of calystegine B2 was not catabolized by R . meliloti, and it inhibited alpha-galactosidase, but not beta-glucosidase, whereas the parent alkaloid inhibits both enzymes . This N-methyl analog therefore could serve to construct a cellular or animal model for Fabry's disease, which is caused by a lack of alpha-galactosidase activity.

Plant Mol Biol, 1996 Dec, 32(5), 809 - 21
A novel type of DNA-binding protein interacts with a conserved sequence in an early nodulin ENOD12 promoter; Christiansen H et al.; The pea genes PsENOD12A and PsENOD12B are expressed in the root hairs shortly after infection with the nitrogen-fixing bacterium Rhizobium leguminosarum bv . viciae or after application of purified Nod factors . A 199 bp promoter fragment of the PsENOD12B gene contains sufficient information for Nod factor-induced tissue-specific expression . We have isolated a Vicia sativa cDNA encoding a 1641 amino acid protein, ENBP1, that interacts with the 199 bp ENOD12 promoter . Two different DNA-binding domains were identified in ENBP1 . A domain containing six AT-hooks interacts specifically with an AT-rich sequence located between positions -95 and -77 in the PsENOD12B promoter . A second domain in ENBP1 is a cysteine-rich region that binds to the ENOD12 promoter in a sequence non-specific but metal-dependent way . ENBP1 is expressed in the same cell types as ENOD12 . However, additional expression is observed in the nodule parenchyma and meristem . The presence of three small overlapping ORFs in the 5'-untranslated region of the ENBP1 cDNA indicates that ENBP1 expression might be regulated at the translational level . The interaction of ENBP1 with a conserved AT-rich element within the ENOD12 promoter and the presence of the ENBP1 transcript in cells expressing ENOD12 strongly suggest that ENBP1 is a transcription factor involved in the regulation of ENOD12 . Finally, the C-terminal region of ENBP1 shows strong homology to a protein from rat that is specifically expressed in testis tissue.

Plant Physiol, 1996 Dec, 112(4), 1437 - 46
Gene structure and differential regulation of the Rhizobium-induced peroxidase gene rip1; Peng HM et al.; Primary expression of the Rhizobium meliloti-induced peroxidase gene rip1 occurs prior to nodule morphogenesis, specifically at the site of impending rhizobial infection (D . Cook, D . Dreyer, D . Bonnet, M . Howell, E . Nony, K . VandenBosch {1995} Plant Cell 7: 43-55) . We examined the distribution and structure of rip1 transcript throughout nodule development . We determined that expression of rip1 in root tips is correlated with the competence of this zone for symbiotic association, whereas after rhizobial infection rip1 transcript is specifically associated with the zone of nodule development, including nascent nodule primordia . rip1 transcripts are characterized by multiple polyadenylation sites distributed within 200 to 400 bp of the translation stop site, and a single major transcription initiation site in close proximity to the rip1 open reading frame . Thus, rip1 expression is likely to be mediated through effects on a single transcription unit . Immediately 5' of the rip1 transcription unit DNA sequence analysis identified a 377-bp DNA element containing extensive repeat structure that is widely distributed in the Medicago truncatula genome.

Mol Plant Microbe Interact, 1996 Dec, 9(9), 869 - 73
Deletion analysis of the 5' untranslated region of the Rhizobium meliloti nodF gene; Kalinowski G et al.; Efficient establishment of the symbiosis between rhizobia and their host plants requires precise regulation of bacterial nod genes . The nod gene transcripts in Rhizobium meliloti have approximately 200 nucleotides of untranslated sequence 5' of the start codon (5' UTR) . We measured the significance of this region by constructing fusions between deletion derivatives of nodF and the reporter beta-glucuronidase (GUS) . Flavonoid-inducible expression of the fusions in R . meliloti was evident when extra copies of the positive transcriptional activators NodD1, NodD3, or SyrM were present . The fusions responded normally over a range of inducer concentrations in Rhizobium leguminosarum bv . trifolii . GUS assays in planta showed no significant difference between the deletion constructs and a wild-type fusion . We conclude that the 5' UTRs of the nod gene transcripts are unlikely to have a significant regulatory role.

Mol Plant Microbe Interact, 1996 Dec, 9(9), 843 - 9
Identification and distribution of plasmid-type A replicator region in rhizobia; Burgos PA et al.; Sinorhizobium meliloti strain GR4 harbors two cryptic plasmids, named pRmeGR4a and pRmeGR4b in addition to the symbiotic megaplasmids . The replicator region of plasmid pRmeGR4a has been recently cloned and sequenced . By DNA hybridization, homology to former replicator region was found on plasmid pRmeGR4b as well as on other plasmids harbored by S . meliloti and S . fredii strains . In these former bacteria, a PCR product of 362 bp was generated using pRmeGR4a-repC derived primers (C1 and C2) . DNA sequence analysis showed that the amplified repC fragments were closely related being classified into two subgroups designated A(I) and A(II) . Similarly, a PCR product of 482 bp was obtained when primers (C3 and C5) derived from pRmeGR4a repC-upstream non-coding DNA region (IR) were used . DNA sequence analysis of the corresponding amplified products showed that, as occurs, with repC the IRs were also conserved . In addition, we designed a set of primers (P2/P4), derived from the S . meliloti and S . fredii consensus sequence encompassing the IRs and repC loci, that are able to recognize homologous plasmid-type A replicator regions in Rhizobium . By using these primers, we determined that the former replicator region is widespread in S . meliloti indigenous populations and that its frequency within the infective isolates depends on the host plant . Furthermore, it is shown that the replicator region type A is linked to cryptic plasmids in S . meliloti and S . fredii, whereas it is located in the pSym of R . tropici strains.

J Bacteriol, 1996 Dec, 178(24), 7265 - 75
The Bradyrhizobium japonicum fegA gene encodes an iron-regulated outer membrane protein with similarity to hydroxamate-type siderophore receptors; LeVier K et al.; Iron is important in the symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, yet little is known about rhizobial iron acquisition strategies . Analysis of outer membrane proteins (OMPs) from B . japonicum 61A152 identified three iron-regulated OMPs in the size range of several known receptors for Fe(III)-scavenging siderophores . One of the iron-regulated proteins, FegA, was purified and microsequenced, and a reverse genetics approach was used to clone a fegA-containing DNA fragment . Sequencing of this fragment revealed a single open reading frame of 750 amino acids . A putative N-terminal signal sequence of 14 amino acids which would result in a mature protein of 736 amino acids with a molecular mass of 80,851 Da was predicted . FegA shares significant amino acid similarity with several Fe(III)-siderophore receptors from gram-negative bacteria and has greater than 50% amino acid similarity and 33% amino acid identity with two {corrected} bacterial receptors for hydroxamate-type Fe(III)-siderophores . A dendrogram describing total inferred sequence similarity among 36 TonB-dependent OMPs was constructed; FegA grouped with Fe(III)-hydroxamate receptors . The transcriptional start site of fegA was mapped by primer extension analysis, and a putative Fur-binding site was found in the promoter . Primer extension and RNA slot blot analysis demonstrated that fegA was expressed only in cells grown under iron-limiting conditions . This is the first report of the cloning of a gene encoding a putative Fe(III)-siderophore receptor from nitrogen-fixing rhizobia.

J Bacteriol, 1996 Dec, 178(24), 7159 - 66
Use of green fluorescent protein to visualize the early events of symbiosis between Rhizobium meliloti and alfalfa (Medicago sativa); Gage DJ et al.; A gene encoding a variant of green fluorescent protein (GFP) of Aequorea victoria was put under the control of a promoter which is constitutive in Rhizobium meliloti . The heterologous GFP gene was expressed at high levels during all stages of symbiosis, allowing R . meliloti cells to be visualized as they grew in the rhizosphere, on the root surface, and inside infection threads . In addition, nodules that were infected with bacteria which were synthesizing GFP fluoresced when illuminated with blue light . GFP-tagged bacteria could be seen inside infection threads, providing the opportunity to measure the growth rate and determine the patterns of growth of R . meliloti residing inside its host plant.

J Bacteriol, 1996 Dec, 178(24), 7099 - 105
Utilization of orotate as a pyrimidine source by Salmonella typhimurium and Escherichia coli requires the dicarboxylate transport protein encoded by dctA; Baker KE et al.; Mutants deficient in orotate utilization (initially termed out mutants) were isolated by selection for resistance to 5-fluoroorotate (FOA), and the mutations of 12 independently obtained isolates were found to map at 79 to 80 min on the Salmonella typhimurium chromosome . A gene complementing the mutations was cloned and sequenced and found to possess extensive sequence identity to characterized genes for C4-dicarboxylate transport (dctA) in Rhizobium species and to the sequence inferred to be the dctA gene of Escherichia coli . The mutants were unable to utilize succinate, malate, or fumarate as sole carbon source, an expected phenotype of dctA mutants, and introduction of the cloned DNA resulted in restoration of both C4-dicarboxylate and orotate utilization . Further, succinate was found to compete with orotate for entry into the cell . The S . typhimurium dctA gene encodes a highly hydrophobic polypeptide of 45.4 kDa, and the polypeptide was found to be enriched in the membrane fraction of minicells harboring a dctA+ plasmid . The DNA immediately upstream of the deduced -35 region contains a putative cyclic AMP-cyclic AMP receptor protein complex binding site, thus affording an explanation for the more effective utilization of orotate with glycerol than with glucose as carbon source . The E . coli dctA gene was cloned from a lambda vector and shown to complement C4-dicarboxylate and orotate utilization in FOA-resistant mutants of both E . coli and S . typhimurium . The accumulated results demonstrate that the dctA gene product, in addition to transporting C4-dicarboxylates, mediates the transport of orotate, a cyclic monocarboxylate.

J Bacteriol, 1996 Dec, 178(23), 6802 - 9
Cloning and characterization of the katA gene of Rhizobium meliloti encoding a hydrogen peroxide-inducible catalase; Herouart D et al.; To investigate the involvement of bacterial catalases of the symbiotic gram-negative bacterium Rhizobium meliloti in the development of Medicago-Rhizobium functional nodules, we cloned a putative kat gene by screening a cosmid library with a catalase-specific DNA probe amplified by PCR from the R . meliloti genome . Nucleotide sequence analysis of a 1.8-kb DNA fragment revealed an open reading frame, called katA, encoding a peptide of 562 amino acid residues with a calculated molecular mass of 62.9 kDa . The predicted amino acid sequence showed a high homology with the primary structure of monofunctional catalases from eucaryotes and procaryotes . The katA gene was localized on the chromosome, and the katA gene product was essentially found in the periplasmic space . A katA::Tn5 mutant was obtained and showed a drastic sensitivity to hydrogen peroxide, indicating an essential protective role of KatA . However, neither Nod nor Fix phenotypes were impaired in the mutant, suggesting that KatA is not essential for nodulation and establishment of nitrogen fixation . Exposure to a sublethal concentration of H2O2 enhanced KatA activity (100-fold) and also increased survival to subsequent H2O2 exposure at higher concentrations . No protection is observed in katA::Tn5, indicating that KatA is the major component of an adaptive response.

J Bacteriol, 1996 Dec, 178(23), 6706 - 13
Stigmatella aurantiaca fruiting body formation is dependent on the fbfA gene encoding a polypeptide homologous to chitin synthases; Silakowski B et al.; Stigmatella aurantiaca is a prokaryotic organism that undergoes a multicellular cycle of development resulting in the formation of a fruiting body . For analyzing this process, mutants defective in fruiting body formation have been induced by transposon mutagenesis using a Tn5-derived transposon . About 800 bp upstream of the transposon insertion of mutant AP182 which inactivates a gene (fbfB) involved in fruiting, a further gene (fbfA) needed for fruiting body formation was detected . Inactivation of fbfA leads to mutants which form only non-structured clumps instead of the wild-type fruiting body . The mutant phenotype of fbfA mutants can be partially suppressed by mixing the mutant cells with cells of some independent mutants defective in fruiting body formation . The fbfA gene is transcribed after 8 h of development as determined by measuring the induction of beta-galactosidase activity of a fbfA-delta(trp)-lacZ fusion gene and by Northern (RNA) analysis using an insertion encoding a stable mRNA . The predicted polypeptide FbfA shows a homology of about 30% to NodC of rhizobia, an N-acetylglucosamine-transferase which is involved in the synthesis of the sugar backbone of lipo-oligosaccharides . These induce the formation of the root nodules in the Papilionaceae . Besides the predicted molecular mass of 45.5 kDa, the hydropathy profile reveals a structural relationship to the NodC polypeptide.

Curr Microbiol, 1996 Dec, 33(6), 371 - 6
Roles of Exopolysaccharides and Lipopolysaccharides in the Adsorption of the Siphovirus Phage NM8 to Rhizobium meliloti M11S Cells
Defives C, Werquin M, Mary P, Hornez JP.
The exopolysaccharides produced by Rhizobium meliloti M11S inhibited nonspecifically the adsorption of phage NM8 by coating the cells . But lipopolysaccharides (LPS) had a specific inhibitory effect . Only the polysaccharide moiety of LPS, composed of glucose, glucosamine, galactose, 3-deoxy-D-manno-octulosonic acid (KDO), and large amounts of sialic acid, inhibited phage adsorption; neither the lipid A moiety nor a cellular glucan was involved . Rhizobium strains lacking sialic acids did not bind phage NM8 . Inhibition of phage binding by lectin specific for N-acetylneuraminic acid demonstrated that phage NM8 bound to sialic acids . Preincubation of the phage with monosaccharides showed that inactivation of phage was very stereospecific for N-acetylneuraminic acid . Phage adsorption was also strongly inhibited by N-acetylglucosamine, which is not present in the LPS . Therefore, the receptor for phage NM8 appears to be a saccharide site, probably involving the acetyl groups of sialic acids.

FEMS Microbiol Lett, 1996 Nov 15, 145(1), 33 - 40
Symbiotic nitrogen fixation does not require adenylylation of glutamine synthetase I in Rhizobium meliloti; Arcondeguy T et al.; Symbiotic nitrogen fixation is accompanied by a shift of Rhizobium nitrogen metabolism from ammonium assimilation to ammonium export, which probably involves genetic or metabolic regulation of glutamine synthetase activity . In free-living Rhizobium meliloti glutamine synthetase I (GSI) is regulated post-translationally by reversible adenylylation in response to ammonium addition . Moreover, full expression of the GSI gene glnA requires the transcriptional activator, NtrC . A glnA1 mutant synthesizing a non-adenylylatable GSI produces normal nitrogen-fixing nodules on alfalfa: GSI adenylylation is dispensable for symbiotic nitrogen fixation . This is rationalized by the observation that less GS protein is present in R . meliloti bacteroids than in free-living bacterial cells.

FEMS Microbiol Lett, 1996 Nov 15, 145(1), 1 - 8
Molecular and immunological characterization of the major outer membrane proteins of Brucella; Cloeckaert A et al.; The major outer membrane proteins (OMPs) of Brucella spp . were initially identified in the early 1980s by selective extraction techniques and classified according to their apparent molecular mass as 36-38 kDa OMPs or group 2 porin proteins and 31-34 kDa and 25-27 kDa OMPs which belong to the group 3 proteins . Variation in apparent molecular mass is essentially due to association with peptidoglycan subunits of different sizes . Two genes, omp2a and omp2b, which are closely linked in the Brucella genome, and which share a great degree of homology (> 85%), encode the 36 kDa porin proteins, now named Omp2a and Omp2b proteins respectively . Two genes code for the group 3 OMPs and are named omp25 and omp31 . The predicted amino acid sequences of omp25 and omp31 share 34% identity . Furthermore, all Brucella major OMPs share amino acid sequence homology with the major OMPs RopA or RopB of Rhizobium leguminosarum, which supports the close genetic relationship of brucellae with members of the alpha-2 subdivision of the class Proteobacteria . Another characteristic common to the major OMPs of R . leguminosarum and Brucella is that they are tightly, probably covalently, associated with the peptidoglycan . The major OMP genes display diversity among Brucella species, biovars and strains allowing their differentiation, and the polymorphic markers identified have brought new insights into the evolutionary development of the genus Brucella, antigenic variability of brucellae, and future prospects in the field of vaccine development.

Proc Natl Acad Sci U S A, 1996 Nov 12, 93(23), 12998 - 3003
Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli cells by using green fluorescent protein; Ma X et al.; In the current model for bacterial cell division, FtsZ protein forms a ring that marks the division plane, creating a cytoskeletal framework for the subsequent action of other proteins such as FtsA . This putative protein complex ultimately generates the division septum . Herein we report that FtsZ and FtsA proteins tagged with green fluorescent protein (GEP) colocalize to division-site ring-like structures in living bacterial cells in a visible space between the segregated nucleoids . Cells with higher levels of FtsZ-GFP or with FtsA-GFP plus excess wild-type FtsZ were inhibited for cell division and often exhibited bright fluorescent spiral tubules that spanned the length of the filamentous cells . This suggests that FtsZ may switch from a septation-competent localized ring to an unlocalized spiral under some conditions and that FtsA can bind to FtsZ in both conformations . FtsZ-GFP also formed nonproductive but localized aggregates at a higher concentration that could represent FtsZ nucleation sites . The general domain structure of FtsZ-GFP resembles that of tubulin, since the C terminus of FtsZ is not required for polymerization but may regulate polymerization state . The N-terminal portion of Rhizobium FtsZ polymerized in Escherichia coli and appeared to copolymerize with E . coli FtsZ, suggesting a degree of interspecies functional conservation . Analysis of several deletions of FtsA-GFP suggests that multiple segments of FtsA are important for its localization to the FtsZ ring.

Gene, 1996 Nov 7, 179(1), 141 - 6
Rhizobium meliloti exopolysaccharides: synthesis and symbiotic function; Gonzalez JE et al.; Bacterial exopolysaccharide (EPS) is required for establishment of the nitrogen-fixing symbiosis between Rhizobium meliloti and its host plant, Medicago sativa (alfalfa), but the precise role of EPS in this interaction is not well defined . Bacterial mutants which fail to produce EPS induce nodules on the roots of the host plant, but fail to invade these root nodules . Research conducted in our lab and others suggests that EPS plays a specific role in the R . meliloti-M . sativa symbiosis . A common theme emerging from these studies is that small quantities of low-molecular-weight (LMW) EPS are sufficient to mediate successful invasion by R . meliloti mutants which fail to produce EPS, implying that LMW EPS may act as a signaling molecule during this process.

Gene, 1996 Nov 7, 179(1), 61 - 71
Metabolic engineering: prospects for crop improvement through the genetic manipulation of phenylpropanoid biosynthesis and defense responses--a review; Dixon RA et al.; In leguminous plants such as the forage legume alfalfa, products of the phenylpropanoid pathway of secondary metabolism are involved in interactions with beneficial microorganisms (flavonoid inducers of the Rhizobium symbiosis), and in defense against pathogens (isoflavonoid phytoalexins) . In addition, the phenylpropane polymer lignin is a major structural component of secondary vascular tissue and fibers in higher plants . the recent isolation of genes encoding key enzymes of the various phenylpropanoid branch pathways opens up the possibility of engineering important crop plants such as alfalfa for: (a) improved forage digestibility, by modification of lignin composition and/or content; (b) increased or broader-spectrum disease resistance, by introducing novel phytoalexins or structural variants of the naturally occurring phytoalexins, or by modifying expression of transcriptional regulators of phytoalexin pathways; and (c) enhanced nodulation efficiency, by engineering over-production of flavonoid nod gene inducers . The basic biochemistry and molecular biology underlying these strategies is briefly reviewed, and recent progress with transgenic plants summarized . The potential importance of metabolic compartmentation for attempts to engineer phenylpropanoid biosynthetic pathways is also discussed . Over-expression of an alfalfa glucanase-encoding gene confers significant protection against Phytophthora in alfalfa, possibly via indirect effects on phenylpropanoid metabolism.

Mol Microbiol, 1996 Nov, 22(3), 427 - 36
Different roles of CheY1 and CheY2 in the chemotaxis of Rhizobium meliloti; Sourjik V et al.; Cells of Rhizobium meliloti swim by the unidirectional, clockwise rotation of their right-handed helical flagella and respond to tactic stimuli by modulating the flagellar rotary speed . We have shown that wild-type cells respond to the addition of proline, a strong chemoattractant, by a sustained increase in free-swimming speed (chemokinesis) . We have examined the role of two response regulators, CheY1 and CheY2, and of CheA autokinase in the chemotaxis and chemokinesis of R . meliloti by comparing wild-type and mutant strains that carry deletions in the corresponding genes . Swarm tests, capillary assays, and computerized motion analysis revealed that (i) CheY2 alone mediates 60 to 70% of wild-type taxis, whereas CheY1 alone mediates no taxis, but is needed for the full tactic response; (ii) CheY2 is the main response regulator directing chemokinesis and smooth swimming in response to attractant, whereas CheY1 contributes little to chemokinesis, but interferes with smooth swimming; (iii) in a CheY2-overproducing strain, flagellar rotary speed increases upon addition and decreases upon removal of attractant; (iv) both CheY2 and CheY1 require phosphorylation by CheA for activity . We conclude that addition of attractant causes inhibition of CheA kinase and removal causes activation, and that consequent production of CheY1-P and CheY2-P acts to slow the flagellar motor . The action of the chief regulator, CheY2-P, on flagellar rotation is modulated by CheY1, probably by competition for phosphate from CheA.

Appl Environ Microbiol, 1996 Nov, 62(11), 4191 - 4
Simultaneous detection of different Rhizobium strains marked with either the Escherichia coli gusA gene or the Pyrococcus furiosus celB gene; Sessitsch A et al.; A new marker system for gram-negative bacteria was developed on the basis of the celB gene from the hyperthermophilic archaeon Pyrococcus furiosus, which encodes a thermostable beta-glucosidase with a high level of beta-galactosidase activity . The celB gene is highly suitable as a marker for studying plant-bacterium interaction because endogenous background beta-glucosidase and beta-galactosidase enzyme activity can readily be inactivated by heat and because inexpensive substrates for detection are commercially available . Two celB-expressing transposons were constructed for use in ecological studies of a variety of gram-negative bacteria . The combined use of the gusA marker gene and celB allowed the simultaneous detection of several Rhizobium strains on a plant, and multiple-strain occupancy of individual modules also could be easily detected.

J Bacteriol, 1996 Nov, 178(21), 6403 - 6
Genetic and chemical characterization of a mutant that disrupts synthesis of the lipopolysaccharide core tetrasaccharide in Rhizobium leguminosarum; Allaway D et al.; A 2-kb region that complements the Tn5-derived lipopolysaccharide (LPS) rough mutant Rhizobium leguminosarum RU301 was sequenced . Two open reading frames (ORFs) were identified . The first ORF (lpcA) is homologous to a family of bacterial sugar transferases involved in LPS core tetrasaccharide biosynthesis . ORF2 (lpcB), in which Tn5 transposed, has no significant homology to any DNA in the GenBank-EMBL databases . Chemical characterization of LPS produced by strain RU301 demonstrated that the 3-deoxy-D-manno-2-octulosonic acid (Kdo) residue which normally attaches the core tetrasaccharide to the O chain was missing, suggesting that IpcB may encode a CMP-Kdo:LPS Kdo transferase.

Mol Plant Microbe Interact, 1996 Nov, 9(8), 720 - 8
Characterization and mutational analysis of nodHPQ genes of Rhizobium sp . strain N33; Cloutler J et al.; We have shown, by sequencing the nodulation gene region of Rhizobium sp . strain N33 previously isolated from the Canadian high arctic, that the nodHPQ genes are located in a 4.8-kb region downstream of nodBCIJ . The open reading frames of nodHPQ are 747, 906, and 1941 nucleotides long, respectively . The strain N33 genome contains one copy of nodH and two copies of nodPQ that are homologous to those genes in Rhizobium meliloti . Tn5 insertions in the nodHPQ genes of strain N33 did not affect the formation of nodules on the two homologous hosts, Astragalus cicer and Onobrychis viciifolia . Since strain N33 contains the nodBCIJHPQ genes and the recently sequenced nodAFEG genes, we looked for similar host range with R . meliloti . Strain N33 and R . meliloti strains A2 and RCR2011 were shown to induce the formation of root nodules on plants of O . viciifolia . However, strain N33, compared with R . meliloti strains, was able to elicit a few, white, empty, root nodules on Medicago sativa . R . meliloti strains, compared with strain N33, were shown to induce only few nodules containing bacteria on A . cicer . Induction of nod genes transcription in strain N33 was shown to be induced by a variety of flavonoid compounds that are different from those inducing nod genes from R . meliloti.

Mol Plant Microbe Interact, 1996 Nov, 9(8), 689 - 95
Expression of cysteine protease genes in pea nodule development and senescence; Kardailsky IV et al.; Coding sequences for two cysteine proteases were amplified from cDNA derived from pea nodule mRNA using primers based on conserved regions of plant cysteine proteases . One of the amplified cDNA sequences corresponded to a previously described cysteine protease gene, Cyp15a, expressed in pea shoots in response to dehydration (J.T . Jones and J.E . Mullet, Plant Mol . Biol . 28:1055-1065, 1995) . Inside the pea root nodule, in situ hybridization revealed that this gene is expressed strongly in the apical region and more weakly in the uninfected cortex and in the central infected tissue where nitrogen fixation takes place . The complete sequence of the cDNA corresponding to the other gene, PsCyp1, was obtained . Expression of this gene, which was studied both on RNA blots and in situ, showed good correlation with the onset of nodule senescence . In situ hybridization studies revealed that PsCyp1 was expressed in senescent infected tissue at the base of the nodule . This signal was just detectable in normal symbiotically wild-type nodules but was much stronger in the early senescing nodules formed by a symbiotically defective mutant of Rhizobium leguminosarum.

Mol Plant Microbe Interact, 1996 Nov, 9(8), 671 - 80
Analysis of the C-terminal secretion signal of the Rhizobium leguminosarum nodulation protein NodO; a potential system for the secretion of heterologous proteins during nodule invasion; Sutton JM et al.; We used deletions to analyze the domains required for secretion of the Rhizobium leguminosarum bv . viciae nodulation protein, NodO, by the sec-independent pathway . Deletion of the C-terminal 24 amino-acids (residues 261 to 284) reduced secretion by at least 95% . A monoclonal antibody that recognizes the C-terminal domain of NodO was used to identify four nested deletions that retained the C-terminal 24 residues of NodO but had lost up to 133 residues (amino acids 128 to 259); all four proteins were secreted into the growth medium with an efficiency between 50 and 90% of normal . A deleted derivative of NodO that retained residues 1 to 21 and 167 to 284 (and therefore lacked most of the N-terminal Ca(2+)-binding domain) was secreted at around 80% of normal efficiency . Taken together, these observations indicate that the C-terminal 24 amino acids are sufficient for NodO secretion although the region adjacent to this domain appears to affect secretion efficiency . A derivative of the Escherichia coli alkaline phosphatase (phoA) gene was cloned into two derivatives of nodO such that PhoA (lacking the N-terminal transit peptide) was in-frame at both ends, with the C terminus fused to either the last 24 or 50 amino acids of NodO . These fusion proteins were secreted at 40 and 80% of the wild-type level, respectively, and the larger of the two retained alkaline phosphatase activity . A hybrid protein, containing E . coli beta-glucuronidase (GUS) fused to the N terminus of NodO, was not secreted, and it reduced the levels of wild-type NodO secreted by R . leguminosarum bv . viciae . The nature of the NodO C-terminal secretion signal is discussed with regard to its use as a delivery system for heterologous proteins useful for investigating the Rhizobium-legume interaction.

J Biol Chem, 1996 Oct 18, 271(42), 26435 - 42
Cooperative binding of DctD to the dctA upstream activation sequence of Rhizobium meliloti is enhanced in a constitutively active truncated mutant; Scholl D et al.; DctD, a sigma54-dependent, two-component regulator, binds to promoter distal (A) and promoter proximal (B) sites in an activation sequence located upstream of the dctA promoter . We report gel filtration and quantitative DNase I footprint experiments supporting a model in which DctD2 binds to these sites cooperatively . The global analysis of upstream activation sequences containing sites A and B, A and B one-half helical turn out of phase, and only B yielded values for the intrinsic and cooperative binding free energies of DeltaG0A = -9.5 +/- 0.3, DeltaG0B = -11.2 +/- 0.2, and DeltaG0AB = -2.5 +/- 0.5 . A separate analysis of data from upstream activation sequences containing site A and a point mutant of site B, and site A and mutant site B one-half helical turn out of phase confirmed the estimate of cooperativity, yielding free energy values of DeltaG0A = -9.4 +/- 0.2, DeltaG0B(G-->C) = -10.0 +/- 0.2, and DeltaG0AB(G-->C) = -2.2 +/- 0.4 . We previously showed that removing the two-component receiver domain from DctD, making DctDDelta(1-142), yields a constitutively active truncated protein . Global analysis of binding data for DctDDelta(1-142) showed that this constitutively active mutant has intrinsic binding energies equal to that of the inactive DctD protein, but that it displays significantly higher cooperativity (DeltaG0A = -9.4 +/- 0.6, DeltaG0B = -11.1 +/- 0.3, and DeltaG0AB = -3.8 +/- 0.6.).

Curr Opin Genet Dev, 1996 Oct, 6(5), 631 - 8
The role of lipochitooligosaccharides in root nodule organogenesis and plant cell growth; Schultze M et al.; Lipochitooligosaccharides (Nod signals) excreted by rhizobia induce the formation of symbiotic root nodules in leguminous plants . This process is host plant specific, depending on the structural modifications of Nod signals . Rapid responses of plant roots in single cell assays have provided powerful tools in dissecting Nod signal transduction pathways and in elucidating the molecular basis of host specificity . Recent findings indicate that lipochitooligosaccharides, as well as symbiosis-related genes, also function in non legumes, pointing to a general role for these elements in plant morphogenesis.

Chem Biol, 1996 Oct, 3(10), 841 - 50
Nonsteric factors dominate binding of nitric oxide, azide, imidazole, cyanide, and fluoride to the rhizobial heme-based oxygen sensor FixL; Winkler WC et al.; BACKGROUND: The FixL protein is a heme-based sensor . Binding of oxygen to a unique heme domain inhibits a kinase domain of the type found in two-component regulators . Oxygen association is slow, but the dissociation rate is comparable to that of myoglobins . We have probed the size and chemistry of the FixL heme pocket by measuring the affinites, on rates and off rates for a wide variety of ferric heme ligands . Cyanide, but not fluoride, regulates the kinase activity . To examine how the sensory heme domain interacts with the kinase, we asked how the presence of the kinase domain affects ligand binding . RESULTS: The affinities of ferric FixL for heme ligands follow the same trend as their pKa values: cyanide > 4-methyl imidazole > imidazole > fluoride > azide >> thiocyanate . The association rates follow the reverse trend . Striking differences from myoglobin include a 6-fold greater affinity for, and faster binding to, the bulky ligand imidazole, a 14-fold faster on rate for nitric oxide, a 2 800-fold lower affinity for azide, and a complete failure to bind thiocyanate . The presence of the kinase domain does not alter the affinity or binding kinetics of the high-spin ligand fluoride, but affects the off rates of other ligands . The EPR spectrum shows a characteristic pentacoordinate nitrosyl heme, indicating that the Fe-His bond in FixL is strained . CONCLUSIONS: The importance of ligand deprotonation to the on rates and the fact that large ligands bind readily indicate that the heme pocket is open and apolar . Ligand basicity strongly influences the strength of binding . The destabilization of inhibitory ligands by the presence of the kinase domain is consistent with a 'load' imposed by coupling to the inactivating mechanism.

Mol Microbiol, 1996 Oct, 22(2), 303 - 14
The NodA proteins of Rhizobium meliloti and Rhizobium tropici specify the N-acylation of Nod factors by different fatty acids; Debelle F et al.; Rhizobia synthesize mono-N-acylated chitooligosaccharide signals, called Nod factors, that are required for the specific infection and nodulation of their legume hosts . The biosynthesis of Nod factors is under the control of nodulation (nod) genes, including the nodABC genes present in all rhizobial species . The N-acyl substitution can vary between species and can play a role in host specificity . In Rhizobium meliloti, an alfalfa symbiont, the acyl chain is a C16 unsaturated or a (omega-1) hydroxylated fatty acid, whereas in Rhizobium tropici, a bean symbiont, it is vaccenic acid (C18:1) . We constructed R . meliloti derivatives having a non-polar deletion of nodA, and carrying a plasmid with either the R . meliloti or the R . tropici nodA gene . The strain with the R . tropici nodA gene produced Nod factors acylated by vaccenic acid, instead of the C16 unsaturated or hydroxylated fatty acids characteristic of R . meliloti Nod factors, and infected and nodulated alfalfa with a significant delay . These results show that NodA proteins of R . meliloti and R . tropici specify the N-acylation of Nod factors by different fatty acids, and that allelic variation of the common nodA gene can contribute to the determination of host range.

Int J Syst Bacteriol, 1996 Oct, 46(4), 1016 - 24
Classification of bacteria nodulating Lathyrus japonicus and Lathyrus pratensis in northern Quebec as strains of Rhizobium leguminosarum biovar viciae; Drouin P et al.; The diversity of two populations of rhizobia isolated from Lathyrus japonicus (30 strains) and Lathyrus pratensis (49 strains) growing in northern regions of Quebec, Canada, was determined on the basis of phenotypic characteristics, multilocus enzyme electrophoresis, DNA-DNA homology, and 16S ribosomal DNA sequencing . According to numerical analysis of phenotypic characteristics, strains were divided into four groups . Strains isolated from L . pratensis fell in groups I to III; the latter included reference strains of Rhizobium leguminosarum . All strains isolated from L . japonicus were included in group IV . All strains had nodulation characteristics similar to those of R . leguminosarum bv . viciae . Strains isolated from L . japonicus originating from an arctic region were usually able to grow at 5 degrees C and were more likely to be tolerant to copper (CuCl2.H2O, 100 micrograms/ml) and lead {Pb(CH3COO)2, 500 micrograms/ml} than strains isolated from L . pratensis from a boreal zone . However, both populations of Lathyrus strains were adapted to the cold in comparison to reference strains from temperate regions . Each population had similar genetic diversity (H = 0.45), determined by multilocus enzyme electrophoresis of the loci encoding eight enzymes, but the diversity obtained by analyzing all strains including the reference strains (H = 0.58) was higher . Representative strains of both populations showed high levels of DNA homology among themselves and with R . leguminosarum . Partial sequences of the 16S ribosomal RNA genes were similar to those reported for R . leguminosarum bv . viciae . We conclude that the strains isolated from L . japonicus and L . pratensis belong to R . leguminosarum bv . viciae but are distinguishable by growth at 5 degrees C, which is a characteristic related to their geographic origin.

Int J Syst Bacteriol, 1996 Oct, 46(4), 972 - 80
Sinorhizobium medicae sp . nov., isolated from annual Medicago spp; Rome S et al.; The taxonomic position of isolates of a new genomic species (designated genomic species 2) obtained from several annual Medicago species and originating from different geographical locations was established through the results of phenotypic tests (including the results of auxanographic and biochemical tests and symbiotic properties) and 16S rRNA phylogenetic inferences . A comparison of the complete 16S rRNA sequence of a representative of genomic species 2 (strain A 321T {T = type strain}) with the 16S rRNA sequences of other members of the Rhizobiaceae and closely related taxa showed that genomic species 2 was phylogenetically related to Sinorhizobium meliloti, Sinorhizobium fredii, Sinorhizobium saheli, and Sinorhizobium teranga . The levels of sequence similarity and observed numbers of nucleotide substitutions in Sinorhizobium strains indicated that A 321T and S . meliloti exhibited the highest level of sequence similarity (99.7%), with four nucleotide substitutions and one deletion . The results of a numerical analysis based on data from 63 auxanographic and biochemical tests clearly separated genomic species 2 isolates from S . meliloti . Genomic species 2 isolates nodulated and fixed nitrogen with Medicago polymorpha, whereas S . meliloti isolates were ineffective and formed rudimentary nodules on this host plant . On the basis of phenotypic and 16S sequence analysis data, genomic species 2 isolates cannot be assigned to a previously described species . We propose that these isolates belong to a new species, Sinorhizobium medicae.

J Bacteriol, 1996 Oct, 178(20), 5960 - 70
Pyruvate carboxylase from Rhizobium etli: mutant characterization, nucleotide sequence, and physiological role; Dunn MF et al.; Pyruvate carboxylase (PYC), a biotin-dependent enzyme which catalyzes the conversion of pyruvate to oxaloacetate, was hypothesized to play an important anaplerotic role in the growth of Rhizobium etli during serial subcultivation in minimal media containing succinate (S . Encarnacion, M . Dunn, K . Willms, and J . Mora, J . Bacteriol . 177:3058-3066, 1995) . R . etli and R . tropici pyc::Tn5-mob mutants were selected for their inability to grow in minimal medium with pyruvate as a sole carbon source . During serial subcultivation in minimal medium containing 30 mM succinate, the R . etli parent and pyc mutant strains exhibited similar decreases in growth rate with each subculture . Supplementation of the medium with biotin prevented the growth decrease of the parent but not the mutant strain, indicating that PYC was necessary for the growth of R . etli under these conditions . The R . tropici pyc mutant grew normally in subcultures regardless of biotin supplementation . The symbiotic phenotypes of the pyc mutants from both species were similar to those of the parent strains . The R . etli pyc was cloned, sequenced, and found to encode a 126-kDa protein of 1,154 amino acids . The deduced amino acid sequence is highly homologous to other PYC sequences, and the catalytic domains involved in carboxylation, pyruvate binding, and biotinylation are conserved . The sequence and biochemical data show that the R . etli PYC is a member of the alpha4, homotetrameric, acetyl coenzyme A-activated class of PYCs.

J Biol Chem, 1996 Sep 20, 271(38), 23400 - 6
Molecular cloning and characterization of a putative mouse hyaluronan synthase; Spicer AP et al.; We report the isolation of a novel mouse gene which encodes a putative hyaluronan synthase . The cDNA was identified using degenerate reverse transcriptase-polymerase chain reaction . Degenerate primers were designed based upon an alignment of the amino acid sequences of Streptococcus pyogenes HasA, Xenopus laevis DG42, and Rhizobium meliloti NodC . A mouse embryo cDNA library was screened with the resultant polymerase chain reaction product, and multiple cDNA clones spanning 3 kilobase pairs (kb) were isolated . The open reading frame predicted a 63-kDa protein with several transmembrane sequences, multiple consensus phosphorylation sites, and four putative hyaluronan binding motifs . The amino acid sequence displayed 55% identity to mouse HAS, 56% identity to Xenopus DG42, and 21% identity to Streptococcus HasA . Northern analysis identified transcripts of 4.8 kb and 3.2 kb, which were expressed highly in the developing mouse embryo and at lower levels in adult mouse heart, brain, spleen, lung, and skeletal muscle . Transfection experiments demonstrated that mouse Has2 could direct hyaluronan coat biosynthesis in transfected COS cells, as evidenced by a classical particle exclusion assay . These results suggest that mammalian HA synthase activity is regulated by at least two related genes . Accordingly, we propose the name Has2 for this gene.

Gene, 1996 Sep 16, 173(2), 183 - 7
The role of the nodI and nodJ genes in the transport of Nod metabolites in Rhizobium etli; Cardenas L et al.; A kinetic analysis of secretion of lipo-chitin oligosaccharides (LCO) produced by Rhizobium etli (Re) wild-type (wt) strain and derivatives carrying disrupted nodI or nodJ genes was performed . LCO were detected in the growth media of the wt strain as early as 1 h after nod gene induction . In contrast, strains carrying nodI or nodJ mutations secreted less LCO, and accumulated LCO metabolites intracellularly after 4 h of induction . These Re mutants presented a delayed nodulation phenotype and a reduction in the maximum number of nodules formed in Phaseolus vulgaris roots.

FEBS Lett, 1996 Sep 16, 393(2-3), 273 - 9
Rhizobium fredii synthesizes an array of lipooligosaccharides, including a novel compound with glucose inserted into the backbone of the molecule; Bec-Ferte MP et al.; Flavonoid cues from the plant host cause symbiotic, nitrogen-fixing rhizobia to synthesize lipochitooligosaccharides (LCOs), which act as return signals to initiate the nodulation process . Rhizobium fredii strain USDA257 is known to produce four LCOs, all substituted with vaccenic acid (C18:1) . We show here that a mutant strain can replace vaccenic acid with a C16:0 side chain, and that strain USDA191 synthesizes additional LCOs that differ from one another in the length and unsaturation of their fatty acyl substituents . USADA191 and 257DH4 both produce a novel LCO with glucose substituted for N-acetyl-D-glucosamine in the backbone of the molecule.

J Biol Chem, 1996 Sep 13, 271(37), 22563 - 9
Rhizobium leguminosarum bv . trifolii produces lipo-chitin oligosaccharides with nodE-dependent highly unsaturated fatty acyl moieties . An electrospray ionization and collision-induced dissociation tandem mass spectrometric study; van der Drift KM et al.; The lipo-chitin oligosaccharides (LCO) or nodulation factors synthesized by Rhizobium leguminosarum bv . trifolii were analyzed using positive mode fast atom bombardment and positive and negative mode electrospray ionization mass spectrometry . From their mass spectrometric behavior it is clearly possible to distinguish between the {M + Na}+ pseudomolecular ion of the nodE-independent molecule IV(C18:1,Ac) and the {M + H}+ pseudomolecular ion of the nodE-dependent molecule IV(C20:4,Ac), although they both have the same mass value . The results unequivocally show that the bacterial strain investigated produces nodE-dependent LCOs with highly unsaturated fatty acyl moieties . We further demonstrate that the interpretation of the mass spectrometric data by Philip-Hollingsworth et al . (Philip-Hollingsworth, S., Orgambide, G . G., Bradford, J . J., Smith, D . K., Hollingsworth, R . I., and Dazzo, F . B . (1995) J . Biol . Chem . 270, 20968) is incorrect and that their data do not contradict our hypothesis that the nodE gene determines the host specificity of R . leguminosarum bv . trifolii.

Trends Microbiol, 1996 Sep, 4(9), 364 - 8
Biogenesis of the peribacteroid membrane in root nodules; Verma DP et al.; An infected root nodule cell may contain several thousand rhizobial symbionts, each enclosed in a membrane envelope, the peribacteroid membrane (PBM) . The PBM is derived from the host plasma membrane, but shares properties with the vacuolar membrane and contains several nodule-specific proteins (nodulins) that perform unique functions for symbiosis.

Can J Microbiol, 1996 Sep, 42(9), 903 - 10
A 150-megadalton plasmid in Rhizobium etli strain TAL182 contains genes for nodulation competitiveness on Phaseolus vulgaris L; Borthakur D et al.; Rhizobium etli TAL182, a competitive strain for the nodulation of Phaseolus beans, occupied more than 99% of the nodules when co-inoculated in various proportions with Rhizobium TAL1145 or Rhizobium tropici CIAT899 . Two overlapping cosmid clones, pUHR68 and pUHR69, containing genes for nodulation competitiveness from TAL182, were isolated by functional complementation of strain TAL1145 . Using one of these cosmid clones, we constructed two Tn5-insertion mutants of TAL182 defective in nodulation competitiveness . The Tn5 insertions in both mutants were localized in identical positions within a 4.6-kb HindIII fragment . One mutant, RUH120, was complemented for nodulation competitiveness by this HindIII fragment . The cloned DNA in pUHR68 is a part of a plasmid, 150 MDa in size, in TAL182 and does not show homology with TAL1145 genomic DNA . The 4.6-kb HindIII fragment contains a gene(s) required for nodulation competitiveness on beans, which is present only in some R . etli strains and absent in other Rhizobium spp.

Microbiology, 1996 Sep, 142 ( Pt 9), 2603 - 12
Aspartate transport by the Dct system in Rhizobium leguminosarum negatively affects nitrogen-regulated operons; Reid CJ et al.; Amino acid uptake by the general amino acid permease (Aap) of Rhizobium leguminosarum strain 3841 was severely reduced by the presence of aspartate in the growth medium when glucose was the carbon source . The reduction in transport by the Aap appeared to be caused by inhibition of uptake and not by transcriptional repression . However, as measured with lacZ fusions, the Ntr-regulated gene glnII was repressed by aspartate . The negative regulatory effect on both the Aap and glnII was prevented by mutation of any component of the dicarboxylate transport (Dct) system or by the inclusion of a C4- dicarboxylate in the growth medium, including the non-metabolizable analogue 2-methylsuccinate . As measured by total uptake and with a dctA-lacZ fusion, aspartate was an efficient inducer of the Dct system, but slightly less so than succinate alone or succinate and aspartate together . Thus, aspartate does not cause overexpression of DctA leading to improper regulation of other operons . Transport measurements revealed that the Dct system has an apparent Km for succinate of 5 microM and an apparent Ki for aspartate inhibition of succinate uptake of 5 mM . These data imply that the Dct-mediated accumulation of aspartate causes an unregulated build-up of aspartate or a metabolic product of it in the cell . This accumulation of aspartate is prevented either by mutation of the dct system or by the presence of a higher affinity substrate that will reduce access of aspartate to the carrier protein . Elevation or disruption of the intracellular aspartate pool is predicted to disrupt N-regulated operons and nitrogen fixation.

FEMS Microbiol Lett, 1996 Sep 1, 142(2-3), 271 - 6
Bacillus polymyxa stimulates increased Rhizobium etli populations and nodulation when co-resident in the rhizosphere of Phaseolus vulgaris; Petersen DJ et al.; Microbial competition for carbon sources is a primary determinant of rhizosphere ecology . We employed the PCR to examine the population fluctuations of a symbiotic nitrogen-fixing bacterium (Rhizobium etli) during the first 11 days following inoculation of Phaseolus vulgaris seedlings grown in the presence or absence of a common asymbiotic rhizosphere resident (Bacillus polymyxa) . When B . polymyxa was applied as a co-inoculant, increases in both early rhizobial root populations and final root population densities were observed as compared to single inoculation with R . etli . Modifications to host plant growth (including increased lateral root formation and nodules number) were found concomitant with elevations in R . etli populations on plants co-inoculated with both bacterial genera . In contrast to the in planta results, population enhancements were not observed when R . etli and B . polymyxa were co-cultured in vitro using minimal media in the absence of the seedling . Addition of seed exudate to the growth media also failed to stimulate the population increases observed during co-release in planta . These results suggest that B . polymyxa acts indirectly (i.e., via the plant host) to increase R . etli populations . Our observed synergism among co-resident bacteria supports the hypothesis that microbial communities which colonize the spermosphere may play a significant role in plant development and rhizosphere ecology.

Mol Plant Microbe Interact, 1996 Sep, 9(7), 600 - 7
Gnotobiotic system for studying rhizosphere colonization by plant growth-promoting Pseudomonas bacteria; Simons M et al.; A gnotobiotic system for studying tomato rhizosphere colonization by Pseudomonas bacteria was developed . The system is based on sterile seedlings that are inoculated with one or two strains and subsequently grown in a sterile glass tube containing quartz sand . After 7 days of growth in a climate-controlled growth chamber, the number of bacteria present on the root tip was analyzed . The system was optimized with respect to root morphology, inoculation of the seedling, and isolation of root tip bacteria . With this system, rhizosphere colonization on tomato, radish, wheat, and potato was analyzed . For detailed analysis of tomato rhizosphere colonization by some representative plant growth-promoting rhizo-bacteria, the colonization of known poor, moderate, and good potato root-colonizing Pseudomonas strains and of four Rhizobium strains was determined . All strains colonized the root tips when inoculated as single strains . When inoculated in competition with the efficient root colonizer P . fluorescens strain WCS365, many strains were outcompeted . Mutants of Pseudomonas biocontrol bacteria lacking flagella or the O-antigen of lipopolysaccharide (LPS), which were isolated in previous studies and shown to be impaired in potato rhizosphere colonization in field soil systems, showed a reduced colonization ability in the gnotobiotic system also . The gnotobiotic system was used to screen a collection of 300 random P . fluorescens WCS365::Tn5 mutants for colonization-impaired mutants . Three novel mutants were found that were outcompeted by the wild-type strain in tomato root tip colonization but were not impaired in known colonization traits such as motility, amino acid auxotrophy, and presence of the O-antigenic side chain of LPS . One strain appeared to be a thiamine auxotroph, suggesting that the root does not secrete a sufficient amount of thiamine to support growth of this strain . The other two mutants had a reduced growth rate in laboratory media, suggesting that growth rate is an important colonization factor . As the system is gnotobiotic and devoid of field-soil variables, it can also be used to study the effects of selected biotic and abiotic factors on colonization.

J Appl Bacteriol, 1996 Sep, 81(3), 319 - 28
Fate of genetically modified Rhizobium leguminosarum biovar viciae during long-term storage of commercial inoculants; Corich V et al.; A study was carried out to assess the behaviour, in terms of strain survival and genetic stability, of genetically modified micro-organisms (GEMs) during their storage in commercial-type agricultural inoculants . Three genetically modified Rhizobium leguminosarum biovar viciae strains were constructed, using a gene cassette containing an inducible lacZ gene from Escherichia coli and mercury resistance determinants from transposon Tn 1831 . In the first case the genes have been integrated into the chromosome, the second strain contains the inducible cassette on a plasmid, in the third case the cassette is carried by the same plasmid, but the lacZ is constitutively expressed at high levels, due to the removal of the regulatory structure (lac operator) between the gene and its promoter . Three inoculum formulations, based on liquid, vermiculite and peat carriers, were prepared using the genetically modified strains, and were monitored during a period of up to 16 months . Results indicate a high stability of the chromosomally integrated markers . The plasmid-borne modification also was very stable, though the presence of the plasmid affected the strain growth kinetics . In contrast, the strain containing the highly expressed lacZ showed dramatic marker instability . Strain behaviour in stored inoculant packages reflected that observed in batch cultures; moreover, prolonged storage appeared to magnify differences found in in vitro cultures.

J Bacteriol, 1996 Sep, 178(18), 5529 - 32
The "missing" typical Rhizobium leguminosarum O antigen is attached to a fatty acylated glycerol in R . leguminosarum bv . trifolii 4S, a strain that also lacks the usual tetrasaccharide "core" component; Cedergren RA et al.; Rhizobium leguminosarum bv . trifolii 4S has a lipopolysaccharide O antigen that lacks galactose and many of the typical glycosyl components found in related strains . Here, we show that it also lacks the typical core tetrasaccharide but synthesizes an alternative glycolipid that contains galactose and the typical O-antigen glycosyl components, suggesting that in this strain, the O antigen is transferred to an alternative lipid acceptor.

Appl Environ Microbiol, 1996 Sep, 62(9), 3333 - 8
Recombinant Rhizobium meliloti strains with extra biotin synthesis capability; Streit WR et al.; The growth of Rhizobium meliloti 1021 in an experimental alfalfa (Medicago sativa L.) rhizosphere was stimulated by adding nanomolar amounts of biotin . To overcome this biotin limitation, R . meliloti strains were constructed by conjugating the Escherichia coli biotin synthesis operon into biotin auxotroph R . meliloti 1021-B3 . Transconjugant strains Rm1021-WS10 and Rm1021-WS11 grew faster in vitro and achieved a higher cell density than did R . meliloti 1021 and overproduced biotin on a defined medium . The increase in cell yield was associated with as much as a 99% loss in viability for Rm1021-WS11, but data suggested that a separate stabilizing factor in the E . coli DNA reduced cell death in Rm1021-WS10 . In rhizosphere tests, the recombinant strains showed delayed growth and competed poorly against Rm1021.

Infect Immun, 1996 Sep, 64(9), 3744 - 51
Cloning, nucleotide sequence, and expression of the Brucella melitensis omp31 gene coding for an immunogenic major outer membrane protein; Vizcaino N et al.; The gene coding for the major outer membrane protein (OMP) of 31 to 34 kDa, now designated Omp31, of Brucella melitensis 16M was cloned and sequenced . A B . melitensis 16M genomic library was constructed in lambda GEM-12 XhoI half-site arms, and recombinant phages expressing omp31 were identified by using the anti-Omp31 monoclonal antibody (MAb) A59/10F09/G10 . Subcloning of insert DNA from a positive phage into pGEM-7Zf allowed the selection of a plasmid bearing a 4.4-kb EcoRI fragment that seemed to contain the entire omp31 gene under control of its own promoter . omp31 was localized within a region of the EcoRI insert of approximately 1.1 kb . Sequencing of this region revealed an open reading frame of 720 bp encoding a protein of 240 amino acids and a predicted molecular mass of 25,307 Da . Cleavage of the first 19 amino acids, showing typical features of signal peptides for protein export, leaves a mature protein of 221 amino acids with a predicted molecular mass of 23,412 Da . The predicted amino acid sequence of B . melitensis 16M Omp31 showed 35.2% identity with the RopB OMP of Rhizobium leguminosarum bv . viciae 248 and 34.3% identity with Omp25 of B . abortus 544 . As in Brucella spp., Omp31 was located in the outer membrane of recombinant Escherichia coli, but its reported peptidoglycan association in Brucella cells was not detected in E . coli . The ability of Omp31 to form oligomers resistant to sodium dodecyl sulfate denaturation at low temperatures, a characteristic described for several bacterial porins, was observed in both B . melitensis and recombinant E . coli . The epitope recognized by the anti-Omp31 MAb A59/10F09/G10, for which a protective activity has been suggested, has been delimited to a region of 36 amino acids of Omp31 covering the most hydrophilic part of the protein . The availability of recombinant Omp31 and the identification of the antigenic determinant recognized by MAb A59/10F09/G10 will allow the evaluation of their potential protective activity and their potential for the development of subcellular vaccines against brucellosis.

Carbohydr Res, 1996 Aug 19, 289, 115 - 36
Structural determination of symbiotic nodulation factors from the broad host-range Rhizobium species NGR234; Price NP et al.; Nod factors are secreted lipo-oligosaccharides produced by symbiotic nitrogen-fixing Rhizobium bacteria that induce nodule formation on the roots of host leguminous plants . Two biologically active fractions (NodNGRA and NodNGRB) were isolated by reversed-phase HPLC from the culture supernatant of a Nod factor overproducing strain of Rhizobium sp . NGR234 . NodNGRA and NodNGRB are heterogeneous mixtures of N-acylated 2-O-methylfucosylated chitomers, in which the fucosyl residue may be either 3-sulfated (NodNGRA), or 4-O-acetylated or nonsubstituted (NodNGRB) . Structurally analogous series of compounds occur with either N-vaccenic (C18:1) or N-palmitic (C16:0) substituents . The presence of 6-O-carbamoyl groups on the GlcNMe-Acyl residue occurs on some molecules, while others are di-O-carbamoylated . Detailed structural analysis of seventeen Nod factors are reported here.

Proc Natl Acad Sci U S A, 1996 Aug 6, 93(16), 8636 - 41
Low molecular weight EPS II of Rhizobium meliloti allows nodule invasion in Medicago sativa; Gonzalez JE et al.; Effective invasion of alfalfa by Rhizobium meliloti Rm1021 normally requires the presence of succinoglycan, an exopolysaccharide (EPS) produced by the bacterium . However, Rm1021 has the ability to produce a second EPS (EPS II) that can suppress the symbiotic defects of succinoglycan-deficient strains . EPS II is a polymer of modified glucose-(beta-1,3)-galactose subunits and is produced by Rm1021 derivatives carrying either an expR101 or mucR mutation . If the ability to synthesize succinoglycan is blocked genetically, expR101 derivatives of Rm1021 are nodulation-proficient, whereas mucR derivatives of Rm1021 are not . The difference in nodulation proficiency between these two classes of EPS II-producing strains is due to the specific production of a low molecular weight form of EPS II by expR101 strains . A low molecular weight EPS II fraction consisting of 15-20 EPS II disaccharide subunits efficiently allows nodule invasion by noninfective strains when present in amounts as low as 7 pmol per plant, suggesting that low molecular weight EPS II may act as a symbiotic signal during infection.

Trends Microbiol, 1996 Aug, 4(8), 317 - 20
Environmental regulation of rhizobial symbiotic nitrogen fixation genes; Fischer HM; Adaptation of rhizobia to a symbiotic life style and synthesis of the nitrogen-fixing apparatus are coordinated with nodule development by the microaerobic conditions prevailing in the central nodule tissue . Sensing and transduction of the "low oxygen' signal involves similar regulatory elements in different rhizobia, yet, these are combined in species-specific circuits.

J Bacteriol, 1996 Aug, 178(15), 4555 - 62
Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids; Haaker H et al.; The relationship between the O2 input rate into a suspension of Rhizobium leguminosarum bacteroids, the cellular ATP and ADP pools, and the whole-cell nitrogenase activity during L-malate oxidation has been studied . It was observed that inhibition of nitrogenase by excess O2 coincided with an increase of the cellular ATP/ADP ratio . When under this condition the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added, the cellular ATP/ADP ratio was lowered while nitrogenase regained activity . To explain these observations, the effects of nitrogenase activity and CCCP on the O2 consumption rate of R . leguminosarum bacteroids were determined . From 100 to 5 microM O2, a decline in the O2 consumption rate was observed to 50 to 70% of the maximal O2 consumption rate . A determination of the redox state of the cytochromes during an O2 consumption experiment indicated that at O2 concentrations above 5 microM, electron transport to the cytochromes was rate-limiting oxidation and not the reaction of reduced cytochromes with oxygen . The kinetic properties of the respiratory chain were determined from the deoxygenation of oxyglobins . In intact cells the maximal deoxygenation activity was stimulated by nitrogenase activity or CCCP . In isolated cytoplasmic membranes NADH oxidation was inhibited by respiratory control . The dehydrogenase activities of the respiratory chain were rate-limiting oxidation at O2 concentrations (if >300 nM . Below 300 nM the terminal oxidase system followed Michaelis-Menten kinetics (Km of 45 +/- 8 nM) . We conclude that (i) respiration in R . leguminosarum bacteroids takes place via a respiratory chain terminating at a high-affinity oxidase system, (ii) the activity of the respiratory chain is inhibited by the proton motive force, and (iii) ATP hydrolysis by nitrogenase can partly relieve the inhibition of respiration by the proton motive force and thus stimulate respiration at nanomolar concentrations of O2.

J Bacteriol, 1996 Aug, 178(15), 4540 - 7
A phosphate transport system is required for symbiotic nitrogen fixation by Rhizobium meliloti; Bardin S et al.; The bacterium Rhizobium meliloti forms N2-fixing root nodules on alfalfa plants . The ndvF locus, located on the 1,700-kb pEXO megaplasmid of R . meliloti, is required for nodule invasion and N2 fixation . Here we report that ndvF contains four genes, phoCDET, which encode an ABC-type transport system for the uptake of Pi into the bacteria . The PhoC and PhoD proteins are homologous to the Escherichia coli phosphonate transport proteins PhnC and PhnD . The PhoT and PhoE proteins are homologous to each other and to the E . coli phosphonate transport protein PhnE . We show that the R . meliloti phoD and phoE genes are induced in response to phosphate starvation and that the phoC promoter contains two elements which are similar in sequence to the PHO boxes present in E . coli phosphate-regulated promoters . The R . meliloti ndvF mutants grow poorly at a phosphate concentration of 2 mM, and we hypothesize that their symbiotic phenotype results from their failure to grow during the nodule infection process . Presumably, the PhoCDET transport system is employed by the bacteria in the soil environment, where the concentration of available phosphate is normally 0.1 to 1 microM.

Mol Plant Microbe Interact, 1996 Aug, 9(6), 523 - 31
Sequence and mutational analysis of the common nodBCIJ region of Rhizobium sp . (Oxytropis arctobia) strain N33, a nitrogen-fixing microsymbiont of both arctic and temperate legumes; Cloutier J et al.; By heterologous hybridization, we have identified the common nodulation genes nodBCIJ of Rhizobium sp . strain N33 within a 8.2-kb PstI fragment . The nodBCIJ genes are located within a 4,620-bp region which also included a consensus nod box promoter . The four open reading frames coding for the nodBCIJ genes contain 657, 1,353, 915, and 789 nucleotides, respectively . We found that the nodA gene was not adjacent to the nodB gene, unlike the situation in many rhizobia . The DNA of the nodBCIJ genes of Rhizobium sp . strain N33 were found to be homologous to the corresponding genes of other rhizobia except for the 3'-coding region of the nodC gene . The deduced NodC protein was the longest of the rhizobia except Bradyrhizobium japonicum . Tn5 mutagenesis of the common nod region of strain N33 revealed that the nodBC genes were essential for nodulation on their temperate hosts Onobrychis viciifolia and Astragalus cicer . By contrast, mutations in the nodI and nodJ genes produced a Nod+ phenotype with a reduced number of nodules on the temperate hosts . Nodules formed on Onobrychis viciifolia by either nodI or nodJ mutants were approximately 10 times smaller than nodules formed by the wild type strain: this reduction in nodule size was not observed on Astragalus cicer.

Mol Plant Microbe Interact, 1996 Aug, 9(6), 492 - 500
Isolation and characterization of Rhizobium tropici Nod factor sulfation genes; Laeremans T et al.; Rhizobium tropici produces a mixture of sulfated and non-sulfated Nod factors . The genes responsible for the sulfation process in R . tropici strain CFN299 were cloned and sequenced . These genes are homologous to the nodP, nodQ, and nodH genes from R . meliloti . The identity among the two species is 75% for nodP, 74% for nodQ, and 69% for nodH . NodH resembles sulfotransferases in general and NodQ has the characteristic purine-binding motifs and the PAPS 3'-phosphoadenosine 5'-phosphosulfate) motif . Mutants of NodP and NodH were obtained by site-directed mutagenesis . They are no longer able to synthesize the sulfated Nod factor, as was demonstrated in high-pressure liquid chromatography and thin-layer chromatography assays . The NodP- mutant had a decreased nodulation capacity in Phaseolus vulgaris Negro Xamapa bean plants . In contrast, NodH- and NodP- mutants acquired an increased capacity to nodulate the high-nitrogen-fixing bean cultivars N-8-116 and BAT-477 . Nodulation was restored to normal levels when the mutants were complemented with a 16-kb clone carrying the wild-type genes . The role of the sulfate on Nod factors in R . tropici was dependent on the bean cultivar and the conditions assayed.

Mol Plant Microbe Interact, 1996 Aug, 9(6), 457 - 63
Proteins from cells of Rhizobium fredii bind to DNA sequences precedingnolX, a flavonoid-inducible nod gene that is not associated with a nod box; Bellato CM et al.; Rhizobium fredii strains USDA257 and USDA191 both contain a set of nodulation genes termed nolXWBTUV . In the USDA257 background, nolX prevents infection of soybean cultivars such as McCall, and in both backgrounds, it blocks nodulation of Erythrina spp . We report here that expression of nolX is differentially responsive to a panel of flavonoids, and that the most potent inducers are also the most active inducers of nodC, a conventional, nod box-associated gene . Cell-free protein extracts from uninduced and flavonoid-induced cells of strains USDA191 and USDA257 retard the electrophoretic mobility of DNA sequences that lie upstream of nolX . Binding is dependent both on nodD1 and nodD2, and it is abolished by the presence of a double-stranded, 23-bp oligonucleotide that lies within a 114-bp TaqI/SacII restriction fragment . This oligomer has significant sequence homology to A3, a putative negative regulatory element from R.leguminosarum bv . viciae . Deletion of the A3-homologous sequences elevates the basal and flavonoid-inducible expression of nolX by about 50%.

Appl Environ Microbiol, 1996 Aug, 62(8), 2818 - 25
Four unnamed species of nonsymbiotic rhizobia isolated from the rhizosphere of Lotus corniculatus; Sullivan JT et al.; Previously, we found that genetically diverse rhizobia nodulating Lotus corniculatus at a field site devoid of naturalized rhizobia had symbiotic DNA regions identical to those of ICMP3153, the inoculant strain used at the site (J . T . Sullivan, H . N . Patrick, W . L . Lowther, D . B . Scott, and C . W . Ronson, Proc . Natl . Acad . Sci . USA 92:8985-8989, 1995) . In this study, we characterized seven nonsymbiotic rhizobial isolates from the rhizosphere of L . corniculatus . These included two from plants at the field site sampled by Sullivan et al . and five from plants at a new field plot adjacent to that site . The isolates did not nodulate Lotus species or hybridize to symbiotic gene probes but did hybridize to genomic DNA probes from Rhizobium loti . Their genetic relationships with symbiotic isolates obtained from the same sites, with inoculant strain ICMP3153, and with R . loti NZP2213T were determined by three methods . Genetic distance estimates based on genomic DNA-DNA hybridization and multilocus enzyme electrophoresis were correlated but were not consistently reflected by 16S rRNA nucleotide sequence divergence . The nonsymbiotic isolates represented four genomic species that were related to R . loti; the diverse symbiotic isolates from the site belonged to one of these species . The inoculant strain ICMP3153 belonged to a fifth genomic species that was more closely related to Rhizobium huakuii . These results support the proposal that nonsymbiotic rhizobia persist in soils in the absence of legumes and acquire symbiotic genes from inoculant strains upon introduction of host legumes.

Appl Environ Microbiol, 1996 Aug, 62(8), 2767 - 72
Root colonization of maize and lettuce by bioluminescent Rhizobium leguminosarum biovar phaseoli; Chabot R et al.; Two strains of Rhizobium leguminosarum bv . phaseoli and three other plant growth-promoting rhizobacteria (PGPR) were examined for the potential of maize and lettuce root colonization . All of these strains were selected in vitro for their phosphate-solubilizing abilities . Maize and lettuce seeds were treated with derivatives of all strains marked with lux genes for bioluminescence and resistance to kanamycin and rifampin prior to planting in nonsterile Promix and natural soil . The introduced bacterial strains were quantified on roots by dilution plating on antibiotic media together with observation of bioluminescence . Rhizobia were superior colonizers compared with other tested bacteria; rhizobial root populations averaged log 4.1 CFU/g (fresh weight) on maize roots 4 weeks after seeding and log 3.7 CFU/g (fresh weight) on lettuce roots 5 weeks after seeding . The average populations of the recovered PGPR strains were log 3.5 and log 3.0 CFU/g (fresh weight) on maize and lettuce roots, respectively . One of the three PGPR was not recovered later than the first week after seeding in Promix . Bioluminescence also permitted visualization of in situ root colonization in rhizoboxes and demonstrated the efficiency of rhizobial strains to colonize and survive on maize and lettuce roots.

Biochim Biophys Acta, 1996 Jul 31, 1308(1), 7 - 11
Identification of a gene for a chemoreceptor of the methyl-accepting type in the symbiotic plasmid of Rhizobium leguminosarum bv . viciae UPM791; Brito B et al.; The 4 kb DNA region located immediately upstream of the Rhizobium leguminosarum bv . viciae UPM791 hydrogen structural genes was sequenced and found to encode a chemoreceptor of the methyl-accepting type, the first to be described in a rhizobial symbiotic plasmid . Two additional open reading frames were found . Their protein products showed sequence homology to dehydrogenases and isomerases involved in the metabolism of aromatic compounds . Mutant analysis showed that this region is not required for hydrogenase activity.

Biochemistry, 1996 Jul 23, 35(29), 9539 - 48
Structural basis for ligand discrimination and response initiation in the heme-based oxygen sensor FixL; Rodgers KR et al.; FixL is a multiple-domain bacterial O2-sensing protein that modulates the activity of its kinase domain in response to O2 concentration . The kinase activity is coupled, via phosphoryl transfer, to transcriptional activation by a response-regulating protein, FixJ . Heme ligation resulting in a transition from high to low spin inhibits the kinase through an, as yet, ill-defined mechanism . This report presents spectroscopic, kinetic, and thermodynamic data on various complexes of two deletion derivatives of Rhizobium meliloti FixL, FixLN (the heme domain) and a functional heme kinase, FixL* . Resonance Raman characterization of metFixLN and metFixL* indicates that the heme core is smaller than that observed in metmyoglobin and is indicative of a five-coordinate high-spin heme in metFixLs . Resonance Raman spectra of FixL-CO adducts reveal that the Fe-C = O unit and/or its electrostatic environment in FixL*-CO is distorted relative to that in FixLN-CO . The 1H NMR spectra of the met forms further support the model of an asymmetric perturbation of the heme pocket structure associated with the presence of the kinase domain in FixL* . Observation of equivalent Fe-imidazole stretching vibrations for deoxyFixLN and deoxyFixL* (212 cm-1) indicates that the source of this perturbation in the heme pocket of FixL* does not lie on the proximal side of the heme . The equivalent Fe-imidazole stretching frequencies for deoxyFixLN and FixL* indicate that the presence of the kinase domain does not alter the relative strength of the proximal Fe-imidazole bond and that the proximal imidazole ligand is weakly H-bonded, probably to a backbone carbonyl group . Kinetic and thermodynamic data for the reactions of cyanide and fluoride ions with FixL are consistent with shape selectivity due to steric and/or an anisotropic electrostatic field in the distal heme pocket being responsible for the unique reactivities (or lack thereof) of FixL with ligands, i.e., O2, CO, CN-, F-, N3-, and SCN- . While the rate constants for binding of CN- to metFixLN and metFixL* are an order of magnitude slower than that for metMb, the stabilities of these complexes and metMb-CN are nearly the same . Neither N3- nor SCN- binds to the heme with measurable affinity . Since other ferric heme proteins form stable adducts with these ligands, the inability of FixL to form analogous complexes suggests that the ligand selectivity of this protein is rooted in insurmountable activation barriers to the binding of ligands containing more than two atoms and for ligands whose lowest-energy coordination geometries are linear . This allows the natural O2 ligand to compete kinetically with other naturally occurring ligands that form stable complexes with unencumbered hemes . Moreover, the rate constant for binding of CN- to the functional heme-kinase (metFixL*) is smaller than its metFixLN counterpart and the stability of metFixL*-CN is measurably lower than that of metFixLN-CN . This indicates that the contacts between the heme and kinase domains of FixL* impose more stringent geometric constraints on ligand binding than FixLN . The kinase is thus implicated in a possible mechanism for phosphate-dependent feedback control over ligand affinity of the heme.

Mol Microbiol, 1996 Jul, 21(2), 397 - 408
NodZ of Bradyrhizobium extends the nodulation host range of Rhizobium by adding a fucosyl residue to nodulation signals; Lopez-Lara IM et al.; The nodulation genes of rhizobia are involved in the production of the lipo-chitin oligosaccharides (LCO), which are signal molecules required for nodule formation . A mutation in nodZ of Bradyrhizobium japonicum results in the synthesis of nodulation signals lacking the wild-type 2-O-methylfucose residue at the reducting-terminal N-acetylglucosamine . This phenotype is correlated with a defective nodulation of siratro (Macroptilium atropurpureum) . Here we show that transfer of nodZ to Rhizobium leguminosarum blovar (bv) viciae, which produces LCOs that are not modified at the reducing-terminal N-acetylglucosamine, results in production of LCOs with a fucosyl residue on C-6 of the reducing-terminal N-acetylglucosamine . This finding, together with in vitro enzymatic assays, indicates that the product of nodZ functions as a fucosyltransferase . The transconjugant R . leguminosarum strain producing fucosylated LCOs acquires the capacity to nodulate M . atropurpureum, Glycine soja, Vigna unguiculate and Leucaena leucocephala . Therefore, nodZ extends the narrow host range of R . leguminosarum bv . viciae to include various tropical legumes . However, microscopic analysis of nodules induced on siratro shows that these nodules do not contain bacteroids, showing that transfer of nodZ does not allow R . leguminosarum to engage in a nitrogen-fixing symbiosis with this plant.

Mol Microbiol, 1996 Jul, 21(2), 267 - 80
Rhizobium leguminosarum bv . viciae contains a second fnr/fixK-like gene and an unusual fixL homologue; Patschkowski T et al.; Genes of Rhizobium leguminosarum bv . viciae VF39 coding for the regulatory elements NifA, FixL and FixK were isolated, sequenced and genetically analysed . The fixK-fixL region is located upstream of the fixNOQP operon on the non-nodulation plasmid pRleVF39c . The deduced amino acid sequence of FixL revealed an unusual structure in that it contains a receiver module (homologous to the N-terminal domain of response regulators) fused to its transmitter domain . An oxygen-sensing haem-binding domain, found in other FixL proteins, is conserved in R, leguminosarum bv . viciae FixL . R . leguminosarum bv . viciae possesses a second fnr-like gene, designated fixK, whose encoded gene product is very similar to Rhizobium meliloti and Azorhizobium caulinodans FixK . Individual R . leguminosarum bv . viciae fixK and fixL insertion mutants displayed a Fix+ phenotype . A reduced nitrogen-fixation activity was found for a R . leguminosarum bv . viciae fnrN-deletion mutant, whereas no nitrogen-fixation activity was detectable for a flxK/fnrN double mutant . The R . leguminosarum bv . viciae nifA gene is expressed independently of FixL and FixK under aerobic and microaerobic conditions, whereas fixL gene expression is induced under microaerobiosis . Another orf was identified down-stream of fixK-fixL and encodes a product which has homology to pseudoazurins from different species . Mutation of this azu gene showed that it is dispensable for nitrogen fixation.

Cell Mol Biol (Noisy-le-grand), 1996 Jul, 42(5), 737 - 58
The in vitro biosynthesis of the exopolysaccharide produced by Rhizobium leguminosarum bv . trifolii, strain NA 30; Bossio JC et al.; Rhizobium leguminosarum bv . trifolii, strain NA 30 nodulates both red (Trifolium pratense) and white (T . repens) clover and produces an acidic exopolysaccharide (EPS) containing glucose, galactose, glucuronic acid, acetate and ketalpyruvate residues in a 5:1:2:1:2 molar ratio . The in vitro synthesis of this EPS as well as the characterization of five structurally related lipid linked oligosaccharides is described employing EDTA treated cells as enzyme preparation and 14C-labelled UDP-Glc, UDP-GlcA, Acetyl CoA and phosphoenol pyruvate (PEP) and doubly labelled 32P UDP-14C-Glc as precursors . The lipidic derivatives, extracted with chloroform, methanol, water (1:2:0,3) had the properties expected for prenyl-diphospho-sugars, as judged by the pattern of labelling, DEAE cellulose column chromatography, catalytic reduction and acid lability, etc . The sugar moieties of these phosphoprenyl derivatives were identified as the acetylated octasaccharide repeating unit, its mono- and di-ketalpyruvate derivatives and two trisaccharides, one of them acetylated, on the basis of specific labelling, gel filtration, paper electrophoresis and chromatography, TLC, permethylation, etc . In vitro polymer synthesis was greatly increased when electroporated cells were substituted for EDTA treated cells as enzyme system.

Cell Mol Biol (Noisy-le-grand), 1996 Jul, 42(5), 729 - 35
Introduction and expression of an anti-dipteran toxin gene from B . thuringiensis in nodulating Rhizobia; Guerchicoff A et al.; Rhizobium-based inoculants are widely used in leguminous crops . Improved strains with dual functions (i.e, nitrogen fixing and insecticidal) could be successfully applied to organic pest management . These could also be used as seed protectants, reducing the use of chemical insecticides . The cryIVB gene from B . thuringiensis israelensis coding for an anti-dipteran toxin was used as a model for expression . A highly stable plasmid vector (pTR101) was chosen in order to carry out nodulation experiments in the absence of selective pressure . Constructions were introduced into Rhizobium fredii 191 and meliloti 1021, through triparental matings with a helper plasmid . Expression was monitored by Western blotting of crude extracts using a specific antibody raised against recombinant CryIVB protein . Although rearrangements leading to deletions were observed, an immunoreactive polypeptide was reproducibly detected in both Rhizobia . The presence of the recombinant plasmids did not affect the nodulative capacity nor the growth of the inoculated plants.

Chem Biol, 1996 Jul, 3(7), 561 - 6
Assignment of the hyperfine-shifted 1H-NMR signals of the heme in the oxygen sensor FixL from Rhizobium meliloti; Bertolucci C et al.; BACJGROUND: The Rhizobial oxygen sensor FixL is a hemoprotein with kinase activity . On binding of strong-field ligands, a change of the ferrous or ferric heme iron from high to low spin reversibly inactivates the kinase . This spin-state change and other information on the heme pocket have been inferred from enzymatic assays, absorption spectra and mutagenesis studies . We set out to investigate the spin-state of the FixL heme and to identify the hyperfine-shifted heme-proton signals by NMR spectroscopy . RESULTS: Using one-dimensional NMR we directly observed the high- and low-spin nature of the met- and cyanomet-FixL heme domain, respectively . We determined the hyperfine-shifted 1H-NMR signals of the heme and the proximal histidine by one- and two-dimensional spectroscopy and note the absence of distal histidine signals . CONCLUSIONS: These findings support the spin-state mechanism of FixL regulation . They establish that the site of heme coordination is a histidine residue and strongly suggest that a distal histidine is absent . With a majority of the heme resonances identified, one- and two-dimensional NMR techniques can be extended to provide structural and mechanistic information about the residues that line the heme pocket.

Genome Res, 1996 Jul, 6(7), 590 - 600
Sequencing the 500-kb GC-rich symbiotic replicon of Rhizobium sp . NGR234 using dye terminators and a thermostable "sequenase": a beginning; Freiberg C et al.; Genomes of the soil-borne nitrogen-fixing symbionts of legumes {Azo(Brady)Rhizobium species} typically have GC contents of 59-65 mol% . As a consequence, compressions (up to 400 per cosmid) are common using automated dye primer shotgun sequencing methods . To overcome this difficulty, we have exclusively applied dye terminators in combination with a thermostable "sequenase" for shotgun sequencing GC-rich cosmids from pNGR234a, the 500-kbp symbiotic replicon of Rhizobium sp . NGR234 . A thermostable sequenase incorporates dye terminators into DNA more efficiently than Taq DNA polymerase, thus reducing the concentrations needed (20- to 250-fold) . Unincorporated dye terminators can simply be removed by ethanol precipitation . Here, we present data of pXB296, one of 23 overlapping cosmids representing pNGR234a . We demonstrate that the greatly reduced number of compressions results in a much faster assembly of cosmid sequence data by comparing assembly of the shotgun data from pXB296 and the data from another pNGR234a cosmid (pXB110) sequenced using dye primer methods . Within the 34,010-bp sequence from pXB296, 28 coding regions were predicted . All of them showed significant homologies to known proteins, including oligopeptide permeases, an essential cluster for nitrogen fixation, and the C4-dicarboxylate transporter DctA.

Appl Environ Microbiol, 1996 Jul, 62(7), 2279 - 85
Genetic diversity of an Italian Rhizobium meliloti population from different Medicago sativa varieties; Paffetti D et al.; We investigated the genetic diversity of 96 Rhizobium meliloti strains isolated from nodules of four Medicago sativa varieties from distinct geographic areas and planted in two different northern Italian soils . The 96 isolates, which were phenotypically indistinguishable, were analyzed for DNA polymorphism with the following three methods: (i) a randomly amplified polymorphic DNA (RAPD) method, (ii) a restriction fragment length polymorphism (RFLP) analysis of the 16S-23S ribosomal operon spacer region, and (iii) an RFLP analysis of a 25-kb region of the pSym plasmid containing nod genes . Although the bacteria which were studied constituted a unique genetic population, a considerable level of genetic diversity was found . The new analysis of molecular variance (AMOVA) method was used to estimate the variance among the RAPD patterns . The results indicated that there was significant genetic diversity among strains nodulating different varieties . The AMOVA method was confirmed to be a useful tool for investigating the genetic variation in an intraspecific population . Moreover, the data obtained with the two RFLP methods were consistent with the RAPD results . The genetic diversity of the population was found to reside on the whole bacterial genome, as suggested by the RAPD analysis results, and seemed to be distributed on both the chromosome and plasmid pSym.

J Bacteriol, 1996 Jul, 178(14), 4150 - 6
A newly discovered gene, tfuA, involved in the production of the ribosomally synthesized peptide antibiotic trifolitoxin; Breil B et al.; Trifolitoxin (TFX) is a gene-encoded, posttranslationally modified peptide antibiotic . Previously, we have shown that tfxABCDEFG from Rhizobium leguminosarum bv . trifolii T24 is sufficient to confer TFX production and resistance to nonproducing strains within a distinct taxonomic group of the alpha-proteobacteria (E . W . Triplett, B . T . Breil, and G . A . Splitter, Appl . Environ . Microbiol . 60:4163-4166, 1994) . Here we describe strain Tn5-2, a Tn5 mutant of T24 defective in the production of TFX, whose insertion maps outside of the tfx cluster . It is not altered in growth compared with T24, nor does it inactivate TFX in its proximity . The wild-type analog of the mutated region of Tn5-2 was cloned . Sequencing, transcriptional fusion mutagenesis, and subcloning were used to identify tfuA, a gene involved in TFX production . On the basis of computer analysis, the putative TfuA protein has a mass of 72.9 kDa and includes a peroxidase motif but no transmembrane domains . TFX production studies show that extra copies of the tfxABCDEFG fragment increase TFX production in a T24 background while additional copies of tfuA do not . Lysate ribonuclease protection assays suggest that tfuA does not regulate transcription of tfxA . Upstream of tfuA are two open reading frames (ORFs) . The putative product of ORF1 shows high similarity to the LysR family of transcriptional regulators . The putative product of ORF2 shows high similarity to the cytosine deaminase (CodA) of Escherichia coli.

Microbiology, 1996 Jul, 142 ( Pt 7), 1693 - 704
Acid tolerance in Rhizobium meliloti strain WSM419 involves a two-component sensor-regulator system; Tiwari RP et al.; An acid-sensitive mutant, TG5-46, derived from Rhizobium meliloti WSM419 by Tn5 mutagenesis, fails to grow below pH 6.0 whereas the parent strain grows at pH 5.7 . The DNA sequence of a 2.2 kb rhizobial DNA region flanking Tn5 in TG5-46 contains two open reading frames, ORF1 (designated actS) and ORF2 (designated actR), having high similarity to the sensor-regulator pairs of the two-component systems involved in signal transduction in prokaryotes . Insertion of an omega interposon into actS in R . meliloti WSM419 resulted in an acid-sensitive phenotype . A DNA fragment from the wild-type complemented the acid-sensitive phenotype of RT295 (ActS-) and TG5-46 (ActR-), while fragments containing only actR or actS complemented TG5-46 and RT295, respectively . The presence of multiple copies of actR complemented not only TG5-46 but also RT295 . Cloning DNA upstream from actR and actS into a broad-host-range lacZ expression vector and measuring beta-galactosidase activities showed that both genes are constitutively expressed regardless of the external pH . Genomic DNA from all strains of R . meliloti, but no other bacteria tested, hybridized with an actRS probe at high stringency . These data implicate a two-component sensor-regulator protein pair in acid tolerance in R . meliloti and suggest their involvement in pH sensing and/or response by these bacteria.

Microb Ecol, 1996 Jul, 32(2), 149 - 69
Surface Properties and Motility of Rhizobium and Azospirillum in Relation to Plant Root Attachment
De Troch P, Vanderleyden J.
Plant growth promotion by rhizobacteria is a widely spread phenomenon . However only a few rhizobacteria have been studied thoroughly . Rhizobium is the best-studied rhizobacterium . It forms a symbiosis with a restricted host range . Azospirillum is another plant-growth-promoting rhizobacterium which forms rhizocoenoses with a wide range of plants . In both bacteria, the interaction with the plant involves the attraction toward the host plant and the attachment to the surface of the root . Both bacteria are attracted to plant roots, but differ in specificity . Attachment to plant roots occurs in two steps for both bacteria: a quick, reversible adsorption, and a slow, irreversible anchoring to the plant root surface . However, for the two systems under study, the bacterial surface molecules involved in plant root attachment are not necessarily the same.

J Bacteriol, 1996 Jul, 178(13), 3967 - 70
Use of PCR to isolate genes encoding sigma54-dependent activators from diverse bacteria; Kaufman RI et al.; Degenerate PCR probes were used to amplify gene fragments encoding the catalytic domain of sigma54-dependent transcription activators . The procedure should be widely applicable, as it recovered both known and novel gene fragments: 5 from Rhizobium meliloti, 13 from Myxococcus xanthus, and 3 from Bacillus subtilis . No fragments were obtained from Synechococcus sp . strain PCC 7002 or Saccharomyces cerevisiae.

Mol Plant Microbe Interact, 1996 Jul, 9(5), 395 - 400
mucS, a gene involved in activation of galactoglucan (EPS II) synthesis gene expression in Rhizobium meliloti; Astete SG et al.; In addition to the exopolysaccharide succinoglycan, Rhizobium meliloti can produce a galactoglucan exopolysaccharide, EPS II . The production of EPS II occurs in certain mutant strains, in strains containing extra copies of EPS II synthesis genes, or in the wild-type strain under phosphate-limiting conditions . We have identified a gene, mucS, that is in a locus required for EPS II induction by extra gene copies and by phosphate limitation, and that activates the expression of at least one other EPS II synthesis gene . mucS lies within a cluster of EPS II synthesis genes and contains an open reading frame of 190 amino acids . MucS does not show any significant similarity to known genes and may represent a new type of regulatory protein.

Mol Plant Microbe Interact, 1996 Jul, 9(5), 330 - 8
Biotin and other water-soluble vitamins are key growth factors for alfalfa root colonization by Rhizobium meliloti 1021; Streit WR et al.; Rhizosphere growth limitations imposed on Rhizobium meliloti by availability of biotin, thiamine, and riboflavin were overcome by adding nanomolar amounts of these vitamins . Studies done with R . meliloti 1021 showed that both synthesis and uptake of biotin promote colonization of alfalfa roots . Two lines of evidence indicated that plant-derived biotin normally promotes root colonization: (i) adding avidin significantly (P < or = 0.01) reduced rhizosphere growth of R meliloti 1021, and (ii) growth of Tn5-induced biotin auxotrophs still increased 10-fold in the rhizosphere . Synthesis, however, is the more important source of biotin for R . meliloti 1021 because in root colonization tests biotin auxotrophs competed very poorly with the parent strain . Mutations conferring biotin auxotrophy were closely linked on a single restriction fragment, and one was complemented with the Escherichia coli bio operon . Initial nucleotide sequencing and DNA-DNA hybridization tests showed the biotin synthesis genes in R . meliloti are quite different from those in E . coli.

J Biol Chem, 1996 Jun 21, 271(25), 14856 - 63
Widespread use of the glu-tRNAGln transamidation pathway among bacteria . A member of the alpha purple bacteria lacks glutaminyl-trna synthetase; Gagnon Y et al.; The expression of the Rhizobium meliloti glutamyl-tRNA synthetase gene in Escherichia coli under the control of a trc promoter results in a toxic effect upon isopropyl-beta-D-thiogalactopyranoside induction, which is probably caused by a misacylation activity . To further investigate this unexpected result, we looked at the pathway of Gln-tRNAGln formation in R . meliloti . No glutaminyl-tRNA synthetase activity has been found in R . meliloti crude extract, but we detected a specific aminotransferase activity that changes Glu-tRNAGln to Gln-tRNAGln . Our results show that R . meliloti, a member of the alpha-subdivision of the purple bacteria, is the first Gram-negative bacteria reported to use a transamidation pathway for Gln-tRNAGln synthesis . A phylogenetic analysis of the contemporary glutamyl-tRNA synthetase and glutaminyl-tRNA synthetase amino acid sequences reveals that a close evolutionary relationship exists between R . meliloti and yeast mitochondrial glutamyl-tRNA synthetases, which is consistent with an origin of mitochondria in the alpha-subdivision of Gram-negative purple bacteria . A 256-amino acid open reading frame closely related to bacterial glutamyl-tRNA synthetases, which probably originates from a glutamyl-tRNA synthetase gene duplication, was found in the 4-min region of the E . coli chromosome . We suggest that this open reading frame is a relic of an ancient transamidation pathway that occurred in an E . coli ancestor before the horizontal transfer of a eukaryotic glutaminyl-tRNA synthetase (Lamour, V., Quevillon, S., Diriong, S., N'Guyen, V . C., Lipinski, M., and Mirande, M.(1994) Proc . Natl . Acad . Sci . U . S . A . 91, 8670-8674) and that it favored its stable acquisition . From these observations, a revisited model for the evolution of the contemporary glutamyl-tRNA synthetases and glutaminyl-tRNA synthetases that differs from the generally accepted model for the evolution of aminoacyl-tRNA synthetases is proposed.

FEBS Lett, 1996 Jun 10, 388(1), 66 - 72
NMR investigations of the structural properties of the nodulation protein, NodF, from Rhizobium leguminosarum and its homology with Escherichia coli acyl carrier protein; Ghose R et al.; Heteronuclear NMR methods have been used to elucidate the secondary structure and the general tertiary fold of the protein NodF from Rhizobium leguminosarum . A similarity to acyl carrier proteins of the fatty acid synthase system had been suggested by the presence of a phosphopantetheine prosthetic group and a short stretch of sequence homology near the prosthetic group attachment site . NMR results suggest that the structural homology extends well beyond this region . Both proteins have three well-formed helices which fold in a parallel-antiparallel fashion and a prosthetic group attachment site near the beginning of the second helix.

FEBS Lett, 1996 Jun 10, 388(1), 53 - 8
In vitro characterization of the ORF1-ntrBC promoter of Rhizobium etli; Martino M et al.; Rhizobium sigma vegetative-dependent promoters are different from those of enteric bacteria and have never been characterized before . We report here the biochemical characterization of the ORF1-ntrBC promoter of Rhizobium etli . The minimal promoter region was located by means of a transcriptional fusion and further characterized by in vitro transcription and gel retardation experiments . Oligonucleotides used as DNA competitors in runoff transcription experiments allowed the precise localisation of the promoter region . Protein extracts from an ntrC+, but not from an ntrC- strain, inhibited in vitro transcription . The NtrC protein was found to bind specifically to the promoter, where an NtrC binding site overlapping the transcription initiation site, is present.

Biochim Biophys Acta, 1996 Jun 3, 1307(1), 26 - 30
Long tandemly repeated repetitive (LTRR) sequences in the filamentous cyanobacterium Anabaena sp . PCC 7120; Masepohl B et al.; Nucleotide sequence analysis of the DNA region carrying transposon Tn5-1087b from the Anabaena 7120 nitrogen fixation-deficient mutant YC16 revealed the presence of a novel repeated DNA element in cyanobacteria designated long tandemly repeated repetitive (LTRR) sequence . The LTRR element is 37 bp long and contains an inverted repeat sequence . 17 copies of the LTRR element, 13 of which were completely identical, were identified within a 1.3 kb DNA fragment, which was flanked by two divergently transcribed genes homologous to bacteriophage T4 'gene 15' and Rhizobium meliloti exoD, respectively . LTRR-like sequences occur in several DNA regions in Anabaena 7120 and in other cyanobacteria . Furthermore, the presence of an LTRR-like DNA region in mitochondrial plasmids of Vicia faba indicates strong conservation of such structures during evolution.

Biochem Mol Biol Int, 1996 Jun, 39(3), 621 - 8
Salinity and osmotic stress-regulated proteins in cowpea Rhizobium 4a (peanut isolate); Wankhade S et al.; The effect of salinity and osmotic stress on protein synthesis was studied in cowpea Rhizobium 4a . Osmotic component of salinity stress has been shown to induce ten proteins in a salt tolerant/osmosensitive cowpea Rhizobium . 4a (groundnut isolate) . Of these seven polypeptides were induced only by salt/osmotic stress while two were induced by heat shock . The results demonstrate a commonality as well as stress specificity of protein synthesis regulation.

Mol Microbiol, 1996 Jun, 20(5), 993 - 1000
Role of nodl and nodJ in lipo-chitooligosaccharide secretion in Azorhizobium caulinodans and Escherichia coli; Fernandez-Lopez M et al.; Lipo-chitooligosaccharide (LCO) Nod factors are produced and secreted by rhizobia and trigger nodule development in leguminous host plants . The products of the bacterial nodlJ genes are related to transporters of capsular polysaccharides and were proposed to be involved in LCO transport . We have studied nodlJ of Azorhizobium caulinodans ORS571 by analysis of cell-associated and secreted radioactively labelled Nod factors in wild-type ORS571, a nodJ mutant and a complemented strain . Secretion was strongly reduced in the nodJ mutant, and restored to wild-type levels after complementation . Constructs were made for expression of combinations of different nod genes in Escherichia coli DH5 alpha . The strain DH5 alpha (pUCNABCSU) synthesized LCOs, but they were only secreted when a plasmid containing both nodl and nodJ was supplied in trans . nodl or nodJ alone was not sufficient . In E . coli as well as in Azorhizobium, the nodlJ-encoded transporter showed a specificity for more hydrophilic LCOs.

Can J Microbiol, 1996 Jun, 42(6), 533 - 8
Lysis and biological control of Aspergillus niger by Bacillus subtilis AF 1; Podile AR et al.; A biocontrol rhizobacterial strain of Bacillus subtilis AF 1 grown for 6 h was coinoculated with Aspergillus niger at different time intervals and microscopic observations revealed adherence of bacterial cells to the fungal mycelium . Bacterial cells multiplied in situ and colonized the mycelial surface . Growth of AF 1 resulted in damage to the cell wall, followed by lysis . AF 1 inoculation into media containing A . niger at 0, 6, and 12 h suppressed > 90% fungal growth, while in 18- and 24-h cultures fungal growth inhibition was 70 and 56%, respectively, in terms of dry weight . In dual culture the fungal growth was not accompanied by formation of spores . The mycelial preparation of A . niger as principal carbon source supported the growth of B . subtilis, as much as chitin . Extracellular protein precipitate from B . subtilis culture filtrate had a significant growth-retarding effect on A . niger . Groundnut seeds bacterized with B . subtilis showed a reduced incidence of crown rot in a niger infested soil, suggesting a possible role of A . subtilis in biological control of A . niger.

Appl Environ Microbiol, 1996 Jun, 62(6), 2029 - 36
Typing of rhizobia by PCR DNA fingerprinting and PCR-restriction fragment length polymorphism analysis of chromosomal and symbiotic gene regions: application to Rhizobium leguminosarum and its different biovars; Laguerre G et al.; Characterization of 43 strains of Rhizobium leguminosarum biovars viciae, trifolii, and phaseoli was performed by two methodologies based on PCR amplification, i.e., PCR DNA fingerprinting of interrepeat sequences and restriction fragment length polymorphism (RFLP) analysis of PCR -amplified chromosomal and symbiotic gene regions . Groupings generated by PCR DNA fingerprinting with either extragenic palindromic repetitive primers or two different single random primers were correlated with similar levels of resolution . Although less discriminating, PCR-RFLP analysis of intergenic spacer between genes coding for 16S and 23S rRNA (16S and 23S rDNA) yielded intraspecific polymorphisms . The classification of strains was independent of the biovar status and was in agreement with those obtained by PCR DNA fingerprinting . Intrabiovar variation within symbiotic gene regions was detected by PCR-RFLP analysis of nifDK and nodD gene regions, but the strains were grouped according to the biovar . The rDNA intergenic spacer and nif primers were verified to be universal for rhizobial species by testing of various reference strains, whereas the nod primers designed in this study were biovar or species specific for R . leguminosarum and Rhizobium etli . Classifications of R . leguminosarum strains by the PCR-based methods were correlated with those previously obtained by conventional total DNA restriction profile comparisons and RFLP analysis using chromosomal and symbiotic gene probes . Ranges of discriminating powers were also equivalent between the two approaches . However, the PCR-based methods are much less time-consuming and are therefore more convenient.

Curr Opin Biotechnol, 1996 Jun, 7(3), 343 - 7
Biological control of plant root pathogens; Thomashow LS; Rhizobacteria introduced to control soil-borne root diseases must establish metabolically active populations that mediate protection either by direct antagonism of pathogens or by stimulation of host plant defenses . Recent interest has focused on the genetic and biochemical basis of disease control and the influence of environmental factors on the expression and activity of biocontrol mechanisms . The cloning and sequencing of genes involved in the production of microbial metabolites playing key roles in plant defense opens new possibilities for improving the performance of biocontrol agents.

J Bacteriol, 1996 Jun, 178(12), 3621 - 7
Modulation of development, growth dynamics, wall crystallinity, and infection sites in white clover root hairs by membrane chitolipooligosaccharides from Rhizobium leguminosarum biovar trifolii; Dazzo FB et al.; We used bright-field, time-lapse video, cross-polarized, phase-contrast, and fluorescence microscopies to examine the influence of isolated chitolipooligosaccharides (CLOSs) from wild-type Rhizobium leguminosarum bv . trifolii on development of white clover root hairs, and the role of these bioactive glycolipids in primary host infection . CLOS action caused a threefold increase in the differentiation of root epidermal cells into root hairs . At maturity, root hairs were significantly longer because of an extended period of active elongation without a change in the elongation rate itself . Time-series image analysis showed that the morphological basis of CLOS-induced root hair deformation is a redirection of tip growth displaced from the medial axis as previously predicted . Further studies showed several newly described infection-related root hair responses to CLOSs, including the localized disruption of the normal crystallinity in cell wall architecture and the induction of new infection sites . The application of CLOS also enabled a NodC- mutant of R . leguminosarum bv . trifolii to progress further in the infection process by inducing bright refractile spot modifications of the deformed root hair walls . However, CLOSs did not rescue the ability of the NodC- mutant to induce marked curlings or infection threads within root hairs . These results indicate that CLOS Nod factors elicit several host responses that modulate the growth dynamics and symbiont infectibility of white clover root hairs but that CLOSs alone are not sufficient to permit successful entry of the bacteria into root hairs during primary host infection in the Rhizobium-clover symbiosis.

J Bacteriol, 1996 Jun, 178(11), 3212 - 20
Thermoregulation of kpsF, the first region 1 gene in the kps locus for polysialic acid biosynthesis in Escherichia coli K1; Cieslewicz M et al.; The kps locus for biosynthesis of the capsular polysialic acid virulence factor in Escherichia coli K1 contains at least two convergently transcribed operons, designated region 1 and regions 2 plus 3 . On the basis of DNA sequence analysis, kpsF appeared to be a good candidate for the first gene of region 1 (M . J . Cieslewicz, S . M . Steenbergen, and E . R . Vimr, J . Bacteriol . 175:8018-8023, 1993) . A preliminary indication that kpsF is required for capsule production is the capsule-negative phenotype of an aph T insertion in the chromosomal copy of kpsF . The present communication describes the isolation and phenotypic characterization of this mutant . Although transcription through kpsF was required for capsule production, complementation analysis failed to indicate a clear requirement for the KpsF polypeptide . However, since E . coli contains at least two other open reading frames that could code for homologs of KpsF, the apparent dispensability of KpsF remains provisional . DNA sequence analysis of 1,100 bp upstream from the kpsF translational start site did not reveal any open reading frames longer than 174 nucleotides, consistent with kpsF being the first gene of region 1 . Since kpsF appeared to be the first gene of a region whose gene products are required for polysialic acid transport and because capsule production is known to be thermoregulated, primer extension analyses were carried out with total RNA isolated from cells grown at permissive (37 degrees C) and nonpermissive (20 degrees C) temperatures . The results revealed a potentially complex kpsF promoter-like region that was transcriptionally silent at the nonpermissive temperature, suggesting that thermoregulation of region 1 may be exerted through variations in kpsF expression . Additional evidence supporting this conclusion was obtained by demonstrating the effects of temperature on expression of the gene kpsE, immediately downstream of kpsF . Chloramphenicol acetyltransferase assays were carried out with constructs containing the kpsF 5' untranslated region fused to a promoterless cat cassette, providing further evidence that kpsF is thermoregulated . Although the function of KpsF is unclear, primary structure analysis indicated two motifs commonly observed in regulatory proteins and homology with glucosamine synthase from Rhizobium meliloti.

J Bacteriol, 1996 Jun, 178(11), 3119 - 26
Regulatory proteins and cis-acting elements involved in the transcriptional control of Rhizobium etli reiterated nifH genes; Valderrama B et al.; In Rhizobium etli the nitrogenase reductase genes are reiterated . Strain CE3 has three copies; nifHa and nifHb form part of nifHDK operons with the nitrogenase structural genes, while nifHc is linked to a truncated nifD homolog . Their sequences are identical up to 6 residues upstream from a sigma54-dependent promoter . A remarkable difference among them is the absence of canonical NifA binding sites upstream of nifHc while a canonical binding site is located 200 bp upstream of nifHa and nifHb . To evaluate the transcriptional regulation of the reiterated nifH genes, we constructed fusions of nifHa and nifHc with the lacZ gene of Escherichia coli . Both genes were expressed at maximum levels under 1% oxygen in free-living cultures, and their expression declined as the oxygen concentration was increased . This expression was dependent on the integrity of nifA, and nifHc was expressed at higher levels than nifHa . The same pattern was observed with root nodule bacteroids . Expression of both genes in E . coli required sigma54 in addition to NifA bound to the upstream activator sequence . In vivo dimethyl sulfate footprinting analyses showed that NifA binds to the canonical site upstream of nifHa and to a TGT half-site 6 nucleotides further upstream . NifA protected an imperfect binding site upstream of nijHc at position 85 from the promoter . The integration host factor stimulated each gene differently, nifHa being more dependent on this protein . The above results correlate the asymmetric arrangement of cis-acting elements with a differential expression of the reiterated nifH genes, both in culture and during symbiosis with bean plants.

Cell, 1996 May 31, 85(5), 673 - 81
Calcium spiking in plant root hairs responding to Rhizobium nodulation signals; Ehrhardt DW et al.; SUMMARY: Rhizobium lipochitooligosaccharide signal molecules stimulate multiple responses in legume host plants, including changes in host gene expression, cell growth, and mitoses leading to root nodule development . The basis for signal transduction in the plant is not known . We examined cytoplasmic free calcium in host root hairs using calcium-sensitive reporter dyes . Image analysis of injected dyes revealed localized periodic spikes in cytoplasmic calcium levels that ensued after a characteristic lag following signal application . Structural features of the signal molecules required to cause nodulation responses in alfalfa are also essential for stimulating calcium spiking . A nonnodulating alfalfa mutant is defective in calcium spiking, consistent with the possibility that this mutant is blocked in an early stage of nodulation signal perception.

J Chromatogr B Biomed Appl, 1996 May 17, 680(1-2), 171 - 81
Purification of pea nodule symbiosomes using an aqueous polymer two-phase system; Hernandez LE et al.; Symbiosomes were obtained from mature pea (Pisum sativum cv . Argona) root nodules infected with Rhizobium leguminosarum strain (biov . viciae 3841) and purified using an aqueous polymer two-phase system (APS) . The APS consists of a mixture of polymers, usually dextran T500 and poly(ethylene glycol) 3350, prepared as aqueous solutions on a weight per weight basis, where each fraction distributes according to their surface characteristics . Results of ATPase activity, cytochrome c oxidase activity, glucan synthase II activity, NAD(P)H-cytochrome c reductase activity, NO3(-)-sensitive ATPase activity, transport of {14C}malate vs . {14C}glutamate and MAC 57 antigen analysis showed that the APS method provided intact symbiosomes with low bacteroid, plasma membrane, endoplasmic reticulum and/or mitochondria contamination . No complicated equipment is needed and the method was simple and fast, compared with other purification techniques.

FEMS Microbiol Lett, 1996 May 15, 139(1), 19 - 25
Modular structure of the Rhizobium meliloti DctB protein; Giblin L et al.; To investigate the modular structure of the Rhizobium meliloti dicarboxylic acid sensor protein, DctB, three truncated DctB proteins (DctB4, DctB5 and DctB4G) were constructed, overproduced in Escherichia coli and purified . The DctB4G protein was composed of 446 amino acids of the DctB C-terminus and displayed strong autophosphorylation activity in vitro . This activity was sustained when a further 120 amino acids at the N-terminus of the polypeptide were deleted (DctB5) . This protein which has an intact transmitter domain exhibits specific but inefficient phospho-transfer capabilities . Removal of 58 amino acids from the DctB4G C-terminus which included blocks F and G2 of the transmitter domain, rendered the resultant protein (DctB4) incompetent in autophosphorylation . Phosphorylation activity was restored to DctB4 through intramolecular complementation with DctB . Therefore, it would appear that the R . meliloti DctB protein is active as a dimer (or higher order oligomer) . Furthermore, the intramolecular complementation experiments indicate that the amino acids 171-291, a predicted periplasmic stretch, play an important role in the dimerization process.

Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 4548 - 53
Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of Nod-like chitin oligosaccharides during early embryogenesis; Semino CE et al.; The Xenopus developmental gene DG42 is expressed during early embryonic development, between the midblastula and neurulation stages . The deduced protein sequence of Xenopus DG42 shows similarity to Rhizobium Nod C, Streptococcus Has A, and fungal chitin synthases . Previously, we found that the DG42 protein made in an in vitro transcription/translation system catalyzed synthesis of an array of chitin oligosaccharides . Here we show that cell extracts from early Xenopus and zebrafish embryos also synthesize chitooligosaccharides . cDNA fragments homologous to DG42 from zebrafish and mouse were also cloned and sequenced . Expression of these homologs was similar to that described for Xenopus based on Northern and Western blot analysis . The Xenopus anti-DG42 antibody recognized a 63-kDa protein in extracts from zebrafish embryos that followed a similar developmental expression pattern to that previously described for Xenopus . The chitin oligosaccharide synthase activity found in extracts was inactivated by a specific DG42 antibody; synthesis of hyaluronic acid (HA) was not affected under the conditions tested . Other experiments demonstrate that expression of DG42 under plasmid control in mouse 3T3 cells gives rise to chitooligosaccharide synthase activity without an increase in HA synthase level . A possible relationship between our results and those of other investigators, which show stimulation of HA synthesis by DG42 in mammalian cell culture systems, is provided by structural analyses to be published elsewhere that suggest that chitin oligosaccharides are present at the reducing ends of HA chains . Since in at least one vertebrate system hyaluronic acid formation can be inhibited by a pure chitinase, it seems possible that chitin oligosaccharides serve as primers for hyaluronic acid synthesis.

Appl Microbiol Biotechnol, 1996 May, 45(4), 509 - 12
Characterization of two bioluminescent Rhizobium meliloti strains constructed for field releases; Dammann-Kalinowski T et al.; The deliberate release of genetically engineered microorganisms requires a thorough characterization of the microbes in question . For the two bioluminescent Rhizobium meliloti strains, L1 and L33 {Selbitschka et al . (1992) Mol Ecol 1:9-19; Selbitschka et al . (1995) FEMS Microbiol Ecol 16:223-232}, designated for field release, the sites of genetic modifications in the chromosomes were sequenced from amplified genomic DNA . This indicated no unexpected alterations in the nucleotide sequence . The bioluminescent phenotype was stably inherited over more than 100 generations in liquid cultures . The presence of the luciferase gene in both strains did not have secondary effects on a variety of metabolic pathways as assessed by the Biolog GN system . A specific polymerase chain reaction amplification, based on the chromosomal insertion site of the luc cassette, allowed the discrimination between the two strains and thus simplifies monitoring . The RecA-deficient strain L1 showed a strongly (more than 90%) reduced ability to nodulate alfalfa in competition with its parent strain R . meliloti 2011 and its RecA+ counterpart L33.

Plant Physiol, 1996 May, 111(1), 49 - 60
Identification of a new pea gene, PsNlec1, encoding a lectin-like glycoprotein isolated from the symbiosomes of root nodules; Kardailsky IV et al.; A 27-kD glycoprotein antigen recognized by monoclonal antibody MAC266 was purified from isolated symbiosomes derived from pea (Pisum sativum) root nodules containing Rhizobium . The N-terminal amino acid sequence was obtained, and the corresponding cDNA clone was isolated by a polymerase chain reaction-based strategy . The clone contained a single open reading frame, and the gene was termed PsNlec1 . Phylogenetic analysis of 31 legume sequences showed that the PsNlec1 protein is related to the legume lectin family but belongs to a subgroup that is very different from pea seed lectin . Expression of the PsNlec1 transcript was much stronger in nodules than in other parts of the plant . It was found in both infected and uninfected cells in the central tissue of the nodule and in the stele of the root near the attachment point of the nodule . When uninfected pea seedlings were grown on medium containing nitrate, weak transcription of PsNlec1 was observed in the root system . The identification of PsNlec1 inside the symbiosome is consistent with the observation that legume lectins are generally vacuolar proteins that may serve as transient storage components.

Can J Microbiol, 1996 May, 42(5), 503 - 6
Isolation and characterization of Azospirillum lipoferum locus that complements Rhizobium meliloti dctA and dctB mutations; Tripathi AK et al.; A DNA probe containing the structural gene for dicarboxylate transport (dct A) of Rhizobium meliloti hybridized strongly with the fragments of Azospirillum lipoferum genomic DNA . A genomic library of A . lipoferum was screened for the dct A gene by complementation of a dct A mutant of Rhizobium meliloti . A recombinant cosmid, p37D, capable of restoring growth of the dct A mutant on dicarboxylates was isolated and found to hybridize to the dctA probe . The ability of p37D to complement the dct B mutant of R . meloliti indicated that dct A and dct B genes in A . lipoferum may be organized adjacent to each other.

Mol Plant Microbe Interact, 1996 May, 9(4), 233 - 42
Use of a subtractive hybridization approach to identify new Medicago truncatula genes induced during root nodule development; Gamas P et al.; We report the identification of new molecular markers associated with different stages of Rhizobium-induced nodule development in the legume Medicago truncatula . A cDNA library was constructed from pre-nitrogen-fixing M . Truncatula nodules, and differentially screened with a polymerase chain reaction-amplified subtracted probe . Twenty-nine new families of nodulin cDNA clones, designated MtN1 to MtN29, were thus identified in addition to clones for several known nodulins . All MtN genes were shown by Northern (RNA) hybridization analysis to be induced during nodulation, some of them well before nodule emergence . The MtN genes were classified into three groups depending on their expression kinetics . The expression of three MtN genes showed a limited induction by Nod factors purified from Rhizobium meliloti . Homologies with a variety of proteins were found for the deduced amino acid sequences of 10 of the MtN genes.

J Bacteriol, 1996 May, 178(10), 2727 - 33
Isolation of monoclonal antibodies reacting with the core component of lipopolysaccharide from Rhizobium leguminosarum strain 3841 and mutant derivatives; Lucas MM et al.; Monoclonal antibodies reacting with the core oligosaccharide or lipid A component of Rhizobium lipopolysaccharide (LPS) could be useful for the elucidation of the structure and biosynthesis of this group of macromolecules . Mutant derivatives of Rhizobium leguminosarum 3841 with LPS structures lacking the major O-antigen moiety were used as immunogens, and eight antibodies were selected for further study . All the antibodies reacted with the fast-migrating species known as LPS-2 following gel electrophoresis of Rhizobium cell extracts . For four of these antibodies, reactivity with affinity-purified LPS was lost after mild acid hydrolysis, indicating that they probably recognized the core oligosaccharide component . The four other antibodies still reacted with acid-treated LPS and may recognize the lipid A moiety, which is stable to mild acid hydrolysis . The pattern of antibody staining after gel electrophoresis revealed differences in LPS-2 epitope structure between each of the mutants and the wild type . Furthermore, for each of the mutants the antibodies crossreacted with a minor band that migrated more slowly than LPS-2; we have termed this more slowly migrating form LPS-3 . The majority of the antibodies also reacted with LPS from strain CE109, a derivative of Rhizobium etli CE3, confirming that the LPS core antigens can be relatively conserved between strains of different Rhizobium species . One of the antibodies isolated in this study (JIM 32) was unusual because it appeared to react with all forms of LPS from strain 3841 (namely, LPS-1, LPS-2, and LPS-3) . Furthermore, JIM 32 reacted positively with the LPS from many strains of Rhizobium tested (excluding the Rhizobium meliloti subgroup) . JIM 32 did not react with representative strains from Bradyrhizobium, Azorhizobium or other related bacterial species.

J Bacteriol, 1996 May, 178(9), 2676 - 87
Linkage of genes essential for synthesis of a polysaccharide capsule in Sphingomonas strain S88; Yamazaki M et al.; Several structurally related capsular polysaccharides that are secreted by members of the genus Sphingomonas are being developed as aqueous rheological control agents for diverse industrial and food applications . They include gellan (S-60), welan (S-130), rhamsan (S-194), S-657, S-88, S-198, S-7, and NW-11 . We refer to these polysaccharides as sphingans, after the genus name . This paper characterizes the first gene cluster isolated from a Sphingomonas species (S88) that is required for capsule synthesis . Overlapping DNA segments which spanned about 50 kbp of S88 DNA restored the synthesis of sphingan S-88 in capsule-negative mutants . The mutations were mapped into functional complementation groups, and the contiguous nucleotide sequence for the 29-kbp cluster was determined . The genetic complementation map and the DNA sequences were interpreted as an extended multicistronic locus containing genes essential for the assembly and secretion of polysaccharide S-88 . Many of the deduced amino acid sequences were similar to gene products from other polysaccharide-secreting bacteria such as Rhizobium meliloti (succinoglycan), Xanthomonas campestris (xanthan gum), and Salmonella enterica (O antigen) . The S88 locus contained a four-gene operon for the biosynthesis of dTDP-L-rhamnose, an essential precursor for the sphingans . Unexpectedly, there were also two genes for secretion of a lytic or toxin-like protein nested within the polysaccharide cluster . The conservation and linkage of genes that code for a defensive capsule and genes for secretion of an offensive lysin or toxin suggest a heretofore unknown pathogenic life history for Sphingomonas strain S88.

Mol Gen Genet, 1996 Apr 24, 251(1), 44 - 51
Rhizobium nodulation protein NodA is a host-specific determinant of the transfer of fatty acids in Nod factor biosynthesis; Ritsema T et al.; In the biosynthesis of lipochitin oligosaccharides (LCOs) the Rhizobium nodulation protein NodA plays an essential role in the transfer of an acyl chain to the chitin oligosaccharide acceptor molecule . The presence of nodA in the nodABCIJ operon makes genetic studies difficult to interpret . In order to be able to investigate the biological and biochemical functions of NodA, we have constructed a test system in which the nodA, nodB and nodC genes are separately present on different plasmids . Efficient nodulation was only obtained if nodC was present on a low-copy-number vector . Our results confirm the notion that nodA of Rhizobium leguminosarum biovar viciae is essential for nodulation on Vicia . Surprisingly, replacement of R . l . by viciae nodA by that of Bradyrhizobium sp . ANU289 results in a nodulation-minus phenotype on Vicia . Further analysis revealed that the Bradyrhizobium sp . ANU289 NodA is active in the biosynthesis of LCOs, but is unable to direct the transfer of the R . l . by, viciae nodFE-dependent multi-unsaturated fatty acid to the chitin oligosaccharide acceptor . These results lead to the conclusion that the original notion that nodA is a common nod gene should be revised.

Mol Plant Microbe Interact, 1996 Apr, 9(3), 187 - 97
Novel and complex chromosomal arrangement of Rhizobium loti nodulation genes; Scott DB et al.; A mutational and structural analysis of Rhizobium loti nodulation genes in strains NZP2037 and NZP2213 was carried out . Unlike the case with other Rhizobium strains examined to date, nodB was found on an operon separate from nodACIJ . Sequence analysis of the nodACIJ and nodB operon regions confirm that R . loti common nod genes have a gene organization different from that of other Rhizobium spp . At least 4 copies of nodD-like sequences were identified in R . loti . The complete nucleotide sequence of one of these, nodD3, was determined . A new host-specific nod gene, nolL, was identified adjacent to nodD3 . NolL shares homology with NodX and other O-acetyl transferases . Mutational analysis of the nod regions of strains NZP2037 and NZP2213 showed that nodD3, nodI, nodJ, and nolL were all essential for R . loti strains to effectively nodulate the extended host Lotus pedunculatus, but were not necessary for effective nodulation of the less restrictive host, Lotus corniculatus . Both nodD3 and nolL were essential for R . loti strains to nodulate Leucaena leucocephala.

Mol Plant Microbe Interact, 1996 Apr, 9(3), 151 - 63
Characterization of Rhizobium tropici CIAT899 nodulation factors: the role of nodH and nodPQ genes in their sulfation; Folch-Mallol JL et al.; We have purified and characterized the nodulation factors produced by Rhizobium tropici CIAT899 . This strain produces a large variety of nodulation factors, these being a mixture of sulfated or nonsulfated penta- or tetra-chito-oligosaccharides to which any of six different fatty acyl moieties may be attached to nitrogen of the nonreducing terminal residue . In this mixture we have also found methylated or nonmethylated lipo-chitin oligosaccharides . Here we describe a novel lipo-chitin-oligosaccharide consisting of a linear backbone of 4 N-acetylglucosamine residues and one mannose that is the reducing-terminal residue and bearing a C18:1 fatty acyl moiety on the nonreducing terminal residue . In addition, we have identified, cloned, and sequenced R . tropici nodH and nodPQ genes, generated mutations in the nodH and nodQ genes, and tested the mutant strains for nodulation in Phaseolus and Leucaena plants . Our results indicate that the sulfate group present in wild-type Nod factors plays a major role in nodulation of Leucaena plants by strain CIAT899 of R . tropici.

Biosci Biotechnol Biochem, 1996 Apr, 60(4), 717 - 20
Cloning and sequencing of trehalose biosynthesis genes from Rhizobium sp . M-11; Maruta K et al.; The two genes encoding maltooligosyl trehalose synthase and maltooligosyl trehalose trehalohydrolase, which are related to biosynthesis of alpha, alpha-trehalose, were cloned from Rhizobium sp . M-11 . Sequence analysis showed that the synthase gene composed of 2316 bp was connected with the hydrolase gene of 1788 bp by an overlap of one nucleotide . The deduced amino acid sequences of both enzymes have several regions common to amylolytic enzymes belonging to an "alpha-amylase family".

Plant Mol Biol, 1996 Apr, 31(1), 169 - 73
Insertion of pea lectin into a phospholipid monolayer; Booij P et al.; Pea lectin (PSL) is a secretory sugar-binding protein, readily soluble in aqueous solutions of low osmolarity . However, PSL also appears to be associated with the plasma membrane at the tip of young pea root hairs . By using the Wilhelmy plate method, we found that PSL can insert into a lipid monolayer . This property appeared to be independent of the sugar-binding ability of the protein . This result suggests that PSL may be directly involved in membrane-mediated interactions with saccharide ligands, for example during root hair infection by symbiotic rhizobia.

Plant Mol Biol, 1996 Apr, 31(1), 149 - 56
The pea early nodulin gene PsENOD7 maps in the region of linkage group I containing sym2 and leghaemoglobin; Kozik A et al.; The early nodulin gene, PsENOD7, is expressed in pea root nodules induced by Rhizobium leguminosarum bv . viciae, but not in other plant organs . In situ hybridization showed that this gene is transcribed during nodule maturation in the infected cells of the proximal part of the prefixation zone II . At the transition of zone II into interzone II-III, the level of PsENOD7 mRNA drops markedly . PsENOD7 has no significant homology to other genes . RFLP mapping studies have shown that PsENOD7 is located in linkage group I between the leghaemoglobin genes and sym2.

Mol Ecol, 1996 Apr, 5(2), 177 - 86
A hierarchical analysis of population genetic structure in Rhizobium leguminosarum bv . trifolii; Hagen MJ et al.; Little is known about the population processes that shape the genetic diversity in natural populations of rhizobia . A sample of 912 Rhizobium leguminosarum biovar trifolii isolates were collected from naturalized red clover populations (Trifolium pratense) and analysed for 15 allozyme loci to determine the levels and distribution of genetic diversity . Hierarchical analyses compared different sampling levels, geographical separation, and temporal separation . Total genetic diversity across all isolates was H = 0.426, with 57.6% of the total diversity found among isolates obtained from individual red clover plants . Relatively low genetic differentiation among populations and high differentiation among plants within populations was observed; this suggests that gene flow and founder effect act differently at geographical and local scales . Significant differences were observed in (i) allele frequencies among populations and among plants within populations, and (ii) the frequency distribution of the most widespread and the most abundant strains . When multilocus linkage disequilibrium was calculated, significant levels of disequilibrium were observed in the total sample and in three of the eight populations.

Arch Microbiol, 1996 Apr, 165(4), 285 - 8
Evidence that two genomic species of Rhizobium are associated with Medicago truncatula; Rome S et al.; Seventy-three isolates of rhizobia sampled from root nodules of Medicago truncatula were analyzed by restriction fragment length polymorphism (RFLP) of DNA regions amplified by the polymerase chain reaction (PCR) targeting the symbiotic plasmid (nifD-K, nodD1, and nodD2 genes) and the chromosome (16S rDNA plus intergenic spacer) . Two genotypic groups were found, regardless of the DNA region targeted . These two groups were given the status of genomic species based on results of DNA/DNA hybridization.

J Bacteriol, 1996 Apr, 178(8), 2224 - 31
NADP+ -dependent malic enzyme of Rhizobium meliloti; Driscoll BT et al.; The bacterium Rhizobium meliloti, which forms N2-fixing root nodules on alfalfa, has two distinct malic enzymes; one is NADP+ dependent, while a second has maximal activity when NAD+ is the coenzyme . The diphosphopyridine nucleotide (NAD+)-dependent malic enzyme (DME) is required for symbiotic N2 fixation, likely as part of a pathway for the conversion of C4-dicarboxylic acids to acetyl coenzyme A in N2-fixing bacteroids . Here, we report the cloning and localization of the tme gene (encoding the triphosphopyridine nucleotide {NADP+}-dependent malic enzyme) to a 3.7-kb region . We constructed strains carrying insertions within the tme gene region and showed that the NADP+ -dependent malic enzyme activity peak was absent when extracts from these strains were eluted from a DEAE-cellulose chromatography column . We found that NADP+ -dependent malic enzyme activity was not required for N2 fixation, as tme mutants induced N2-fixing root nodules on alfalfa . Moreover, the apparent NADP+ -dependent malic enzyme activity detected in wild-type (N2-fixing) bacteroids was only 20% of the level detected in free-living cells . Much of that residual bacteroid activity appeared to be due to utilization of NADP+ by DME . The functions of DME and the NADP+ -dependent malic enzyme are discussed in light of the above results and the growth phenotypes of various tme and dme mutants.

J Bacteriol, 1996 Apr, 178(7), 1782 - 7
Transcription start sites for syrM and nodD3 flank an insertion sequence relic in Rhizobium meliloti; Barnett MJ et al.; In Rhizobium meliloti the syrM regulatory gene positively controls nod D3 and syrA, and nodD3 positively controls syrM and nod regulon genes such as nodABC, syrM and nodD3 are divergently transcribed and are separated by approximately 2.8 kb of DNA . The 885-bp SphI DNA fragment between syrM and nodD3 was subcloned and sequenced . Analysis of this intergenic region showed two open reading frames similar to those found in insertion elements of the IS3 family . We determined transcription initiation sites for both syrM and nodD3 using primer extension . The syrM transcription initiation site is 499 bp upstream of the syrM protein-coding region and downstream of a nod box which shows several differences from the R . meliloti nod box consensus sequence . We demonstrated binding of NodD3 to DNA containing the syrM nod box . The nodD3 start site maps 659 bp upstream of the nodD3 translation initiation site . A putative SyrM binding site was identified upstream of the nodD3 start site on the basis of sequence similarity to the upstream region of syrA, another locus regulated by SyrM.

Mol Gen Genet, 1996 Mar 7, 250(4), 437 - 46
Sucrose synthase and enolase expression in actinorhizal nodules of Alnus glutinosa: comparison with legume nodules; van Ghelue M et al.; Two different types of nitrogen-fixing root nodules are known- actinorhizal nodules induced by Frankia and legume nodules induced by rhizobia . While legume nodules show a stem-like structure with peripheral vascular bundles, actinorhizal nodule lobes resemble modified lateral roots with a central vascular bundle . To compare carbon metabolism in legume and actinorhizal nodules, sucrose synthase and enolase cDNA clones were isolated from a cDNA library, obtained from actinorhizal nodules of Alnus glutinosa . The expression of the corresponding genes was markedly enhanced in nodules compared to roots . in situ hybridization showed that, in nodules, both sucrose synthase and enolase were expressed at high levels in the infected cortical cells as well as in the pericycle of the central vascular bundle of a nodule lobe . Legume sucrose synthase expression was studied in indeterminate nodules from pea and determinate nodules from Phaseolus vulgaris by using in situ hybridization.

Protein Sci, 1996 Mar, 5(3), 538 - 41
Crystallization and preliminary diffraction studies of NodL, a rhizobial O-acetyl-transferase involved in the host-specific nodulation of legume roots; Dunn SM et al.; The NodL specified O-acetyltransferase from the microbial symbiont Rhizobium leguminosarum has been over-expressed in Escherichia coli and purified using affinity-elution dye chromatography as the key step . The protein has been crystallized at 20 degrees C in 18% PEG 600, 0.1 M Tris/HCl buffer, pH 8.5, containing 1% dioxane, 0.25% octyl-beta-glucoside, and 5 mM coenzyme A using the hanging drop vapor diffusion method . Ambient temperature X-ray diffraction studies reveal the space group to be hexagonal (P6(3)22) with lattice constants a = b = 77.08 A, c = 160.6 A, and alpha = beta = 90 degrees, gamma = 120 degrees . Crystals that are flash-frozen to 120 K diffract beyond 2.7 A.

Microbiology, 1996 Mar, 142 ( Pt 3), 601 - 10
An essential role for actA in acid tolerance of Rhizobium meliloti; Tiwari RP et al.; The actA gene, which is disrupted by Tn5 in the acid-sensitive mutant of Rhizobium meliloti TG2-6, was cloned and sequenced . It encodes a protein of 541 amino acids with a calculated molecular mass of 57,963 Da and an estimated pl of 9.0 . The ActA protein sequence has 30% identity, and much higher similarity (69%), with the CutE protein of Escherichia coli . Like the cutE mutant of E . coli, TG2-6 is sensitive to copper . The reconstructed wild-type actA gene complemented the low pH- and copper-sensitive phenotype of TG2-6 . Studies with an actA-lacZ gene fusion showed that actA is constitutively expressed at pH 5.8 and 7.0 . The actA gene appears to be chromosomal and is present in all seven strains of R . meliloti tested.

Can J Microbiol, 1996 Mar, 42(3), 279 - 83
Rhizobium leguminosarum as a plant growth-promoting rhizobacterium: direct growth promotion of canola and lettuce; Noel TC et al.; Early seedling root growth of the nonlegumes canola (Brassica campestris cv . Tobin, Brassica napus cv . Westar) and lettuce (Lactuca sativa cv . Grand Rapids) was significantly promoted by inoculation of seeds with certain strains of Rhizobium leguminosarum, including nitrogen- and nonnitrogen-fixing derivatives under gnotobiotic conditions . The growth-promotive effect appears to be direct, with possible involvement of the plant growth regulators indole-3-acetic acid and cytokinin . Auxotrophic Rhizobium mutants requiring tryptophan or adenosine (precursors for indole-3-acetic acid and demonstrate a new facet of the Rhizobium-plant relationship and that Rhizobium leguminosarum can be considered a plant growth-promoting rhizobacterium (PGPR).

Can J Microbiol, 1996 Mar, 42(3), 234 - 42
Modulation of carbohydrate-binding capacities and attachment ability of Bradyrhizobium sp . (lupinus) to white lupin roots; Wisniewski JP et al.; The attachment of Bradyrhizobium sp . (Lupinus) strain MSDJ718 to excised roots of white lupin was examined . Maximal attachment occurred at early to middle log phases of bacterial growth . This binding was pH dependent, with an optimal value reached at 6.6 . Irrespective of the culture age, the attachment was strongly affected by the calcium concentration of the growth medium: a Ca2+ limitation in the Bergersen medium led to optimal attachment of the bacteria . When L-fucose was added during the attachment assay, a significant inhibition was observed . The binding was stimulated when bacteria were cultivated with lupin root extracts, genistein and genistin (two lupin isoflavonoids), and some monosaccharides . In addition, with a spectrofluorimetric method using fluoresceinylated neoglycoproteins, it was shown that the increase of the attachment of bacteria to host cells was correlated to the increase of the L-fucoside binding capacity of the rhizobial cells . Taken together, the results obtained in the present study evidenced a possible role of the L-fucose specific bacterial lectin previously described in the Rhizobium-lupin host cell recognition.

Plasmid, 1996 Mar, 35(2), 121 - 30
A region of a Sym plasmid of Rhizobium leguminosarum biovar phaseoli has similarity to prokaryotic insertion sequences and to eukaryotic integrases; Yeoman KH et al.; Near the nod and nif genes of the Sym plasmid pRP2JI of Rhizobium leguminosarum biovar phaseoli are three open reading frames whose deduced polypeptide products have similarities to those of genes in bacterial insertion sequences . The similarity of one of these ORFs was significantly greater to that of the integrase region of pol proteins of eukaryotic retroviruses and transposable elements in animals and plants than it was to the transposases of prokaryotic insertion sequences . In the noncoding region of the IS-like element, there was a sequence similar to that which had been identified close to nod genes in Azorhizobium caulinodans.

J Bacteriol, 1996 Mar, 178(6), 1646 - 54
Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate; Cevallos MA et al.; Rhizobium etli accumulates poly-beta-hydroxybutyrate (PHB) in symbiosis and in free life . PHB is a reserve material that serves as a carbon and/or electron sink when optimal growth conditions are not met . It has been suggested that in symbiosis PHB can prolong nitrogen fixation until the last stages of seed development, but experiments to test this proposition have not been done until now . To address these questions in a direct way, we constructed an R . etli PHB-negative mutant by the insertion of an Omega-Km interposon within the PHB synthase structural gene (phaC) . The identification and sequence of the R . etli phaC gene are also reported here . Physiological studies showed that the PHB-negative mutant strain was unable to synthesize PHB and excreted more lactate, acetate, pyruvate, beta-hydroxybutyrate, fumarate, and malate than the wild-type strain . The NAD+/NADH ratio in the mutant strain was lower than that in the parent strain . The oxidative capacity of the PHB-negative mutant was reduced . Accordingly, the ability to grow in minimal medium supplemented with glucose or pyruvate was severely diminished in the mutant strain . We propose that in free life PHB synthesis sequesters reductive power, allowing the tricarboxylic acid cycle to proceed under conditions in which oxygen is a limiting factor . In symbiosis with Phaseolus vulgaris, the PHB-negative mutant induced nodules that prolonged the capacity to fix nitrogen.

J Bacteriol, 1996 Mar, 178(6), 1532 - 8
A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum; Preisig O et al.; It has been a long-standing hypothesis that the endosymbiotic rhizobia (bacteroids) cope with a concentration of 10 to 20 nM free O2 in legume root nodules by the use of a specialized respiratory electron transport chain terminating with an oxidase that ought to have a high affinity for O2 . Previously, we suggested that the microaerobically and anaerobically induced fixNOQP operon of Bradyrhizobium japonicum might code for such a special oxidase . Here we report the biochemical characteristics of this terminal oxidase after a 27-fold enrichment from membranes of anaerobically grown B . japonicum wild-type cells . The purified oxidase has TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity as well as cytochrome c oxidase activity . N-terminal amino acid sequencing of its major constituent subunits confirmed that presence of the fixN,fixO, and fixP gene products . FixN is a highly hydrophobic, heme B-binding protein . FixO and FixP are membrane-anchored c-type cytochromes (apparent Mrs of 29,000 and 31,000, respectively), as shown by their peroxidase activities in sodium dodecyl sulfate-polyacrylamide gels . All oxidase properties are diagnostic for it to be a member of the cbb3-type subfamily of heme-copper oxidases . The FixP protein was immunologically detectable in membranes isolated from root nodule bacteroids, and 85% of the total cytochrome c oxidase activity in bacteroid membranes was contributed by the cbb3-type oxidase . The Km values for O2 of the purified enzyme and of membranes from different B . japonicum wild-type and mutant strains were determined by a spectrophotometric method with oxygenated soybean leghemoglobin as the sole O2 delivery system . The derived Km value for O2 of the cbb3-type oxidase in membranes was 7 nM, which is six- to eightfold lower than that determined for the aerobic aa3-type cytochrome c oxidase . We conclude that the cbb3-type oxidase supports microaerobic respiration in endosymbiotic bacteroids.

Microbiology, 1996 Feb, 142 ( Pt 2), 321 - 30
The fructokinase from Rhizobium leguminosarum biovar trifolii belongs to group I fructokinase enzymes and is encoded separately from other carbohydrate metabolism enzymes; Fennington GJ Jr et al.; The Rhizobium leguminosarum bv . trifolii BAL fructokinase (frk) gene was isolated on a 2 center dot 4 kb BamHI fragment from the cosmid pLA72 by complementation analysis of the Tn5-induced frk mutant BAL79, and confirmed by hybridization analysis . The nucleotide sequence of the frk gene was found to contain an open reading frame consisting of 978 bp encoding 326 amino acids, which was then compared to known fructokinase sequences . The fructokinase gene was not contained in an operon and is encoded separately from other enzymes of carbohydrate metabolism . Its product is therefore assigned to the group I fructokinases . A putative promoter (TTGACA-N16-GTTGAT), ribosome-binding site and termination sequence were identified . The Frk protein contained several motifs conserved in other known fructokinase sequences, including an ATP-binding and a substrate-binding motif . The hydropathy plot derived from the frk gene sequence data revealed the fructokinase as a hydrophilic protein . The fructokinase protein was purified to electrophoretic homogeneity by a three-step method using chromatofocusing, affinity chromatography and gel filtration . Its purity was confirmed by SDS-PAGE and it was visualized as a single band by silver staining . The N-terminal amino acid sequence of the purified fructokinase confirmed the proposed open reading frame of the frk gene . The purified fructokinase had a molecular mass of 36 center dot 5 kDa, pl of 4 center dot 65, pH activity range of 6 center dot 0-9 center dot 0 (maximum activity at pH 8 center dot 0) and a Mg2+ requirement . It had a Km of 0 center dot 31 mM and a Vmax of 31 mumol fructose 6-phosphate (mg protein)-1 min-1 with fructose as substrate . The R . leguminosarum bv . trifolii BAL fructokinase was biochemically and molecularly similar to other bacterial fructokinases.

Mol Microbiol, 1996 Feb, 19(3), 443 - 53
The C-terminal domain of the Rhizobium leguminosarum chitin synthase NodC is important for function and determines the orientation of the N-terminal region in the inner membrane; Barny MA et al.; The nodC genes from rhizobia encode an N-acetylglucosaminyl transferase (chitin synthase) involved in the formation of lipo-chito-oligosaccharide Nod factors that initiate root nodule morphogenesis in legume plants . NodC proteins have two hydrophobic domains, one of about 21 residues at the N-terminus and a longer one, which could consist of two or three transmembrane spans, near the C-terminus . These two hydrophobic domains flank a large hydrophilic region that shows extensive homology with other beta -glycosyl transferases . The topology NodC in the inner membrane of Rhizobium leguminosarum biovar viciae was analysed using a series of gene fusions encoding proteins in which NodC was fused to alkaline phosphatase (PhoA) lacking an N-terminal transit sequence or to beta-galactosidase (LacZ) . Our data support a model in which the N-terminal hydrophobic domain spans the membrane in a Nout-Cin orientation, with the adjacent large hydrophilic domain being exposed to the cytoplasm . This orientation appears to depend upon the presence of the hydrophobic region near the C-terminus . We propose that this hydrophobic region contains three transmembrane spans, such that the C-terminus of NodC is located in the periplasm . A short region of about 40 amino acids, encompassing the last transmembrane span, is essential for the function of NodC . Our model for NodC topology suggests that most of NodC, including the region showing most similarity to other beta-glycosyl transferases, is exposed to the cytoplasm, where it is likely that polymerization of N-acetyl glucoasamine occurs . Such a model is incompatible with previous reports suggesting that NodC spans both inner and other membranes.

Plant Foods Hum Nutr, 1996 Feb, 49(2), 93 - 105
Protein quality of peas as influenced by location, nitrogen application and seed inoculation; Igbasan FA et al.; A study was conducted to evaluate the contribution of location, N application and Rhizobium seed inoculation to variations in seed protein content and amino acid (AA) composition of field peas . The magnitude of AA variations with protein level and the nature of the relationships that are involved were determined . Regression equations to predict AAs from protein were developed for the cultivar Bohatyr . The experiments were carried out at two locations in southern Manitoba in 1994 . The levels of N fertilization investigated were: 56, 75, 100, 125, 150, 200, 250 and 300 kg/ha . At each level of N application, seeds planted were either Rhizobium inoculated or not inoculated . The combination of location, fertilizer treatments and inoculation yielded 192 samples for chemical analyses . The samples were analyzed for dry matter (DM), N and AA contents . Location and N fertilization had significant (p < or = 0.001) effects on seed protein content and AA composition . Seed protein content increased with increasing levels of N application . The response of protein to fertilization was not the same in both locations as evidenced from the presence of interaction (p < or = 0.01) between location and N application . Except for methionine and cystine, percent AAs in DM increased with increasing levels of N application . The effects of N application on the concentrations of methionine and cystine were not consistent . On protein basis, the concentrations of AAs decreased with increasing levels of N application . The only exception was arginine which strongly increased in concentration . There was no effect (p > or = 0.05) of seed inoculation observed in this study . Strong positive correlations (r > 0.80) between seed protein content and AA concentrations expressed as percent of DM were found for all AAs except for methionine (r = 0.76) and cystine (r = 0.51) . When AA concentrations are expressed as g per 16 g N, 15 of the 17 AAs were negatively correlated to seed protein content . Only arginine (r = 0.78) and aspartic acid (r = 0.17) had positive correlations . The regression equations developed from this study could be used to predict the concentrations of AAs except methionine and cystine for the cultivar Bohatyr once the protein content is known.

Eur J Biochem, 1996 Feb 1, 235(3), 744 - 9
Cloning, sequencing and expression in Escherichia coli of two Rhizobium sp . genes encoding haloalkanoate dehalogenases of opposite stereospecificity; Cairns SS et al.; A 6.5-kp EcoRI fragment of genomic DNA from a Rhizobium sp . cloned into pUC19 was able to endow Escherichia coli K-12 with the novel ability to grow at the expense of 2-chloropropionic acid . Subcloning showed that this property was a consequence of two dehalogenases encoded on a 2.2-kb PstI fragment . Further subcloning of the PstI fragment led to two constructs that encoded, separately, dehalogenase activity that acted stereospecifically on D-2-chloropropionic acid and L-2-cloropropionic acid, respectively . The genes encoding these two stereospecific dehalogenases have been sequenced and shown to be separated by 177 bp of non-coding DNA . Expression of the dehalogenase genes involved the vector promoter, suggesting that the anticipated Rhizobium sp . regulatory sequences were not functional in E . coli . Comparison of the deduced amino acid sequences of the two dehalogenases (18% identity) indicated with any other 2-chloropropionic acid dehalogenase studied so far.

Plant Mol Biol, 1996 Feb, 30(3), 403 - 17
Phaseolus ENOD40 is involved in symbiotic and non-symbiotic organogenetic processes: expression during nodule and lateral root development; Papadopoulou K et al.; ENOD40 is an early nodulin gene, recently isolated from legume species forming nodules either after Rhizobium infection or spontaneously . ENOD40 cDNAs from Phaseolus plants were isolated and nucleotide sequence determination revealed 85% and 88.5% homology with the reported soybean cDNA clones . The putative polypeptide deduced coincides with the soybean one but a stop codon, almost in the middle of the respective ORF, renders it much shorter . This polypeptide was overexpressed as a fusion protein in Escherichia coli . Although the spatial expression pattern of the gene in the root pericycle and nodule primordium at early stages of development as well as in the pericycle of the vascular bundles and uninfected cells in mature nodules is comparable to the gene's expression pattern in soybean, differences in developmental regulation are evident . We have shown that ENOD40 transcripts are also detected at very early stages of lateral root development, in the dividing pericycle cells of the root stele that give rise to the lateral root primordia . The presence of Rhizobium causes an enhancement of the gene's expression and also induction of the gene in the vascular tissues of developed lateral roots . Interestingly, a discrimination on the gene's expression level in adventious and acropetal incipient lateral root primordia, emerging in infected and uninfected roots, is observed . This indicates that the gene's product may be involved in the hormonal status of the plant and that ENOD40 may be used as a molecular marker in lateral root initiation.

Appl Environ Microbiol, 1996 Feb, 62(2), 685 - 93
Characterization, distribution, and localization of ISRl2, and insertion sequence element isolated from Rhizobium leguminosarum bv . viciae; Mazurier SI et al.; An insertion sequence (IS) element, ISR12, from Rhizobium leguminosarum bv . viciae strain MSDJ4184 was isolated by insertional inactivation of the sacRB gene of pSUP104-sac, which allows positive selection . ISRl2 is 932 bp long, is flanked by 17-bp imperfect terminal inverted repeats, and generated a 3-bp target site duplication . ISRl2 was found to be 63 to 77% homologous to insertion elements of the IS5 group of the IS4 superfamily . A probe incorporating a full-length copy of ISRl2 was used to screen genomic DNAs from a collection of strains and from two field populations of R . leguminosarum to detect and estimate the copy numbers of homologous sequences . Among the collection of 63 strains representing the different species and genera of members of the family Rhizobiaceae, homology to ISRl2 was found within strains belonging to Sinorhizobium meliloti and S . fredii; within four of the six recognized Rhizobium species . R . leguminosarum, R . tropici, R . etli, and R . galegae; and within Rhizobium sp . (Phaseolus) genomic species 2 . The apparent copy numbers of ISRl2 varied from one to eight . Among 139 isolates of R . leguminosarum from two field populations, homology to ISRl2 was detected in 91% of the isolates from one site and in 17% from the other . Analysis of the 95 isolates that hybridize to ISRl2 revealed a total of 20 distinct hybridization patterns composed of one to three bands . Probing blots of Eckhardt gels showed that sequences with homology to ISRl2 may be found on plasmids or the chromosome . Analysis of their genomic distribution demonstrated relationships and diversity among the R . leguminosarum isolates tested.

Appl Environ Microbiol, 1996 Feb, 62(2), 529 - 35
Assessment of competitiveness of rhizobia infecting Galega orientalis on the basis of plant yield, nodulation, and strain identification by antibiotic resistance and PCR; Tas E et al.; Competition between effective and ineffective Rhizobium galegae strains nodulating Galega orientalis was examined on the basis of plant growth, nodulation, antibiotic resistance, and PCR results . In a preliminary experiment in Leonard's jars, ineffective R . galegae strains HAMBI 1207 and HAMBI 1209 competed in similar manners with the effective strain R . galegae HAMBI 1174 . In a pot experiment, soil was inoculated with 0 to 10(5) HAMBI 1207 cells per g before G . orientalis was sown . Seeds of G . orientalis were surface inoculated with 2 x 10(4) and 2 x 10(5) cells of HAMBI 1174 per seed (which represent half and fivefold the commercially recommended amount of inoculant, respectively) . Plant yield and nodulation by the effective strain were significantly reduced, with as few as 10(2) ineffective rhizobia per g of soil, and the inoculation response was not improved by the 10-fold greater dose of the inoculant . Bacteria occupying the nodules were identified by antibiotic resistance and PCR with primers specific for R . galegae HAMBI 1174, R . galegae, and genes coding for bacterial 16S rRNA (bacterial 16S rDNA) . Sixty-two large nodules examined were occupied by the effective strain HAMBI 1174, as proven by antibiotic resistance and amplification of the strain-specific fragment . From 20 small nodules, only the species-specific fragment could be amplified, and isolated bacteria had the same antibiotic resistance and 16S PCR restriction pattern as strain HAMBI 1207 . PCR with our strain-specific and species-specific primers provides a powerful tool for strain identification of R . galegae directly from nodules without genetic modification of the bacteria.

J Bacteriol, 1996 Feb, 178(3), 745 - 52
Genetic analysis of Rhizobium meliloti bacA-phoA fusion results in identification of degP: two loci required for symbiosis are closely linked to degP; Glazebrook J et al.; The function of the Rhizobium meliloti bacA gene, which is a homolog of the Escherichia coli sbmA gene, is required for an intermediate step in nodule development . A strain carrying the bacA386::TnphoA fusion was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, and three mutants that had higher levels of alkaline phosphatase activity were identified . The mutations in these strains were recessive and mapped to the same genetic locus . The gene affected by these mutations was identified and sequenced and was found to be a homolog of the E . coli degP gene, which encodes a periplasmic endopeptidase . Although degP function is important for the virulence of certain intracellular pathogens of mammals, it is not required for the R . meliloti-alfalfa symbiosis . The genetic analyses involving degP were complicated by the presence of a locus immediately upstream of depP that was lethal when present in multiple copies in a DegP- background . R . meliloti derivatives carrying insertion mutations in this locus displayed an N,N,N',N'-tetramethyl-p-phenylenediamine oxidase-negative phenotype, elicited the formation of white cylindrical nodules that did not fix nitrogen, and grew slowly in rich medium, suggesting that the locus was a cyc gene encoding a protein involved in the biosynthesis of a component or components of a respiratory chain . The previously identified fix-382::TnphoA, which similarly causes the formation of white cylindrical nodules that do not fix nitrogen, was shown to affect a gene that is separate from this cyc gene but extremely closely linked to it.

Biochem Biophys Res Commun, 1996 Jan 26, 218(3), 908 - 15
Biodegradation of polychlorinated biphenyls by rhizobia: a novel finding; Damaj M et al.; Metabolism of simple aromatic compounds in rhizobial strains has been a subject of study for a few decades, due either to the significance of nutritional diversity in the inoculum survival during agricultural applications or to the importance of plant phenolics in the microbe-plant cross-talk and signal-transduction . Here, we report the capability of rhizobial strains to catabolize polychlorinated biphenyls (PCBs) . In order to identify the genes in these strains that mediate the catabolism of PCBs we used the bphABC genes from Comamonas testosteroni strain B-356 . Our results showed that genomic DNAs from all four rhizobial strains studied hybridized strongly with the Comamonas-derived probe, indicating the presence of a similar genetic system . This is a novel and interesting finding indicating for the first time, perhaps, of a role of rhizobia in recycling of aromatic compounds in nature and, certainly, opening a new avenue to be explored in the field of bioremediation.

DNA Seq, 1996, 6(5), 275 - 84
Genetic analysis of the acetan biosynthetic pathway in Acetobacter xylinum: nucleotide sequence analysis of the aceB, aceC, aceD and aceE genes; Griffin AM et al.; Sequence analysis of a 5.323 kb chromosomal DNA fragment from Acetobacter xylinum involved in the biosynthesis of the exopolysaccharide acetan, revealed the presence of four ace genes designated aceB, aceC, aceD and aceE . Comparison of translated gene sequences to the databanks was used to assign putative gene functions . AceB displayed strong homology to a glucose-diphosphoprenyl beta, D-glucose transferase from Xanthomonas campestris, while AceC was homologous to a cellobiosyl-diphosphoprenyl alpha, D-mannose transferase from the same organism . Thus these genes encode enzymes catalyzing the second and third steps of the acetan biosynthetic pathway . AceD and AceE were homologous to ExoP and ExoT respectively from Rhizobium meliloti and are likely to be involved in acetan polymerization and export.

Crit Rev Biotechnol, 1996, 16(1), 1 - 51
The genetic and biochemical basis for nodulation of legumes by rhizobia; Pueppke SG; Soil bacteria of the genera Azorhizobium, Bradyrhizobium, and Rhizobium are collectively termed rhizobia . They share the ability to penetrate legume roots and elicit morphological responses that lead to the appearance of nodules . Bacteria within these symbiotic structures fix atmosphere nitrogen and thus are of immense ecological and agricultural significance . Although modern genetic analysis of rhizobia began less than 20 years ago, dozens of nodulation genes have now been identified, some in multiple species of rhizobia . These genetic advances have led to the discovery of a host surveillance system encoded by nodD and to the identification of Nod factor signals . These derivatives of oligochitin are synthesized by the protein products of nodABC, nodFE, NodPQ, and other nodulation genes; they provoke symbiotic responses on the part of the host and have generated immense interest in recent years . The symbiotic functions of other nodulation genes are nonetheless uncertain, and there remain significant gaps in our knowledge of several large groups of rhizobia with interesting biological properties . This review focuses on the nodulation genes of rhizobia, with particular emphasis on the concept of biological specificity of symbiosis with legume host plants.

J Basic Microbiol, 1996, 36(5), 319 - 26
Induction of Rhizobium meliloti nodC gene by Alnus incana compounds; Malek W; An expression of nodC promoter of R . meliloti 1021, cloned in front of the Escherichia coli lacZ gene, was used to study the presence of R . meliloti nodC gene-inducing compound(s) in extracts and exudates of A . incana seeds and roots . The regulatory gene nodD was expressed at comparable level in bacterial culture of R . meliloti with and without the studied plant extracts and exudates, whereas the nodC-lacZ fusion expression was increased 4 times by seed exudates of grey alder and 2 times by seed extracts and root ingredients in comparison to the nodC-lacZ fusion expression in R . meliloti grown in a medium free of plant compounds . Induction of R . meliloti nodC gene expression by A . incana substances was also supported in plant test . Sterile filtrate of coculture of R . meliloti with seed exudates of A . incana induced root hair deformations and nodule-like structures on Medicago sativa.

Annu Rev Microbiol, 1996, 50, 101 - 36
Osmoadaptation by rhizosphere bacteria; Miller KJ et al.; The osmolality of rhizosphere soil water is expected to be elevated in relation to bulk-soil water osmolality as a result of the exclusion of solutes by plant roots during water uptake, the release of plant root exudates, and the production of exopolymers by plant roots and rhizobacteria . In contrast, the osmolality of water within highly hydrated bulk soil is low (less than 50 Osm/kg); thus the ability to adapt to elevated osmolality is likely to be important for successful rhizosphere colonization by rhizobacteria . The present review focuses on the osmoadaptive responses of three gram-negative rhizobacterial genera: Rhizobium, Azospirillum, and Pseudomonas . Specifically, we examine the compatible solutes and osmoprotectants utilized by various species within these genera . The adaptation of rhizobacteria to hypoosmotic environments is also examined in the present review . In particular, we focus on the biosynthesis and accumulation of periplasmic glucans by rhizobacteria . Finally, the relationship between rhizobacterial osmoadaptation and selected plant-microbe interactions is considered.

Rev Argent Microbiol, 1996 Jan-Mar, 28(1), 31 - 8
{Infectivity and effectiveness of different strains of Frankia spp . on Atriplex cordobensis plants}; Fabri S et al.; Frankia is an actinomycete that fixes atmospheric nitrogen in actinorhizal symbiosis (in non-legume plants) . Atriplex cordobensis is an important Chaco Arido forage species due to the high nitrogen content . The aim of this paper was to isolate plant endophytes to be utilized as an inoculum in adapted species . A . cordobensis seedlings of 30 days were inoculated with isolated strains of Atriplex cordobensis, Colletia hystrix, Trevoa trinervis, Talguenea quinquenervia and 4-6 strains of Retanilla ephedra, courtesy of the University of Chile . A complete randomized design with 9 replications was performed . The infective ability of the strains was established through the re-isolation of the endophytes in QMOD artificial medium . The infectivity was evaluated by means of: plant aerial part length, leaf number, number and length of internodes, dry weight and total nitrogen content . The symbiotic effectivity, inoculation response and fixed nitrogen quantity were calculated . All strains treated on A . cordobensis presented Frankia typical morphological characteristics . A . cordobensis and C . hystrix showed the best nitrogen fixing effectivity, T . quinquenervia had a good aerial development but its fixing nitrogen ability was low, as a plant growth promoting rhizobacteria (PGPR) . All strains were infective and A . cordobensis and C . hystrix apported the highest nitrogen amount to the plants.

Annu Rev Biochem, 1996, 65, 503 - 35
Rhizobium lipo-chitooligosaccharide nodulation factors: signaling molecules mediating recognition and morphogenesis; Denarie J et al.; Rhizobia elicit on their specific leguminous hosts the formation of new organs, called nodules, in which they fix nitrogen . The rhizobial nodulation genes specify the synthesis of lipo-chitooligosaccharide signals, the Nod factors (NFs) . Each rhizobial species has a characteristic set of nodulation genes that specifies the length of the chitooligosaccharide backbone and the type of substitutions at both ends of the molecule, thus making the NFs specific for a given plant host . At extremely low concentrations, purified NFs are capable of eliciting on homologous legume hosts many of the plant developmental responses characteristic of the bacteria themselves, including cell divisions, and the triggering of a plant organogenic program . This review summarizes our current knowledge on the biosynthesis, structure, and function of this new class of signaling molecules . Finally we discuss the possibility that these signals could be part of a new family of plant lipo-chitooligosaccharide growth regulators.

Arch Microbiol, 1996 Jan, 165(1), 26 - 33
Characterization of the lipopolysaccharides from Rhizobium meliloti strain 102F51 and its nonnodulating mutant WL113; Russa R et al.; Lipopolysaccharides from the Rhizobium meliloti wild-type strain 102F51, which is effective in symbiosis with alfalfa, and from the nonnodulating mutant WL113, defective in root hair adhesion, derived thereof, were isolated and comparatively analyzed . Both preparations were composed of galactose, glucose, glucuronic acid, galacturonic acid, glucosamine, 3-deoxyheptulosaric acid, and 2-keto-3-deoxyoctonic acid as the major sugar constitutents . After a modified methylation analysis (consisting of the following consecutive steps: methylation, carboxyl reduction, remethylation, mild acid hydrolysis, reduction, and trideuterio-methylation), all of the 3-deoxyheptulosaric and some of the 2-keto-3-deoxyoctonic acid residues were converted into their corresponding 3-deoxyalditol derivatives, which carried trideuteriomethyl groups at positions C-2, C-4, and C-6 . Another part of the permethylated 3-deoxyoctitol was also found as 2,5,6- and 2,6,8-tri-O-trideuteriomethyl derivatives . NMR data obtained with the separated oligosaccharides and the results of methylation analysis indicated that the majority of 2-keto-3-deoxyoctonate was present in the fraction of permethylated disaccharide alditols, namely as 6-O-CD3-alpha Glc(1-->5)3-deoxyoctitol, 6-O-CD3-beta GlcNMeAcyl(1-->4)-3-deoxyoctitol, and as the permethylated trisaccharide alditol, alpha GalA(1-->3)-{6-O-CD3}-beta-Glc(1-->5)-{4-O-CD3}-3-deoxyoctitol . The presence of trideuteriomethyl groups at C-4 of both 3-deoxyalditols and at C-6 of the glucosaminyl or glycosyl residues indicated the linkage points of the released acid-labile ketosidic substituents, such as 3-deoxyheptulosarate and 2-keto-3-deoxyoctonate, in these oligosaccharides . The main differences between the preparations from the wild-type 102F51 and its mutant strain WL 113 were found in the higher content (in strain 102F51) of the following oligosaccharides: alpha-glucuronosyl(1-->4)2-keto-3-deoxyoctonate and alpha-galacturonosyl-(1-->3)alpha-glucosyl-(1-->5)2-keto-3-deoxyoc tonate and in the decreased content of beta-glucosaminyl(1-->4)2-keto-3-deoxy-octonate.

Mol Plant Microbe Interact, 1996 Jan, 9(1), 74 - 7
Isolation and characterization of a pSym locus of Rhizobium sp . BR816 that extends nodulation ability of narrow host range Phaseolus vulgaris symbionts to Leucaena leucocephala; van Rhijn P et al.; Introduction of a cosmid library of megaplasmid DNA of Rhizobium sp . BR816, a broad host range Rhizobium strain, into R . etli CE3, a narrow host range bean symbiont, resulted in the isolation of a transconjugant that could effectively nodulate Leucaena leucocephala . Analysis of the corresponding cosmid, pBRF2, revealed the presence of genes required for elicting nitrogen-fixing nodules on L . leucoephala . Subcloning and Tn5 tagging identified a locus responsible for the host range extension . Sequence analysis of this locus revealed an ORF that shows significant identity with NodO of R . leguminosarum bv . viciae.

Int J Syst Bacteriol, 1996 Jan, 46(1), 240 - 4
Phylogenetic relationships among Rhizobium species nodulating the common bean (Phaseolus vulgaris L.); van Berkum P et al.; The phylogenetic relationships among Rhizobium species that nodulate Phaseolus vulgaris (common bean) were determined by directly sequencing the amplified 16S ribosomal DNA genes of these organisms . The bean strains formed four separate clusters . One cluster was composed of Rhizobium leguminosarum bv . trifolii, R . leguminosarum bv . viciae, and R . leguminosarum bv . phaseoli . Two other clusters comprised Rhizobium etli and Rhizobium tropici, and the fourth cluster contained a single bean-nodulating strain . Data for species identification were obtained from DNA-DNA reassociation experiments . The levels of DNA relatedness among strains belonging to the three biovars of R . leguminosarum ranged from 58 to 67% . The levels of DNA relatedness between R . leguminosarum bv . phaseoli and R . etli and R . tropici ranged from 43 to 45% and 13 to 16%, respectively . The levels of DNA relatedness between the strain belonging to the fourth cluster and strains of the other three Rhizobium species that nodulate beans were less than 10%.

J Bacteriol, 1996 Jan, 178(2), 372 - 6
Cell-to-cell signaling in the symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum: autoinduction of a stationary phase and rhizosphere-expressed genes; Gray KM et al.; The Sym plasmid pRL1JI encodes functions for the formation of nitrogen-fixing pea root nodules by Rhizobium leguminosarum . Some of the nodulation genes are involved in recognition of chemical signals produced by the plant root, and others are required for production of chemical signals recognized by the plant . pRL1JI also contains a regulatory gene, rhiR, that is homologous to luxR, the transcriptional activator of luminescence genes in Vibrio fischeri . LuxR requires a signal compound, an autoinducer, for its activity . We have identified an R . leguminosarum autoinducer that, together with RhiR, is required to activate both the rhizosphere-expressed rhiABC operon and a growth-inhibiting function encoded by pRL1JI . This intercellular signal is an N-acylated homoserine lactone structurally related to the V . fischeri and other autoinducers . These findings indicate a new level of intercellular communication in root nodule formation.

Biochem Biophys Res Commun, 1995 Dec 26, 217(3), 796 - 801
Increase in osmotolerance of Rhizobium fredii soybean isolate BD32 by the proB proA operon of Escherichia coli; Neumivakin LV et al.; The proB proA operons (which are blocked by the feedback inhibition of proline production) of Escherichia coli wild type or with the mutation proBosm, blocking feedback inhibition effect of proline production, were cloned in a broad host range shuttle vector pVA 12-2 . The hybrid plasmids pLVA(proB+A+) and pNSA(proBosm proA), were transferred into a low level osmotolerance Rhizobium fredii strain BD32 . Both types of transconjugants were characterised by increased osmotolerance in a minimal medium supplied with 0.4-0.8 M NaCl but in the case of pNSA the effect was more significant . The strain BD32/pNSA had an increased level of intracellular proline concentration . Practical application of the increase in Rhizobium resistance to the stress factors is discussed.

Mol Gen Genet, 1995 Dec 15, 249(5), 487 - 97
Extension of the Rhizobium meliloti succinoglycan biosynthesis gene cluster: identification of the exsA gene encoding an ABC transporter protein, and the exsB gene which probably codes for a regulator of succinoglycan biosynthesis; Becker A et al.; Two new genes, designated exsA and exsB, were identified adjacent to the 24 kb exo gene cluster of Rhizobium meliloti, which is involved in succinoglycan (EPS I) biosynthesis . The derived amino acid sequence of ExsA displayed significant homologies to ATP binding cassette (ABC) transporter proteins . R . meliloti strains mutated in exsA were characterized by a decreased ratio of HMW to LMW EPS I, indicating a function for ExsA in EPS I biosynthesis . The R . meliloti NdvA protein, which is involved in the transport of cyclic beta-(1,2)-glucans, was identified as the closest homologue of ExsA . R . meliloti exsB mutants produced a three-fold increased amount of EPS I in comparison to the wild-type strain . In contrast, high copy number of exsB resulted in a decrease in the EPS I level to 20% of wild type, indicating that the exsB gene product can negatively influence EPS I biosynthesis . It was demonstrated that this influence is not due to transcriptional regulation of the exo genes by the exsB gene product . By plasmid integration it was shown that exsA and exsB represent monocistronic transcription units.

Int J Biol Macromol, 1995 Dec, 17(6), 369 - 72
Cyclic (1-->2)-beta-D-glucans excreted by the glucuronan-producing strain Rhizobium meliloti M5N1CS (NCIMB 40472) and by the succinoglycan-producing strain Rhizobium meliloti M5N1; Michaud P et al.; During fermentation, the mutant strain Rhizobium meliloti M5N1CS, which induces nodule formation on alfalfa roots, produces a partially acetylated (1-->4)-beta-D-glucuronan . In addition to this exopolysaccharide of high molecular weight, the mutant strain produces oligoglucoronates and cyclic (1-->2)-beta-D-glucans with degrees of polymerization from 17 to 30 . Under the conditions applied, magnesium has no effect on cyclic glucan production by the mutant strain, but the succinoglycan production by the wild-type strain Rhizobium meliloti M5N1 increases.

Int J Biol Macromol, 1995 Dec, 17(6), 365 - 8
Effects of salts on production and on O-acetylation of glucuronan excreted by the Rhizobium meliloti M5N1 CS strain; Roblot C et al.; Acetylation determined by 1H-nuclear magnetic resonance spectroscopy and production of the glucuronan excreted by the Rhizobium meliloti M5N1CS strain during cultivation in RCS medium with and without added magnesium salts have been studied . These salts induce an increase in the degree of substitution and the molar ratio of 2,3-di-O-acetyl residues . A decrease in production is observed after 75 h of fermentation as the magnesium salt concentration increases . The presence of manganese and sodium salts in the culture induces inhibition of exopolysaccharide (EPS) production . However, the structure of the EPS is similar to that of the EPS produced by standard fermentation, without modification in the degree of substitution.

Eur J Cell Biol, 1995 Dec, 68(4), 463 - 9
Nod factors produced by Rhizobium leguminosarum biovar viciae induce ethylene-related changes in root cortical cells of Vicia sativa ssp . nigra; van Spronsen PC et al.; Vicia sativa ssp . nigra plants develop the "Thick short root" (Tsr) phenotype when both (i) the roots are inoculated with the root nodule inducing bacterium Rhizobium leguminosarum biovar viciae, and (ii) the plants, including the roots, are grown in the light . Tsr roots have a reduced length, are locally twice as thick as normal roots and have an increased number of root hairs . Development of the Tsr phenotype is correlated with the presence of nod (nodulation) genes in the rhizobia . Nod factors (lipochitin oligosaccharides), products of these nod genes, can induce the Tsr phenotype in the absence of rhizobia . The Tsr phenotype can be mimicked by addition of the ethylene-releasing compound ethephon . Using several microscopical techniques, we compared roots showing the Tsr phenotype (Tsr roots) with normal roots and roots grown in the presence of the ethylene inhibitor aminoethoxyvinylglycine (AVG) . The thickening of Tsr roots appeared to be caused by a swelling of the cortical cells, which corresponded with (i) a reorientation of the interphase cortical microtubules from a transverse to a longitudinal direction, (ii) general cell wall modifications, (iii) frequent absence of middle lamellae, and (iv) local maceration . The same changes could be induced by ethephon and were inhibited by AVG . This strongly suggests that the Tsr phenotype is caused by excessive ethylene production . The ethylene-related changes mentioned above are also seen during infection thread formation, but only very locally . Apparently, Vicia roots when grown in the light overrespond to Nod factors leading to overproduction of ethylene and to a non-local "ripening" process . These phenomena inhibit nodulation of the main root by preventing formation of pre-infected threads and by reducing formation of root nodule primordia . Local controlled production of ethylene, as induced by Nod factors, may, however, be an essential element of the nodulation process.

FEMS Microbiol Lett, 1995 Dec 1, 134(1), 19 - 25
A new root-nodulating symbiont of the tropical legume Sesbania, Rhizobium sp . SIN-1, is closely related to R . galegae, a species that nodulates temperate legumes; Rana D et al.; Rhizobium sp . SIN-1, isolated in India from root nodules on the tropical legume Sesbania aculeata, also induces nitrogen-fixing nodules on roots of S . macrocarpa, S . speciosa, S . procumbens, S . punicea, S . rostrata, and Vigna unguiculata . Unlike Azorhizobium caulinodans, SIN-1 does not induce stem nodules on S . rostrata . The nodules induced by SIN-1 develop exclusively at the bases of secondary roots . Electron microscopic studies of mature nodule sections revealed rhizobia within intercellular spaces, indicating a 'crack entry' mechanism of root infection . SIN-1 is a fast-growing, acid-producing, salt-tolerant Rhizobium that utilizes a wide variety of carbon sources . The nodulation (nod) genes of this strain are located on a 300-MDa symbiosis (sym) plasmid . Fatty acid profile and sequence comparison of a 260-bp conserved region of the 16S rRNA gene demonstrated that SIN-1 is phylogenetically closely related to R . galegae, a species that nodulates temperate legumes.

FEMS Microbiol Lett, 1995 Nov 15, 133(3), 285 - 91
The Rhizobium meliloti region located downstream of the nod box n6 is involved in the specific nodulation of Medicago lupulina; Plazanet C et al.; Six nod box regulatory sequences are present in the Rhizobium meliloti genome . We have analysed the DNA region located downstream of nod box n6, and identified three open reading frames, designated nolQa, nolQb and nolS . LacZ fusions in these ORFs are not induced by classical nod gene inducers, which indicates that their expression either is not under the control of the nod box, or involves another regulatory mechanism acting in conjunction with the NodD/nod box regulatory circuit . Mutations in this n6 locus result in a delay in nodule formation on a particular host, Medicago lupulina . As this region is not strictly conserved among different R . meliloti strains, nolQa, nolQb and nolS may constitute auxiliary nodulation genes, for which the selection pressure is limited to particular host plants.

Mol Gen Genet, 1995 Nov 1, 249(1), 117 - 26
Structural and functional analysis of the fixLJ genes of Rhizobium leguminosarum biovar phaseoli CNPAF512; D'hooghe I et al.; The fixLJ genes of Rhizobium leguminosarum biovar phaseoli CNPAF512 were identified by DNA hybridization of a genomic library with an internal fragment of the Rhizobium meliloti fixJ gene . The nucleotide sequence was determined and the corresponding amino acid sequence was aligned with the amino acid sequences of the FixL proteins of R . meliloti, Bradyrhizobium japonicum and Azorhizobium caulinodans . While the FixJ protein and the carboxy-terminal part of the FixL protein are highly homologous to the other FixL and FixJ proteins, the homology in the central heme-binding, oxygen-sensing domain and in the amino-terminal domain of FixL is very low . The R . leguminosarum bv . phaseoli FixL protein does not contain the heme-binding motif defined for the previously described FixL proteins . R . leguminosarum bv . phaseoli fixLJ and fixJ mutants were constructed . These mutants can still fix nitrogen, albeit at a reduced level . Expression analysis of nifA-gusA and nifH-gusA fusions in the constructed mutants revealed that the R . leguminosarum bv . phaseoli fixLJ genes are involved in microaerobic nifH expression but not in nifA expression.

Microbiology, 1995 Nov, 141 ( Pt 11), 2883 - 9
The role of glutaminase in Rhizobium etli: studies with a new mutant; Duran S et al.; In order to examine the role of glutaminase in Rhizobium etli, we isolated and characterized a R . etli glutaminase mutant (LM16) . This mutant was selected for its impaired ability to grow on glutamine as nitrogen and carbon source while retaining the ability to grow on other nitrogen and carbon sources . The mutant showed very low levels of glutaminase activity under various growth conditions in comparison with the wild-type strain . With glutamine as the only nitrogen and carbon source, LM16 showed poor growth, with a very high content of glutamine, low glutamate content, and reduced ammonium excretion and 14CO2 evolution from {U-14C}glutamine compared to the wild-type strain . This indicates that the main role of R . etli glutaminase is in the use of glutamine as carbon source . R . etli glutaminase also plays a role in maintaining the balance between glutamate and glutamine, as shown by the accumulation of glutamine and the low glutamate content of the mutant under different growth conditions . These results also indicate that glutaminase participates in a glutamine cycle in which it degrades glutamine which is then resynthesized by glutamine synthetase . The higher glutamine and lower glutamate content found in bacteroids of LM16 in comparison with bacteroids of the wild-type strain indicate that glutamine degradation by glutaminase plays an important role during the symbiosis between R . etli and Phaseolus vulgaris.

Plant Mol Biol, 1995 Nov, 29(3), 465 - 77
Induction of nodule primordia on Phaseolus and Acacia by lipo-chitin oligosaccharide nodulation signals from broad-host-range Rhizobium strain GRH2; Lopez-Lara IM et al.; Rhizobium wild-type strain GRH2 was originally isolated from the tree, Acacia cyanophylla, and has a broad host-range which includes herbaceous legumes, such as Phaseolus and Trifolium species . Here we show that strains of Rhizobium sp . GRH2, into which heterologous nodD alleles have been introduced, produce a large diversity of both sulphated and non-sulphated lipo-chitin oligosaccharides (LCOs) . Most of the molecular species contain an N-methyl group on the reducing-terminal N-acetyl-glucosamine . The LCOs vary in the nature of the fatty acyl chain and in the length of the chitin backbone . The majority of the LCOs have an oligosaccharide chain length of five GlcNAc residues, but a few are oligomers having six GlcNAc units . LCOs purified from GRH2 are able to induce root hair formation and deformation on Acacia cyanophylla and A . melanoxylon plants . We show that an N-vaccenoyl-chitopentaose bearing an N-methyl group is able to induce nodule primordia on Phaseolus vulgaris, A . cyanophylla, and A . melanoxylon, indicating that for these plants an N-methyl modification is sufficient for nodule primordia induction.

Plant Mol Biol, 1995 Nov, 29(3), 453 - 64
Isolation, chemical structures and biological activity of the lipo-chitin oligosaccharide nodulation signals from Rhizobium etli; Cardenas L et al.; Rhizobium etli is a microsymbiont of plants of the genus Phaseolus . Using mass spectrometry we have identified the lipo-chitin oligosaccharides (LCOs) that are produced by R . etli strain CE3 . They are N-acetylglucosamine pentasaccharides of which the non-reducing residue is N-methylated and N-acylated with cis-vaccenic acid (C18:1) or stearic acid (C18:0) and carries a carbamoyl group at C4 . The reducing residue is substituted at the C6 position with O-acetylfucose . Analysis of their biological activity on the host plant Phaseolus vulgaris shows that these LCOs can elicit the formation of nodule primordia which develop to the stage where vascular bundles are formed . The formation of complete nodule structures, including an organized vascular tissue, is never observed . Considering the very close resemblance of the R . etli LCO structures to those of R . loti (I . M . Lopez-Lara, J . D . J . van den Berg, J . E . Thomas Oates, J . Glushka, B . J . J . Lugtenberg, H . P . Spaink, Mol Microbiol 15: 627-638, 1995) we tested the ability of R . etli strains to nodulate various Lotus species and of R . loti to nodulate P . vulgaris . The results show that R . etli is indeed able to nodulate Lotus plants . However, several Lotus species are only nodulated when an additional flavonoid independent transcription activator (FITA) nodD gene is provided . Phaseolus plants can also be nodulated by R . loti bacteria, but only when the bacteria contain a FITA nodD gene . Apparently, the type of nod gene inducers secreted by the plants is the major basis for the separation of Phaseolus and Lotus into different cross inoculation groups.

Plant Mol Biol, 1995 Nov, 29(3), 431 - 9
Sugar-binding activity of pea (Pisum sativum) lectin is essential for heterologous infection of transgenic white clover hairy roots by Rhizobium leguminosarum biovar viciae; van Eijsden R et al.; Legume lectin stimulates infection of roots in the symbiosis between leguminous plants and bacteria of the genus Rhizobium . Introduction of the Pisum sativum lectin gene (psl) into white clover hairy roots enables heterologous infection and nodulation by the pea symbiont R . leguminosarum biovar viciae (R.l . viciae) . Legume lectins contain a specific sugar-binding site . Here, we show that inoculation of white clover hairy roots co-transformed with a psl mutant encoding a non-sugar-binding lectin (PSL N125D) with R.l . viciae yielded only background pseudo-nodule formation, in contrast to the situation after transformation with wild type psl or with a psl mutant encoding sugar-binding PSL (PSL A126V) . For every construct tested, nodulation by the homologous symbiont R.l . trifolii was normal . These results strongly suggest that (1) sugar-binding activity of PSL is necessary for infection of white clover hairy roots by R.l . viciae, and (2) the rhizobial ligand of host lectin is a sugar residue rather than a lipid.

Appl Environ Microbiol, 1995 Nov, 61(11), 3992 - 7
Rhizobium tropici chromosomal citrate synthase gene; Hernandez-Lucas I et al.; Two genes encoding citrate synthase, a key enzyme in the Krebs cycle, have been found in Rhizobium tropici . One of them is in the bacterial chromosome, while the other is in the symbiotic plasmid . We sequenced the chromosomal gene and found that it is very similar to the previously reported plasmidic gene sequence in its structural region but not in its regulatory region . The chromosomal gene is able to complement an Escherichia coli citrate synthase mutant . In R . tropici, a mutant in the chromosomal citrate synthase gene has a diminished citrate synthase activity (in free-living bacteria), a diminished nodulation capacity, and forms nitrogen-fixing nodules . In contrast, the citrate synthase double mutant forms ineffective nodules devoid of bacteroids and forms less nodules than the single chromosomal mutant . It is inferred that both genes are functional and required during the nodulation process in R . tropici.

J Bacteriol, 1995 Nov, 177(22), 6422 - 31
Aerobic and anaerobic regulation in Rhodobacter sphaeroides 2.4.1: the role of the fnrL gene; Zeilstra-Ryalls JH et al.; In Rhodobacter sphaeroides 2.4.1, the cellular requirements for 5-aminolevulinic acid (ALA) are in part regulated by the level of ALA synthase activity, which is encoded by the hemA and hemT genes . Under standard growth conditions, only the hemA gene is transcribed, and the level of ALA synthase activity varies in response to oxygen tension . The presence of an FNR consensus sequence upstream of hemA suggested that oxygen regulation of hemA expression could be mediated, in part, through a homolog of the fnr gene . Two independent studies, one detailed here, identified a region of the R . sphaeroides 2.4.1 genome containing extensive homology to the fix region of the symbiotic nitrogen-fixing bacteria Rhizobium meliloti and Bradyrhizobium japonicum . Within this region that maps to 443 kbp on chromsome I, we have identified an fnr homolog (fnrL), as well as a gene that codes for an anaerobic coproporphyrinogen III oxidase, the second such gene identified in this organism . We also present an analysis of the role of fnrL in the physiology of R . sphaeroides 2.4.1 through the construction and characterization of fnrL-null strains . Our results further show that fnrL is essential for both photosynthetic and anaerobic-dark growth with dimethyl sulfoxide . Analysis of hemA expression, with hemA::lacZ transcriptional fusions, suggests that FnrL is an activator of hemA under anaerobic conditions . On the other hand, the open reading frame immediately upstream of hemA appears to be an activator of hemA transcription regardless of either the presence or the absence of oxygen or FnrL . Given the lack of hemT expression under these conditions, we consider FnrL regulation of hemA expression to be a major factor in bringing about changes in the level of ALA synthase activity in response to changes in oxygen tension.

J Bacteriol, 1995 Nov, 177(22), 6346 - 51
A novel cyclic beta-1,2-glucan mutant of Rhizobium meliloti; Breedveld MW et al.; The periplasmic cyclic beta-1,2-glucans produced by bacteria within the Rhizobiaceae family provide functions during hypo-osmotic adaptation and plant infection . In Rhizobium meliloti, these molecules are highly modified with phosphoglycerol and succinyl substituents, and it is possible that the anionic character of these glucans is important for their functions . In the present study, we have used a thin-layer chromatographic screening method to identify a novel R . meliloti mutant specifically blocked in its ability to transfer phosphoglycerol substituents to the cyclic beta-1,2-glucan backbone . Further analysis revealed that the cyclic glucans produced by this mutant contained elevated levels of succinyl substituents . As a result, the overall anionic charge on the cyclic beta-1,2-glucans was found to be similar to that of wild-type cells . Despite this difference in cyclic beta-1,2-glucan structure, the mutant was shown to effectively nodulate alfalfa and to grow as well as wild-type cells in hypo-osmotic media.

J Bacteriol, 1995 Nov, 177(21), 6282 - 5
Mass spectrometric analysis of chitin oligosaccharides produced by Rhizobium NodC protein in Escherichia coli; Kamst E et al.; A system for studying the in vivo activity of Rhizobium NodC protein in Escherichia coli has been developed . Using thin-layer chromatography, high-performance liquid chromatography, and mass spectrometry, we show that in this system R . leguminosarum bv . viciae NodC protein directs the synthesis of chitinpentaose, chitintetraose, chitintriose, and two as yet unidentified modified chitin oligosaccharides.

J Bacteriol, 1995 Nov, 177(21), 6276 - 81
Rhizobium NodI and NodJ proteins play a role in the efficiency of secretion of lipochitin oligosaccharides; Spaink HP et al.; Thin-layer chromatographic analysis of extracts of D-{1-14C}glucosamine-labelled rhizobia was used to analyze the effects of nodI, nodJ, and nodT on secretion of lipochitin oligosaccharide (LCO) signal molecules . Secretion was analyzed by comparing quantities of radiolabelled LCOs present in the cellular and spent growth medium fractions . A second rapid and sensitive method was introduced to estimate the secreted LCO fractions by using D-{1-14C}glucosamine-labelled cells grown in medium supplemented with chitinase . At various times after induction of LCO synthesis, the quantity of degradation products of LCOs was compared with the amount of nondegraded LCOs . In wild-type strains of Rhizobium leguminosarum biovars viciae and trifolii the nodI and nodJ genes (but not the nodT gene) strongly enhance the secretion of LCOs during the first 5 h after the induction of LCO synthesis . In LCO-overproducing strains the enhancement of secretion was observed only during the first 3 h after induction . At times later than 5 h after induction, a significant influence of the presence of the nodI and nodJ genes on LCO secretion was detectable neither in the wild type nor in LCO-overproducing strains . By using plasmids in which the nodI and nodJ genes are cloned separately under control of a flavonoid-inducible promoter, it was shown that both genes are needed for a wild-type level of LCO secretion . Therefore, these results demonstrate that nodI and nodJ play a role in determining the efficiency of LCO secretion.

J Bacteriol, 1995 Nov, 177(21), 6237 - 45
In vitro sulfotransferase activity of NodH, a nodulation protein of Rhizobium meliloti required for host-specific nodulation; Ehrhardt DW et al.; Early stages of nodulation involve the exchange of signals between the bacterium and the host plant . Bacterial nodulation (nod) genes are required for Rhizobium spp . to synthesize lipooligosaccharide morphogens, termed Nod factors . The common nod genes encode enzymes that synthesize the factor core structure, which is modified by host-specific gene products . Here we show direct in vitro evidence that Rhizobium meliloti NodH, a host-specific nodulation gene, catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the terminal 6-O position of Nod factors, and we show substrate requirements for the reaction . Our results indicate that polymerization of the chitooligosaccharide backbone likely precedes sulfation and that sulfation is not absolutely dependent on the presence or the particular structure of the N-acyl modification . NodH sulfation provides a tool for the enzymatic in vitro synthesis of novel Nod factors, or putative Nod factors intermediates, with high specific activity.

Arch Biochem Biophys, 1995 Oct 20, 323(1), 195 - 204
Adenosine-5'-triphosphate-sulfurylase from Arabidopsis thaliana and Escherichia coli are functionally equivalent but structurally and kinetically divergent: nucleotide sequence of two adenosine-5'-triphosphate-sulfurylase cDNAs from Arabidopsis thaliana and analysis of a recombinant enzyme; Murillo M et al.; ATP-sulfurylase, the first enzyme of sulfate assimilation, catalyzes the formation of adenosine-5'-phosphosulfate from ATP and sulfate . Here we report that the higher plant, Arabidopsis thaliana, contains a three-member, expressed gene family encoding plastid localized forms of ATP sulfurylase . Three cDNAs from A . thaliana, designated APS1, APS2, and APS3, were isolated by their ability to functionally complement a met3 (ATP sulfurylase) mutant strain of Saccharomyces cerevisiae (yeast) . The nucleotide sequence of APS1 was reported previously (1) . APS2 and APS3, reported here, have 476- and 465-codon open-reading frames encoding 53.6- and 52.0-kDa polypeptides, respectively . The translation products of both clones are highly homologous to APS1 (66 and 86% identity, respectively) over their entire lengths, including amino terminal sequences resembling transit peptides for plastid localization . Both clones are less homologous to MET3 (25 and 30% identity, respectively) . Genomic blot analysis of A . thaliana revealed only three genes with homology to the APS cDNAs and RNA blot analysis showed that APS1 is the most highly expressed member of this gene family . The APS polypeptides share homology with ATP-sulfurylases from fungi, a marine worm and a chemoautotrophic bacterium, but, not from Escherichia coli or Rhizobium meliloti . Analysis of recombinant APS3 showed that the protein is structurally and kinetically similar to fungal ATP-sulfurylase, but very different from the E . coli enzyme . The APS3 polypeptide is a homotetramer with specific activities (mumol primary product x mg protein-1 at pH 8.0, 25 degrees C) for 2.9 for APS synthesis, 30.1 for molybdolysis, and 48.7 for ATP synthesis . Despite the sequence, structural, and kinetic differences between higher plant and E . coli ATP-sulfurylases, APS2 and APS3 are able to functionally complement E . coli cysD and cysN (ATP-sulfurylase) mutant strains.

Proc Natl Acad Sci U S A, 1995 Oct 10, 92(21), 9702 - 6
Protein crosslinking studies suggest that Rhizobium meliloti C4-dicarboxylic acid transport protein D, a sigma 54-dependent transcriptional activator, interacts with sigma 54 and the beta subunit of RNA polymerase; Lee JH et al.; Rhizobium meliloti C4-dicarboxylic acid transport protein D (DCTD) activates transcription by a form of RNA polymerase holoenzyme that has sigma 54 as its sigma factor (referred to as E sigma 54) . DCTD catalyzes the ATP-dependent isomerization of closed complexes between E sigma 54 and the dctA promoter to transcriptionally productive open complexes . Transcriptional activation probably involves specific protein-protein interactions between DCTD and E sigma 54 . Interactions between sigma 54-dependent activators and E sigma 54 are transient, and there has been no report of a biochemical assay for contact between E sigma 54 and any activator to date . Heterobifunctional crosslinking reagents were used to examine protein-protein interactions between the various subunits of E sigma 54 and DCTD . DCTD was crosslinked to Salmonella typhimurium sigma 54 with the crosslinking reagents succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N-hydroxysulfosuccinimidyl-4-azidobenzoate . Cys-307 of sigma 54 was identified by site-directed mutagenesis as the residue that was crosslinked to DCTD . DCTD was also crosslinked to the beta subunit of Escherichia coli core RNA polymerase with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, but not with N-hydroxysulfosuccinimidyl-4-azidobenzoate . These data suggest that interactions of DCTD with sigma 54 and the beta subunit may be important for transcriptional activation and offer evidence for interactions between a sigma 54-dependent activator and sigma 54, as well as the beta subunit of RNA polymerase.

Biochemistry, 1995 Oct 3, 34(39), 12712 - 20
Substrate specificity and kinetic studies of nodulation protein NodL of Rhizobium leguminosarum; Bloemberg GV et al.; All lipo-chitin oligosaccharides identified from Rhizobium leguminosarum carry an O-acetyl moiety on C6 of the nonreducing terminal N-acetylglucosamine residue . Previously, we have shown that purified NodL protein, using acetyl-CoA as acetyl donor, in vitro acetylates N-acetylglucosamine, chitin oligosaccharides, and lipo-chitin oligosaccharides . In this paper, the enzymatic properties and substrate specificity of NodL protein were analyzed, using a spectrophotometric assay to quantify NodL transacetylating activity . NodL functions optimally under alkaline conditions . Transacetylating activity has a broad temperature optimum between 28 and 42 degrees C . NodL protein is stable for at least 15 min up to 48 degrees C . Glucosamine, chitosan oligosaccharides, terminally de-N-acetylated chitin derivatives, and cellopentaose were identified as acetyl-accepting substrates for NodL protein . Quantitative substrate specificity studies show that chitin derivatives with a free amino group on the nonreducing terminal residue are the preferred substrates of the NodL protein . Our results strongly indicate that the nonreducing terminally de-N-acetylated chitin oligosaccharides produced by the NodC and NodB enzymes are the in vivo acetyl-accepting substrates for NodL protein.

Carbohydr Res, 1995 Oct 2, 275(2), 285 - 94
Structural analysis of the O-antigen of the lipopolysaccharide of Rhizobium tropici CIAT899; Gil-Serrano AM et al.; The structure of the O-antigen chain of the lipopolysaccharide isolated from Rhizobium tropici CIAT899, by the phenol-water procedure, and recovered from the phenol layer, has been investigated by hydrolysis, methylation analysis and 1D and 2D 1H and 13C NMR spectroscopy of the complete polysaccharide and of oligosaccharides obtained by partial hydrolysis . The O-antigen has the repeating unit {formula: see text}

J Appl Bacteriol, 1995 Oct, 79(4), 425 - 31
Strain-specific fingerprints of Rhizobium galegae generated by PCR with arbitrary and repetitive primers; Selenska-Pobell S et al.; Strain-specific genomic patterns of Rhizobium galegae were generated by PCR using both arbitrary and repetitive (BOX, ERIC and REP) primers . The identification of the strains was achieved also by RFLP analysis . However, the PCR genomic fingerprinting has significant advantages: it is not only simpler and faster, but it is also much more discriminative because it deals with the full bacterial genome and not only with parts of it as is the case with RFLP . In addition, both kinds of PCR fingerprinting (using arbitrary or repetitive primers) generated highly specific and reproducible patterns when parallel reactions with total bacterial DNA, extracted from independent liquid cultures were performed . The latter shows that AP- and rep-PCR are convenient for controlling the production and application of Rhizobium inoculants.

Microbiology, 1995 Oct, 141 ( Pt 10), 2553 - 9
Poly-beta-hydroxybutyrate (PHB) biosynthetic genes in Rhizobium meliloti 41; Tombolini R et al.; Genes encoding beta-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHB-synthase (phaC) from R . meliloti 41, together with a fourth gene, referred to as ORF1, presumed to be involved in PHB biosynthesis, have been cloned and sequenced . phaA, phaB and ORF1 were identified by heterologous hybridization on a cosmid library, while phaC was isolated by cloning the transposon-tagged fragment from a R . meliloti PHB- Tn5 mutant . phaA and phaB were functionally expressed in Escherichia coli while phaC was able to complement a PHB- strain of R . meliloti 41 . The three genes were sufficient to direct the production of polyhydroxyalkanoate in E . coli . The homology of ORF1 with an ORF located near the PHB genes in two phototrophic bacteria suggests its involvement in PHB synthesis.

J Bacteriol, 1995 Oct, 177(19), 5661 - 9
The hypBFCDE operon from Rhizobium leguminosarum biovar viciae is expressed from an Fnr-type promoter that escapes mutagenesis of the fnrN gene; Hernando Y et al.; Pea (Pisum sativum L.) bacteroids produced by Rhizobium leguminosarum bv . viciae UPM791 synthesize a membrane-bound (NiFe) hydrogenase which oxidizes H2 arising from the nitrogen fixation process in root nodules . Synthesis of the active enzyme requires the products of the structural genes hupSL and an array of accessory proteins from at least 15 additional genes, including the gene cluster hypABFCDE, likely involved in nickel metabolism . Unlike the hupSL genes, which are expressed only in symbiosis, the hypBFCDE operon was also activated in vegetative cells in response to low pO2 in the culture medium . In microaerobic cells and in bacteroids, transcription of the hypBFCDE operon occurred from a promoter, P5b, with a transcription initiation site located 190 bp upstream of the ATG start codon of hypB, within the coding sequence of hypA . Transcription start site 5b was preceded by an Fnr box (anaerobox), 5'-TTGAgccatgTCAA-3', centered at position -39.5 . Expression of the P5b promoter in the heterologous Rhizobium meliloti bacterial host was dependent on the presence of an active fixK gene . A 2.6-kb EcoRI fragment was isolated from an R . leguminosarum bv . viciae UPM791 gene bank by complementing an R . meliloti FixK- mutant . Sequencing of this DNA fragment identified an fnrN gene, and cassette insertion mutagenesis demonstrated that R . leguminosarum bv . viciae fnrN is able to replace the R . meliloti fixK gene for activation of both the R . leguminosarum bv . viciae hypBFCDE operon and the R . meliloti fix genes . However, bacteroids from a genomic FnrN- mutant of R . leguminosarum bv . viciae exhibited wild-type levels of hydrogenase activity . Microaerobic expression of P(5b) was reduced to ca . 50% of the wild-type level in the FnrN(-) mutant . These results indicate that hyp gene expression escapes mutagenesis of the fnrN gene and suggest the existence of a second fnr-like gene in R . leguminosarum by . viciae . Southern blot analysis with an fnrN internal probe revealed the presence of a second genomic region with homology to fnrN.

Int J Syst Bacteriol, 1995 Oct, 45(4), 640 - 8
Genomic heterogeneity of strains nodulating chickpeas (Cicer arietinum L.) and description of Rhizobium mediterraneum sp . nov; Nour SM et al.; The genetic diversity of chickpea strains was studied by using 30 isolates obtained from nodules on chickpeas growing in uninoculated fields over a wide geographic range . The following taxonomic approaches were used: DNA-DNA relatedness analysis, restriction fragment length polymorphism analysis of the amplified 16S ribosomal DNA (rDNA) intergenic spacer (IGS), and total 16S rRNA sequence analysis . The division of chickpea-infective strains into two major phylogenetic groups (groups A and B) that has been described previously was confirmed by the polymorphism of the 16S IGS rDNA . We identified a total of five genomic species, including the previously described species Rhizobium ciceri . All of the group B strains except one were homogeneous and belonged to a single genomic species corresponding to R . ciceri . Group A was heterogeneous, containing three genomic species and five strains that remained unclassified, and its members had very different PCR restriction fragment length polymorphism profiles . The complete 16S rRNA sequences of strains representing the two major groups, R . ciceri UPM-Ca7T (T = type strain) and genomic species 2 strain UPM-Ca36T, exhibited 19 mismatches . Both of these strains belonged to the Rhizobium loti-Rhizobium huakuii branch; R . ciceri UPM-Ca7T was closely related to R . loti, and strain UPM-Ca36T was clearly separated from R . ciceri and closely related to R . huakuii . Thus, genomic species 2 could be distinguished from R . ciceri by its 16S rRNA sequence, by DNA relatedness data, by the polymorphism of the 16S IGS rDNAs, and by previously described multilocus enzyme electrophoresis results and phenotypic characteristics . Therefore, we propose that strains belonging to genomic species 2 should be classified in a new species, Rhizobium mediterraneum, and that strain UPM-Ca36 should be the type strain.

Plant Physiol, 1995 Oct, 109(2), 611 - 8
A rapeseed cold-inducible transcript encodes a phosphoenolpyruvate carboxykinase; Saez-Vasquez J et al.; We have isolated a clone corresponding to a new cold-regulated gene from a cDNA library made from rapeseed (Brassica napus cv Samourai) cold-acclimated etiolated seedlings . Sequence analysis and homology searches showed that this clone encodes a protein highly homologous to the ATP-dependent phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.49) from Saccharomyces cerevisiae, Trypanosoma, Rhizobium sp., and Escherichia coli; we refer to the B . napus clone as BnPEPCK . A potential ATP-binding site existing in all PEPCK proteins was also found in BnPEPCK . Although there was a basal expression of BnPEPCK in seedlings grown at control, room temperature, the steady-state level of the transcripts increased at 4 degrees C and decreased to normal levels when the seedlings were returned to control temperature (22 degrees C) . Using antibodies made against a recombinant histidine-BnPEPCK fusion protein, we demonstrated that BnPEPCK protein level is correlated with the accumulation of the BnPEPCK transcript.

J Biol Chem, 1995 Sep 29, 270(39), 22968 - 73
Involvement of nodS in N-methylation and nodU in 6-O-carbamoylation of Rhizobium sp . NGR234 nod factors; Jabbouri S et al.; Although Rhizobium sp . NGR234 and Rhizobium fredii USDA257 share many traits, dysfunctional nodSU genes in the latter prohibit nodulation of Leucaena species . Accordingly, we used R . fredii transconjugants harboring the nodS and nodU genes of NGR234 to study their role in the structural modification of the lipo-oligosaccharide Nod factors . Differences between the Nod factors mainly concern the length of the oligomer (three to five glucosamine residues in USDA257 and five residues only in NGR234) and the presence of additional substituents in NGR234 (N-linked methyl, one or two carbamoyl groups on the non-reducing moiety, acetyl or sulfate groups on the fucose) . R . fredii(nodS) transconjugants produce chitopentamer Nod factors with a N-linked methyl group on the glucosaminyl terminus . Introduction of nodU into USDA257 results in the formation of 6-O-carbamoylated factors . Co-transfer of nodSU directs N-methylation, mono-6-O-carbamoylation, and production of pentameric Nod factors . Mutation of nodU in NGR234 suppresses the formation of bis-carbamoylated species . Insertional mutagenesis of nodSU drastically decreases Nod factor production, but with the exception of sulfated factors (which are partially N-methylated and mono-carbamoylated), they are identical to those of the wild-type strain . Thus, Nod factor levels, their degree of oligomerization, and N-methylation are linked to the activity encoded by nodS.

Gene, 1995 Sep 22, 163(1), 59 - 64
The insertion sequence element ISRm2011-2 belongs to the IS630-Tc1 family of transposable elements and is abundant in Rhizobium meliloti; Selbitschka W et al.; The insertion sequence (IS) element ISRm2011-2 of Rhizobium meliloti (Rm) is characterized by 19-bp imperfect terminal inverted repeats (three mismatches) and a size of 1053 bp . Upon transposition, ISRm2011-2 generates a putative target duplication of 2 bp . ISRm2011-2 carries two major overlapping open reading frames (ORFA and B) with a coding capacity of 135 and 201 amino acids (aa), respectively . A potential translational frameshifting window (5'-AAAAAAAG) is located in the overlapping region of both ORFs . The putative fusion product of both proteins, which probably represents the mature transposase, has a predicted molecular mass of 35.8 kDa and a pI of 10.5 . Comparison of the deduced aa sequence of ORFA with database entries revealed homology to putative transposases of some IS elements of the IS3 family, as well as to eukaryotic transcription factors . The protein encoded by ORFB shows homology to transposases (Tps) of the recently proposed IS630-Tc1 family which includes Tps of both prokaryotic and eukaryotic transposable elements . Analyses of the distribution of ISRm2011-2 in natural Rm populations showed that this IS element is abundant in Rm strains.

Proc Natl Acad Sci U S A, 1995 Sep 12, 92(19), 8985 - 9
Nodulating strains of Rhizobium loti arise through chromosomal symbiotic gene transfer in the environment; Sullivan JT et al.; Rhizobia were isolated from nodules off a stand of Lotus corniculatus established with a single inoculant strain, ICMP3153, 7 years earlier in an area devoid of naturalized Rhizobium loti . The isolates showed diversity in growth rate, Spe I fingerprint of genomic DNA, and hybridization pattern to genomic DNA probes . The 19% of isolates that grew at the same rate as strain ICMP3153 were the only isolates that had the same fingerprint as strain ICMP3153 . Sequencing of part of the 16S rRNA gene of several diverse isolates confirmed that they were not derived from the inoculant strain . Nevertheless, all non-ICMP3153 strains gave EcoRI and Spe I hybridization patterns identical to ICMP3153 when hybridized to nodulation gene cosmids . Hybridization of digests generated by the very rare cutting enzyme Swa I revealed that the symbiotic DNA region (at least 105 kb) was chromosomally integrated in the strains . The results suggest that the diverse strains arose by transfer of chromosomal symbiotic genes from ICMP3153 to nonsymbiotic rhizobia in the environment.

J Biol Chem, 1995 Sep 8, 270(36), 20968 - 77
Mutation or increased copy number of nodE has no effect on the spectrum of chitolipooligosaccharide nod factors made by Rhizobium leguminosarum bv . trifolii; Philip-Hollingsworth S et al.; The bacterial gene nodE is the key determinant of host specificity in the Rhizobium leguminosarum-legume symbiosis and has been proposed to determined unique polyunsaturated fatty acyl moieties in chitolipooligosaccharides (CLOS) made by the bacterial symbiont . We evaluated nodE function by examining CLOS structures made by wild-type R . leguminosarum bv . trifolii ANU843, an isogenic nodE::Tn5 mutant, and a recombinant strain containing multiple copies of the pSym nod region of ANU843 . 1H-NMR, electrospray ionization mass spectrometry, fast atom bombardment mass spectrometry, flame ionization detection-gas chromatography, gas chromatography/mass spectrometry, and high performance liquid chromatography/UV photodiode array analyses revealed that these bacterial strains made the same spectrum of CLOS species . We also found that ions in the mass spectra which were originally assigned to nodE-dependent CLOS species containing unique polyunsaturated fatty acids (Spaink, H . P., Bloemberg, G . V., van Brussel, A . A . N., Lugtenberg, B . J . J., van der Drift, K . M . G . M., Haverkamp, J., and Thomas-Oates, J . E . (1995) Mol . Plant-Microbe Interact . 8, 155-164) were actually due to sodium adducts of the major nodE-independent CLOS species . No evidence for nodE-dependent CLOSs was found for these strains . These results indicate a need to revise the current model to explain how nodE determines host range in the R . leguminosarum-legume symbiosis.

Mol Microbiol, 1995 Sep, 17(5), 923 - 33
Transcriptional organization and expression of noIXWBTUV, a locus that regulates cultivar-specific nodulation of soybean by Rhizobium fredii USDA257; Kovacs LG et al.; Rhizobium fredii is a nitrogen-fixing bacterial symbiont of soybean and a number of other legume species . We have studied the transcriptional organization of a Sym plasmid locus that restricts the host range of R . fredii USDA257 at both the host species and cultivar level . The genes of this host-specificity locus, noIXWBTUV, are transcribed from three promoters . Two of these, which are upstream of noIW and noIBTUV, are oriented face to face and initiate transcription at sites that are 14 bp apart . The third lies upstream from noIX . The noIW promoter is constitutive, whereas the noIB and noIX promoters are inducible by flavonoid signals . We have attempted to express genes from this locus in Escherichia coli systems, both in vivo and in vitro . We detected the insert- and orientation-specific expression of two genes, noIX and noIW, but we were unable to obtain expression of noIBTUV . Antiserum raised against NoIT nevertheless detected an abundantly expressed polypeptide of the predicted size in protein extracts of USDA257 . This observation, as well as RNA dot blot data from a series of mutants, indicates that noIBTUV is expressed as a single transcriptional unit in R . fredii . Immunological detection of NoIT, and of a second protein, NoIX, was strictly dependent on flavonoid induction . The NoIX protein was larger than the size predicted from the previously published nucleotide sequence, and this led to resequencing and revision of the open reading frame.

J Bacteriol, 1995 Sep, 177(17), 5151 - 4
A single rRNA gene region in Bradyrhizobium japonicum; Kundig C et al.; Bradyrhizobium japonicum contains only a single rRNA (rrn) gene region, despite its comparatively large genome size of 8,700 kb . The nucleotide sequence revealed an organization of rRNA and tRNA genes that is frequently found in bacteria: 5'-rrs (16S rRNA)-ileT (tRNA(Ile))-alaT (tRNA(Ala))-rrl (23S rRNA)-rrf (5S rRNA)-3' . The 5' end of the primary transcript, one of the 16S rRNA processing sites, and the 5' end of the mature 16S rRNA were determined by primer extension . DNA hybridization experiments showed that the slowly growing Bradyrhizobium strains generally have only a single copy of the 16S rRNA gene, whereas the faster-growing Rhizobium species contain three rrs copies.

J Bacteriol, 1995 Sep, 177(17), 4927 - 34
Characterization of the cycHJKL genes involved in cytochrome c biogenesis and symbiotic nitrogen fixation in Rhizobium leguminosarum; Delgado MJ et al.; Mutants of Rhizobium leguminosarum bv . viciae unable to respire via the cytochrome aa3 pathway were identified by the inability to oxidize N,N'-dimethyl-p-phenylenediamine . Two mutants which were complemented by cosmid pIJ1942 from an R . leguminosarum clone bank were identified . Although pea nodules induced by these mutants contained many bacteroids, no symbiotic nitrogen fixation was detected . Heme staining of cellular proteins revealed that all cytochrome c-type heme proteins were absent . These mutants lacked spectroscopically detectable cytochrome c, but cytochromes aa3 and d were present, the latter at a higher-than-normal level . DNA sequence analysis of complementing plasmids revealed four apparently cotranscribed open reading frames (cycH, cycJ, cycK, and cycL) . CycH, CycJ, CycK, and CycL are homologous to Bradyrhizobium japonicum and Rhizobium meliloti proteins thought to be involved in the attachment of heme to cytochrome c apoproteins; CycK and CycL are also homologous to the Rhodobacter capsulatus ccl1 and ccl2 gene products and the Escherichia coli nrfE and nrfF gene products involved in the assembly of c-type cytochromes . The absence of cytochrome c heme proteins in these R . leguminosarum mutants is consistent with the view that the cycHJKL operon could be involved in the attachment of heme to apocytochrome c.

Can J Microbiol, 1995 Sep, 41(9), 816 - 25
Detection of Rhizobium meliloti cells in field soil and nodules by polymerase chain reaction; Watson RJ et al.; A genetically marked Rhizobium meliloti strain, R692, was prepared by insertion of a 1.7-kb DNA segment from Tn903 between the nifHDK and fixABC genes in the nod megaplasmid . This DNA was used as a marker, detectable by polymerase chain reaction (PCR), for the specific identification of bacteria in soil samples and alfalfa nodules . This detection technique was tested by applying different titres of the marked strain to field plots seeded with alfalfa . Samples of soil and nodules were assayed for the presence of the marker DNA fragment by PCR using primers specific to the marker sequence . The experiments revealed that the bacteria could be detected directly in soil containing about 10(3)-10(4) bacteria/g, but greater sensitivity was prevented by potent PCR inhibitors present in the samples . The titre of the bacteria in the soil decreased rapidly after inoculation, dropping about 10-fold per week . Tests of vertical location of the bacteria in soil cores showed that the bacteria were initially dispersed to a depth of 18 cm, and subsequently retained viability in the top 2-8 cm . As few as 10 marked R . meliloti per gram of soil resulted in its establishment at detectable levels in nodules . Application of about 10(4)-10(5) bacteria/g soil was sufficient to give the maximum number of nodules per plant and resulted in 70-90% occupancy by the marked strain . Limited movement of the inoculant was detected by analysis of nodules from plants adjacent to the sites where the bacteria were applied, probably by movement in water . The experiments demonstrated the advantages of PCR for the monitoring of marked microorganisms in the environment.

Mol Plant Microbe Interact, 1995 Sep-Oct, 8(5), 733 - 46
The complete hrp gene cluster of Pseudomonas syringae pv . syringae 61 includes two blocks of genes required for harpinPss secretion that are arranged colinearly with Yersinia ysc homologs; Huang HC et al.; Pseudomonas syringae pv . syringae 61 contains a 25-kb hrp cluster that is sufficient to elicit the hypersensitive response (HR) in nonhost plants . Previous studies have shown that mutations in complementation groups VIII, IX, and XI in the hrp cluster abolished the ability of the bacterium to cause the HR . The sequence of a 3.7-kb SmaI-SstI fragment covering groups VIII and IX now reveals five open reading frames (ORFs) in the same transcript, designated as hrpU, hrpW, hrpO, hrpX, and hrpY, and predicted to encode proteins of 14,795, 23,211, 9,381, 28,489, and 39,957 Da, respectively . The hrpU, hrpW, hrpO, hrpX, and hrpY genes are homologous and arranged colinearly with the yscQ/spa33/spaO, yscR/spa24/spaP, yscS/spa9/spaQ, yscT/spa29/spaR, and yscU/spa40/spaS genes of Yersinia spp., Shigella flexneri, and Salmonella typhimurium, respectively . These proteins also show similarity to Fli/Flh proteins of Bacillus and enteric bacteria . The Ysc and Spa proteins are involved in the secretion of virulence factors, like the Yop and Ipa proteins . Fli/Flh proteins are involved in flagellar biogenesis . The sequence of a 2.9-kb EcoRV-EcoRI DNA fragment containing mainly group XI revealed five ORFs, designated hrpC, hrpD, hrpE, hrpF, and hrpG, predicted to encode proteins of 29,096, 15,184, 21,525, 7,959, and 13,919 Da, respectively . The first three genes belong to an operon containing hrpZ, which encodes an extracellular protein that elicits the HR . hrpF and hrpG are two potential ORFs upstream of hrpH in the hrpH operon . HrpC is homologous to Yersinia YscJ, Pseudomonas solanacearum HrpI, Xanthomonas compestris pv . vesicatoria HrpB3, and Rhizobium fredii NolT . HrpE is similar to YscL of Yersinia spp . P . s . pv . syringae 61 Hrp proteins are most similar to Ysc proteins among those homologs . TnphoA insertions in hrpC, hrpE, hrpW, hrpX, and hrpY abolished the ability of P . s.pv . syringae 61 to secrete HrpZ (harpinPss), as determined by immunoblot analysis of cell-bound and culture supernatant fractions . Thus, many of the proteins required for flagellar biogenesis and virulence protein secretion in plant and animal pathogens may have a common ancestry.

Appl Environ Microbiol, 1995 Sep, 61(9), 3422 - 9
Cloning of nod gene regions from mesquite rhizobia and bradyrhizobia and nucleotide sequence of the nodD gene from mesquite rhizobia; Thomas PM et al.; Nitrogen-fixing symbiosis between bacteria and the tree legume mesquite (Prosopis glandulosa) is important for the maintenance of many desert ecosystems . Genes essential for nodulation and for extending the host range to mesquite were isolated from cosmid libraries of Rhizobium (mesquite) sp . strain HW17b and Bradyrhizobium (mesquite) sp . strain HW10h and were shown to be closely linked . All of the cosmid clones of rhizobia that extended the host range of Rhizobium (Parasponia) sp . strain NGR234CS to mesquite also supported nodulation of a Sym- mesquite strain . The cosmid clones of bradyrhizobia that extended the host range of Rhizobium (Parasponia) sp . strain NGR234CS to mesquite were only able to confer nodulation ability in the Sym- mesquite strain if they also contained a nodD-hybridizing region . Subclones containing just the nodD genes of either genus did not extend the host range of Rhizobium (Parasponia) sp . to mesquite, indicating that the nodD gene is insufficient for mesquite nodulation . The nodD gene region is conserved among mesquite-nodulating rhizobia regardless of the soil depth from which they were collected, indicating descent from a common ancestor . In a tree of distance relationships, the NodD amino acid sequence from mesquite rhizobia clusters with homologs from symbionts that can infect both herbaceous and tree legumes, including Rhizobium tropici, Rhizobium leguminosarum bv; phaseoli, Rhizobium loti, and Bradyrhizobium japonicum.

Plant J, 1995 Sep, 8(3), 345 - 58
Alfalfa NADH-dependent glutamate synthase: structure of the gene and importance in symbiotic N2 fixation; Vance CP et al.; Glutamate synthase (GOGAT), a key enzyme in ammonia (NH+4) assimilation, occurs as two forms in plants: a ferredoxin-dependent form (Fd-GOGAT) and an NADH-dependent form (NADH-GOGAT) . These enzymes are encoded by distinct genes as evidenced by their cDNA and deduced amino acid sequences . This paper reports the isolation and characterization of a NADH-GOGAT gene from alfalfa (Medicago sativa L.), the first GOGAT gene to be isolated from a eukaryote . RNase protection and primer extension experiments map the transcription start site of NADH-GOGAT to nearly identical positions . The transcribed region of this gene, 12,214 bp, is comprised of 22 exons separated by 21 introns . The 2.7 kbp region 5' from the translation initiation site confers nodule-specific reporter gene activity when used in a chimeric beta-glucuronidase (GUS) construct and transformed into Lotus corniculatus and Medicago sativa . Both infected and uninfected cells display GUS activity . The abundance of NADH-GOGAT transcripts increases substantially in developing nodules of plants infected with effective rhizobia . However, this increase is not observed when nodules are induced by a variety of ineffective rhizobial strains . Thus, unlike many other plant genes involved in root nodule NH+4 assimilation, high levels of NADH-GOGAT expression are strictly associated with effective nodules indicating that NADH-GOGAT plays a central role in the functioning of effective root nodules . An alfalfa Fd-GOGAT PCR product showing greater than 85% identity to maize Fd-GOGAT was isolated and used to investigate the contribution of this enzyme to NH+4 assimilation in nodules . Fd-GOGAT mRNA was abundant in leaves and cotyledons but was not detected in alfalfa root nodules . Fd-GOGAT in alfalfa does not appear to play a significant role in symbiotic N2 fixation.

Plant Mol Biol, 1995 Sep, 28(6), 1111 - 9
VsENOD5, VsENOD12 and VsENOD40 expression during Rhizobium-induced nodule formation on Vicia sativa roots; Vijn I et al.; We isolated ENOD5, ENOD12 and ENOD40 homologues from Vicia sativa and studied their expression pattern during Rhizobium-induced nodule formation . Comparison of the VsENOD40 nucleotide sequence with the pea, soybean and alfalfa ENOD40 sequences showed that the sequences contain two conserved regions, called region I and region II . Comparison of all the potential open reading frames (ORFs) showed that all the five different ENOD40 clones encode a highly conserved small polypeptide of 12 or 13 amino acids encoded by an ORF located in region I . Furthermore we studied with in situ hybridization the expression pattern of VsENOD5, VsENOD12 and VsENOD40 during Rhizobium-induced nodule formation . Although the expression of these genes is largely similar to that of the pea counterparts, differences where found for the expression of VsENOD12 and VsENOD40 in Vicia . VsENOD12 is expressed in the whole prefixation zone II, whereas in pea ENOD12 is only expressed in the distal part of this zone . VsENOD40 is expressed in the uninfected cells of interzone II-III, while in pea ENOD40 is expressed in both the uninfected and infected cells of this zone.

J Bacteriol, 1995 Sep, 177(17), 4985 - 91
Isolation and characterization of ropA homologous genes from Rhizobium leguminosarum biovars viciae and trifolii; Roest HP et al.; ropA encodes a 36-kDa outer membrane protein of Rhizobium leguminosarum bv . viciae strain 248 which constitutes the low-M(r) part of antigen group III (R.A . de Maagd, I.H.M . Mulders, H.C.J . Canter Cremers, B.J.J . Lugtenberg, J . Bacteriol . 174:214-221, 1992) . We observed that genes homologous to ropA are present in strain 248 as well as in other R . leguminosarum strains, and we describe the cloning and characterization of two of these genes . Sequencing of a 2.2-kb Bg/II fragment from R . leguminosarum bv . viciae strain 248 that hybridizes with ropA revealed one large open reading frame of 1,074 bp encoding a mature protein of 38.096 kDa . Homology between this gene and ropA is 91.8% on the DNA level . Homology on the amino acid level is only 69.9% as a result of a frameshift . On the basis of homology and immunochemical characteristics, we conclude that this gene encodes the high-M(r) part of the outer membrane protein antigen group III that is repressed during symbiosis . We named this gene ropA2 . The second gene that we cloned was the ropA homologous gene of R . leguminosarum bv . trifolii strain LPR5020 . Except for amino acid 43, the N-terminal part of the corresponding protein appeared to be identical to the first 51 amino acids of RopA of strain 248 . The transcription start sites of both genes were determined, and the promoter regions were compared with that of ropA of strain 248 . No clear consensus sequence could be deduced . The relationship of ropA and ropA2 of R . leguminosarum bv . viciae strain 248 with two similar genes from Brucella abortus is discussed.

Gene, 1995 Aug 30, 162(1), 37 - 9
New gentamicin-resistance and lacZ promoter-probe cassettes suitable for insertion mutagenesis and generation of transcriptional fusions; Becker A et al.; A set of antibiotic-resistance and promoter-probe cassettes suitable for insertion mutagenesis and generation of transcriptional fusions was constructed . The cassettes contain the aacC1 gene of transposon Tn1696 conferring resistance to gentamicin in a large variety of Gram- and Gram+ bacteria . In addition to the antibiotic-resistance gene, a promoterless Escherichia coli lacZ gene was included in the cassettes, allowing the determination of the transcriptional activity at the insertion site . The cassettes can be excised from a plasmid mediating ampicillin resistance by many commonly used restriction enzymes . The new constructs have been successfully used for mutagenesis and studies of gene transcription in Rhizobium meliloti.

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