J Bacteriol, 2001 Mar, 183(6), 2117 - 20 Phase-variable expression of an operon encoding extracellular alkaline protease, a serine protease homolog, and lipase in Pseudomonas brassicacearum; Chabeaud P et al.; The rhizobacterium Pseudomonas brassicacearum forms phenotypic variants which do not show extracellular protease and lipase activity . The operon encoding these enzymes, a serine protease homolog, and a type I secretion machinery was characterized . Transcriptional lacZ gene fusions revealed that the expression of the operon is under the control of phase variation. Planta, 2001 Jan, 212(2), 190 - 8 Utilization of mutants to analyze the interaction between Arabidopsis thaliana and its naturally root-associated Pseudomonas; Persello-Cartieaux F et al.; A model system based on the Arabidopsis thaliana (L.) Heynh . Ws ecotype and its naturally colonizing Pseudomonas thivervalensis rhizobacteria was defined . Pseudomonas strains colonizing A . thaliana were found to modify the root architecture either in vivo or in vitro . A gnotobiotic system using bacteria labelled with green fluorescent protein revealed that P . thivervalensis exhibited a colonization profile similar to that of other rhizobacterial species . Mutants of A . thaliana affected in root hair development and possible hormone perception were used to analyze the plant genetic determinants of bacterial colonization . A screen for mutants insensitive to P . thivervalensis colonization yielded two mutants found to be auxin resistant . This further supports a proposed role for bacterial auxin in inducing morphological modifications of roots . This work paves the way for studying the interaction between plants and non-pathogenic rhizobacteria in a gnotobiotic system, derived from a natural association, where interactions between both partners can be genetically dissected. Mol Gen Genet, 2001 Jan, 264(5), 623 - 33 RpoN of Rhizobium leguminosarum bv . viciae strain VF39SM plays a central role in FnrN-dependent microaerobic regulation of genes involved in nitrogen fixation; Clark SR et al.; The rpoN gene, which codes for the alternative transcription factor sigma54, was cloned and sequenced from Rhizobium leguminosarum strain VF39SM . Construction of a rpoN mutant allowed analysis of the role of RpoN as a transcriptional regulator of genes carrying lacZ reporter fusions . Analysis of a rpoN::lacZ transcriptional fusion in the rpoN background revealed that this gene was negatively autoregulated . Site-directed mutagenesis was used to demonstrate that this autoregulation was dependent on a reverse complement RpoN binding site located upstream of the rpoN gene . rpoN was shown to be required for full microaerobic expression of both copies of fixGHIS, as well as of fixNOQP, despite the absence of apparent rpoN binding sites upstream of fixG . Moreover, rpoN was found to be required for full microaerobic expression of fnrN, which in turn is absolutely required for microaerobic induction of fixGHIS . This suggests that the reduced fixG::lacZ expression seen in the rpoN background is due to the dependence of fnrN expression on RpoN. Mol Gen Genet, 2001 Jan, 264(5), 555 - 64 The Rhizobium leguminosarum glnB gene is down-regulated during symbiosis; Ercolano E et al.; Symbiotic nitrogen fixation involves the development, on the legume plant root, of specialised organs called nodules, within which plant photosynthates are exchanged for combined nitrogen of bacterial origin . The glnB gene encodes a signal transduction protein (P(II)) which is a component of the bacterial nitrogen regulation (Ntr) system and an essential regulator of ammonium assimilation . We demonstrate that in Rhizobium leguminosarum the glnB promoter is strongly regulated by nitrogen and NtrC, but still shows a significant level of activity in conditions of nitrogen excess . Expression of genes involved in nitrogen assimilation has been shown to be absent in nitrogen-fixing bacteroids, and, in agreement with this, we find that the glnB promoter is down-regulated during bacteroid differentiation at a time coincident with the arrest of bacterial division in the nodule . This pattern is common to other bacterial genes involved in nitrogen assimilation and it is noteworthy that the zone where the glnB promoter is active is coincident with the region in which NtrC is expressed. Yi Chuan Xue Bao, 2000, 27(11), 1027 - 32 {Analysis of 16S rDNA sequence and DNA-DNA hybridization of rhizobia isolated from Indigofera sp.}; Wei GH et al.; Based on the previous studies on numerical taxonomy and SDS-PAGE of whole-cell protein, the rhizobia strains isolated from Indigofera sp . in loess plateau area of North-west China constituted a new cluster, the 16S rDNA sequence of representative strain SHL042 was tested, and the phylogenetic tree was produced . In this tree, the strain SHL042, R . tropici A, R . tropici B, R . leguminosarum, R . etli, R . hananesis, R . mongolense and R . gallicum constituted a branch of phylogenetic . Within this branch, the similarity values of 16S rDNA sequences were 95.4%, 95.5%, 96.3%, 95.8%, 96.3%, 97.9% and 97.7% respectively . The values were more than 95% . This indicated that these species should belong to the same genus . The values of DNA homology in the new cluster were all more than 80%, but the values between SHL042 and the strains of these species were less than 50% . Thus, the strain SH714 represented a new rhizobia species. Mol Plant Microbe Interact, 2001 Feb, 14(2), 250 - 4 The Rhizobium etli argC gene is essential for Arginine biosynthesis and nodulation of Phaseolus vulgaris; Ferraioli S et al.; A Tn5-induced mutant strain (CTNUX5) of Rhizobium etli unable to grow with ammonium as the sole nitrogen source was isolated and characterized . Sequence analysis showed that Tn5 is inserted into an argC-homologous gene . Unlike its wild-type parent (strain CE3), the mutant strain CTNUX5 had an absolute dependency on arginine to grow . The argC gene was cloned from the wild-type strain CE3, and the resulting plasmid, pAR207, after transformation was shown to relieve the arginine auxotrophy of strain CTNUX5 . Unlike strain CE3 or CTNUX5-pAR207, strain CTNUX5 showed undetectable levels of N-acetyl-gamma-glutamylphosphate reductase activity . Unless arginine was added to the growth medium, strain CTNUX5 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (nodulation factors) and to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris. Mol Plant Microbe Interact, 2001 Feb, 14(2), 173 - 80 Lotus japonicus forms early senescent root nodules with Rhizobium etli; Banba M et al.; Mesorhizobium loti and Rhizobium etli are microsymbionts of the Lotus and Phaseolus spp., respectively, and secrete essentially the same Nod factors . Lotus japonicus efficiently formed root nodules with R . etli CE3, irrespective of the presence or absence of a flavonoid-independent transcription activator nodD gene . On a nitrogen-free medium, however, the host plant inoculated with R . etli showed a severe nitrogen deficiency symptom . Initially, the nodules formed with R . etli were pale pink and leghemoglobin mRNA was detectable at significant levels . Nevertheless, the nodules became greenish with time . Acetylene-reduction activity of nodules formed with R . etli was comparable with that formed by M . loti 3 weeks postinoculation, but thereafter it decreased rapidly . The nodules formed with R . etli contained much more starch granules than those formed with M . loti . R . etli developed into bacteroids in the L . japonicus nodules, although the density of bacteroids in the infected cells was lower than that in the nodules formed with M . loti . The nodules formed with R . etli were of the early senescence type, in that membrane structures were drastically disintegrated in the infected cells of the greenish nodules . Thus, L . japonicus started and then ceased a symbiotic relationship with R . etli at the final stage. Environ Pollut, 2001, 111(2), 311 - 20 Measurement of symbiotic nitrogen-fixation in leguminous host-plants grown in heavy metal-contaminated soils amended with sewage sludge; Obbard JP et al.; Rates of nitrogen fixation by Rhizobium in symbiosis with leguminous host-plants including white clover, broad bean and peas have been established in soils that have been amended experimentally with heavy metal-contaminated sewage sludges . Results from 15N-dilution experiments for the measurement of N2 fixation have shown that adverse heavy metal effects are apparent on symbiotic N2 fixation rates for white clover grown in inter-specific competition with ryegrass under mixed sward conditions, compared to white clover grown in pure sward . Further experiments on broad bean and pea indicated a significant, but minor-inhibitory metal-related effect on the rate of N2 fixation compared to untreated soils and soils amended with a relatively uncontaminated sludge . The implications of the results with respect to sludge utilisation in agriculture are discussed. Mikrobiologiia, 2000 Nov-Dec, 69(6), 844 - 9 {Effect of sodium salicylate on the population dynamics of a rhizospheric strain of Pseudomonas aureofaciens in soil and on wheat roots}; Mordukhova EA et al.; The effect of sodium salicylate on the population dynamics of the rhizobacterium Pseudomonas aureofaciens BS1393 and its variant bearing the naphthalene biodegradation plasmid pBS216 was studied in the wheat rhizoplane and adjacent soil . Optimum salicylate concentration for the maintenance of the plasmid-bearing strain and for the normal growth of wheat was found to be 250 micrograms/g soil . When the soil was supplemented with salicylate, the population of P . aureofaciens BS1393(pBS216) in the wheat rhizoplane and adjacent soil was, respectively, 4- and 20-fold higher than that of the parent strain lacking the plasmid. Mol Plant Microbe Interact, 2001 Jan, 14(1), 86 - 9 Oxidative burst in alfalfa-Sinorhizobium meliloti symbiotic interaction; Santos R et al.; Reactive oxygen species are produced as an early event in plant defense response against avirulent pathogens . We show here that alfalfa responds to infection with Sinorhizobium meliloti by production of superoxide and hydrogen peroxide . This similarity in the early response to infection by pathogenic and symbiotic bacteria addresses the question of which mechanism rhizobia use to counteract the plant defense response. Genetika, 2000 Dec, 36(12), 1573 - 87 {Evolutionary genetics of rhizobia: molecular and population aspects}; Provorov NA et al.; The molecular analysis of the genetic systems controlling the main stages of nodule bacteria (rhizobia) interaction with a legume host (signaling at early stages and symbiotic nitrogen fixation) has shown that the widespread recombination of genetic material in free-living ancestors of rhizobia was an important factor in the evolution of these systems . These recombinations could be conditioned by a high content of repeated DNA sequences and the IS elements in the rhizobial genome . A high recombination activity of rhizobia is manifested in the panmictic structure of their populations, which is associated with frequency-dependent selection favoring rare recombinants . This selection is realized through the competition of virulent strains for the nodule formation and can be controlled by the genes whose expression depends on population density (via the quorum sensing mechanism) . A high degree of panmixia in rhizobial populations is associated with their ecotypic polymorphism, manifested as the coexistence of symbiotic and nonsymbiotic strains . This type of polymorphism is caused by individual selection during the periodic changes of ecological niches (soil-plant host) in the rhizobia life cycle . The rhizobia-plant interaction stimulates selection in bacterial populations, which results in the increased levels of their heterogeneity and panmixia . The combination of individual and frequency-dependent selection types resulted in the high rates of symbiosis evolution and polyphyletic origin of diverse rhizobial species. Wei Sheng Wu Xue Bao, 1997 Dec, 37(6), 411 - 6 {Sequencing the 16S rDNA of representative strain of new rhizobial group and determining of its phylogenetic relationship}; Tan Z et al.; The full-length 16S rDNA sequence of representative strain SH2672 were sequenced by the dideoxy-mediated chain-termination method . This sequence was compared with that of type strains of all known rhizobia and related bacteria . An unrooted phylogenetic tree was produced . In this tree, strain SH2672, Mesorhizobium loti, M . huakuii, M . tianshanense, M . mediterraneum, and M . ciceri constituted a branch . Within this branch, the similarity values of 16S rDNA sequences between strain SH2672 and M . loti, M . huakuii, M . tianshanense, M . mediterraneum and M . ciceri were 96.3%, 96.4%, 97.2%, 95.1% and 95.6% respectively . All similarity values was more than 95%, this indicated that these species should belong to the same genus . The values of DNA-DNA homology between type strains of these species were less than 70%, which showed that strain SH2672 (representative strain of new group) represented a new rhizobial species. Wei Sheng Wu Xue Bao, 1997 Oct, 37(5), 355 - 61 {Nucleotide sequence of the Rhizobium huakuii common nodution genes nodA and nodBC}; Shen S et al.; An another clone pRaN109 which contains nod genes was isolated and identified from the clone bank of R . huakuii 159 DNA using the 32P-labeled 2.3 kb R . meliloti nod DNA fragment as a hybridization probe . Analysis of homologous DNA-DNA hybridization and nucleotide sequence indicated that the 9 kb EcoRI fragment of pRaN109 DNA carried the nodD1BC genes and the 18 kb EcoRI fragment of pRaN109 DNA contained nodD2A genes . The common nodA gene was separated by 6.7 kb from the nodBC genes . Both the nodA and nodB-nodC cistrons showed nod box upstream of these genes . Nucleotide sequence analysis of the genes describes a obviously different organization of common nod genes in R . huakuii 159 compared with that reported for most of the strains from different genera described up to now. J Bacteriol, 2001 Feb, 183(3), 854 - 64 Enhanced symbiotic performance by Rhizobium tropici glycogen synthase mutants; Marroqui S et al.; We isolated a Tn5-induced Rhizobium tropici mutant that has enhanced capacity to oxidize N,N-dimethyl-p-phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase . The mutant had increased levels of the cytochromes c(1) and CycM and a small increase in the amount of cytochrome aa(3) . In plant tests, the mutant increased the dry weight of Phaseolus vulgaris plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance . The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen . The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from Escherichia coli: glycogen phosphorylase (glgP), glycogen branching enzyme (glgB), ADP glucose pyrophosphorylase (glgC), glycogen synthase (glgA), phosphoglucomutase (pgm), and glycogen debranching enzyme (glgX) . All six genes are transcribed in the same direction, and analysis with lacZ gene fusions suggests that the first five genes are organized in one operon, although pgm appears to have an additional promoter; glgX is transcribed independently . Surprisingly, the glgA mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose . A deletion mutant was constructed in order to generate a nonpolar mutation in glgA . This mutant had a phenotype similar to that of the Tn5 mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of glgA and not to a polar effect on a downstream gene. Genome Biol . 2000;1(6):RESEARCH0014 . Epub 2000 Dec 04. Genetic snapshots of the Rhizobium species NGR234 genome; Viprey V et al.; BACKGROUND: In nitrate-poor soils, many leguminous plants form nitrogen-fixing symbioses with members of the bacterial family Rhizobiaceae . We selected Rhizobium sp . NGR234 for its exceptionally broad host range, which includes more than I 12 genera of legumes . Unlike the genome of Bradyrhizobium japonicum, which is composed of a single 8.7 Mb chromosome, that of NGR234 is partitioned into three replicons: a chromosome of about 3.5 Mb, a megaplasmid of more than 2 Mb (pNGR234b) and pNGR234a, a 536,165 bp plasmid that carries most of the genes required for symbioses with legumes . Symbiotic loci represent only a small portion of all the genes coded by rhizobial genomes, however . To rapidly characterize the two largest replicons of NGR234, the genome of strain ANU265 (a derivative strain cured of pNGR234a) was analyzed by shotgun sequencing . RESULTS: Homology searches of public databases with 2,275 random sequences of strain ANU265 resulted in the identification of 1,130 putative protein-coding sequences, of which 922 (41%) could be classified into functional groups . In contrast to the 18% of insertion-like sequences (ISs) found on the symbiotic plasmid pNGR234a, only 2.2% of the shotgun sequences represent known ISs, suggesting that pNGR234a is enriched in such elements . Hybridization data also indicate that the density of known transposable elements is higher in pNGR234b (the megaplasmid) than on the chromosome . Rhizobium-specific intergenic mosaic elements (RIMEs) were found in 35 shotgun sequences, 6 of which carry RIME2 repeats previously thought to be present only in Rhizobium meliloti . As non-overlapping shotgun sequences together represent approximately 10% of ANU265 genome, the chromosome and megaplasmid may carry a total of over 200 RIMEs . CONCLUSIONS: 'Skimming' the genome of Rhizobium sp . NGR234 sheds new light on the fine structure and evolution of its replicons, as well as on the integration of symbiotic functions in the genome of a soil bacterium . Although most putative coding sequences could be distributed into functional classes similar to those in Bacillus subtilis, functions related to transposable elements were more abundant in NGR234 . In contrast to ISs that accumulated in pNGR234a and pNGR234b, the hundreds of RIME elements seem mostly attributes of the chromosome. Plant J, 2001 Jan, 25(1), 55 - 65 Nod factor-induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation; den Hartog M et al.; Rhizobium-secreted nodulation factors are lipochitooligosaccharides that trigger the initiation of nodule formation on host legume roots . The first visible effect is root hair deformation, but the perception and signalling mechanisms that lead to this response are still unclear . When we treated Vicia sativa seedlings with mastoparan root hairs deformed, suggesting that G proteins are involved . To investigate whether mastoparan and Nod factor activate lipid signalling pathways initiated by phospholipase C (PLC) and D (PLD), seedlings were radiolabelled with {(32)P}orthophosphate prior to treatment . Mastoparan stimulated increases in phosphatidic acid (PA) and diacylglycerol pyrophosphate, indicative of PLD or PLC activity in combination with diacylglycerol kinase (DGK) and PA kinase . Treatment with Nod factor had similar effects, although less pronounced . The inactive mastoparan analogue Mas17 had no effect . The increase in PA was partially caused by the activation of PLD that was monitored by its in vivo transphosphatidylation activity . The application of primary butyl alcohols, inhibitors of PLD activity, blocked root hair deformation . Using different labelling strategies, evidence was provided for the activation of DGK . Since the PLC antagonist neomycin inhibited root hair deformation and the formation of PA, we propose that PLC activation produced diacylglycerol (DAG), which was subsequently converted to PA by DGK . The roles of PLC and PLD in Nod factor signalling are discussed. Plant Sci, 2000 Dec 7, 160(1), 67 - 75 The promoter of the Vicia faba L . gene VfEnod12 encoding an early nodulin is active in cortical cells and nodule primordia of transgenic hairy roots of Vicia hirsuta as well as in the prefixing zone II of mature transgenic V . hirsuta root nodules; Fruhling M et al.; A full-length cDNA encoding the Vicia faba L . early nodulin VfEnod12 was isolated . The deduced protein sequence specified a 90 amino acid protein with a MW of 10206 and contained a putative signal peptide sequence followed by PPX(3) repeats characteristic of Enod12 proteins . The VfEnod12 gene was found to be expressed specifically in root nodules as early as 3 days post inoculation with Rhizobium leguminosarum bv . viciae . In mature nodules, VfEnod12 transcripts were confined to the prefixing zone II . A 3.3 kb genomic fragment carrying the complete VfEnod12 coding region was isolated . No intervening sequences were identified in the coding region . A promoter fragment carrying the -692/-41 region mediated reporter gene expression in root cortical cells, nodule primordia and the prefixing zone II of transgenic Vicia hirsuta root nodules . This fragment contained a putative binding site for the transcription factor ENBP1 . In contrast to the highly conserved terminal AATAA motif of the ENBP1 binding site of known Enod12 promoters, the VfEnod12 promoter was characterized by an altered terminal AATAT sequence . This alteration did not interfere with VfEnod12 promoter activity in transgenic roots and nodules of V . hirsuta. Trends Plant Sci, 2001 Jan, 6(1), 24 - 30 Perception of lipo-chitooligosaccharidic Nod factors in legumes; Cullimore JV et al.; Lipo-chitooligosaccharides produced by rhizobia are a class of signalling molecules that mediate recognition and nodule organogenesis in the legume-rhizobia symbiosis . Their synthesis is specified by the nodulation genes of rhizobia and hence they are commonly known as Nod factors . They are amphiphilic molecules and induce a variety of responses in the roots of the legume hosts . Studies using plant and rhizobial mutants and purified molecules suggest that Nod factors are recognized by more than one receptor . In this article, we review evidence about the affinity, specificity and location of these putative receptors and describe recent studies with regard to their identification. J Bacteriol, 2001 Feb, 183(4), 1405 - 12 Potential symbiosis-specific genes uncovered by sequencing a 410-kilobase DNA region of the Bradyrhizobium japonicum chromosome; Gottfert M et al.; The physical and genetic map of the Bradyrhizobium japonicum chromosome revealed that nitrogen fixation and nodulation genes are clustered . Because of the complex interactions between the bacterium and the plant, we expected this chromosomal sector to contain additional genes that are involved in the maintenance of an efficient symbiosis . Therefore, we determined the nucleotide sequence of a 410-kb region . The overall G+C nucleotide content was 59.1% . Using a minimum gene length of 150 nucleotides, 388 open reading frames (ORFs) were selected as coding regions . Thirty-five percent of the predicted proteins showed similarity to proteins of rhizobia . Sixteen percent were similar only to proteins of other bacteria . No database match was found for 29% . Repetitive DNA sequence-derived ORFs accounted for the rest . The sequenced region contained all nitrogen fixation genes and, apart from nodM, all nodulation genes that were known to exist in B . japonicum . We found several genes that seem to encode transport systems for ferric citrate, molybdate, or carbon sources . Some of them are preceded by -24/-12 promoter elements . A number of putative outer membrane proteins and cell wall-modifying enzymes as well as a type III secretion system might be involved in the interaction with the host. J Bacteriol, 2001 Feb, 183(4), 1248 - 58 Genetic characterization of a Sinorhizobium meliloti chromosomal region in lipopolysaccharide biosynthesis; Lagares A et al.; The genetic characterization of a 5.5-kb chromosomal region of Sinorhizobium meliloti 2011 that contains lpsB, a gene required for the normal development of symbiosis with Medicago spp., is presented . The nucleotide sequence of this DNA fragment revealed the presence of six genes: greA and lpsB, transcribed in the forward direction; and lpsE, lpsD, lpsC, and lrp, transcribed in the reverse direction . Except for lpsB, none of the lps genes were relevant for nodulation and nitrogen fixation . Analysis of the transcriptional organization of lpsB showed that greA and lpsB are part of separate transcriptional units, which is in agreement with the finding of a DNA stretch homologous to a "nonnitrogen" promoter consensus sequence between greA and lpsB . The opposite orientation of lpsB with respect to its first downstream coding sequence, lpsE, indicated that the altered LPS and the defective symbiosis of lpsB mutants are both consequences of a primary nonpolar defect in a single gene . Global sequence comparisons revealed that the greA-lpsB and lrp genes of S . meliloti have a genetic organization similar to that of their homologous loci in R . leguminosarum bv . viciae . In particular, high sequence similarity was found between the translation product of lpsB and a core-related biosynthetic mannosyltransferase of R . leguminosarum bv . viciae encoded by the lpcC gene . The functional relationship between these two genes was demonstrated in genetic complementation experiments in which the S . meliloti lpsB gene restored the wild-type LPS phenotype when introduced into lpcC mutants of R . leguminosarum . These results support the view that S . meliloti lpsB also encodes a mannosyltransferase that participates in the biosynthesis of the LPS core . Evidence is provided for the presence of other lpsB-homologous sequences in several members of the family Rhizobiaceae. Appl Environ Microbiol, 2001 Feb, 67(2), 1008 - 10 Characterization of rhizobial isolates of Phaseolus vulgaris by staircase electrophoresis of low-molecular-weight RNA; Velazquez E et al.; Low-molecular-weight (LMW) RNA molecules were analyzed to characterize rhizobial isolates that nodulate the common bean growing in Spain . Since LMW RNA profiles, determined by staircase electrophoresis, varied across the rhizobial species nodulating beans, we demonstrated that bean isolates recovered from Spanish soils presumptively could be characterized as Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum bv . viciae and bv . trifolii, and Sinorhizobium fredii. Appl Environ Microbiol, 2001 Feb, 67(2), 495 - 8 Management of indigenous plant-microbe symbioses aids restoration of desertified ecosystems; Requena N et al.; Disturbance of natural plant communities is the first visible indication of a desertification process, but damage to physical, chemical, and biological soil properties is known to occur simultaneously . Such soil degradation limits reestablishment of the natural plant cover . In particular, desertification causes disturbance of plant-microbe symbioses which are a critical ecological factor in helping further plant growth in degraded ecosystems . Here we demonstrate, in two long-term experiments in a desertified Mediterranean ecosystem, that inoculation with indigenous arbuscular mycorrhizal fungi and with rhizobial nitrogen-fixing bacteria not only enhanced the establishment of key plant species but also increased soil fertility and quality . The dual symbiosis increased the soil nitrogen (N) content, organic matter, and hydrostable soil aggregates and enhanced N transfer from N-fixing to nonfixing species associated within the natural succession . We conclude that the introduction of target indigenous species of plants associated with a managed community of microbial symbionts is a successful biotechnological tool to aid the recovery of desertified ecosystems. Curr Microbiol, 2000 Jul, 41(1), 73 - 5 Effect of agglutinins from Rhizobium leguminosarum strain 252 on the activity of hydrolytic enzymes; Karpunina LV et al.; Cells of the nitrogen-fixing soil bacterium Rhizobium leguminosarum 252 and its hemagglutination-deficient mutant strain 252/7 were found to possess the activities of a variety of hydrolytic enzymes . The agglutinin proteins of rhizobia diminished beta-glucosidase activity, pectinolytic activity, and acid and alkaline phosphatase activities while completely inhibiting proteolytic enzyme activity in the bacterial cell . The results here show that rhizobial agglutinins are capable of affecting enzyme functioning in Rhizobium. J Biol Chem, 2000 Apr 14, 275(15), 11300 - 5 Biosynthesis of vitamin B(6) in rhizobium; Tazoe M et al.; The biosynthetic pathway of pyridoxol (vitamin B(6)) in Rhizobium was clarified by studies on the incorporation of (13)C- or (15)N-labeled precursors into pyridoxol or its biosynthetic intermediates . Pyridoxol was formed by ring closure of two compounds, 1-deoxy-D-xylulose and 4-hydroxy-L-threonine . The former was formed from D-glyceraldehyde and pyruvate through decarboxylation of pyruvate, and the latter from glycine and glycolaldehyde. FEMS Microbiol Lett, 2001 Jan 1, 194(1), 83 - 6 Characterization of the common nodulation genes of the photosynthetic Bradyrhizobium sp . ORS285 reveals the presence of a new insertion sequence upstream of nodA; Chaintreuil C et al.; We isolated and characterized nodA genes from photosynthetic and non-photosynthetic rhizobia nodulating the legume genus Aeschynomene, and found that the nodA sequence from photosynthetic stem-nodulating bacteria was phylogenetically distant from the other already described nodA genes . Characterization of the photosynthetic strain ORS285 common nod gene cluster (nodABC) showed, upstream of nodA, the presence of a new insertion sequence element belonging to the IS3 family and specific to a group of photosynthetic strains from Aeschynomene. J Exp Bot, 2000 Dec, 51(353), 2045 - 51 Differential response of soybean (Glycine max (L.) Merr.) genotypes to lipo-chito-oligosaccharide Nod Bj V (C(18:1) MeFuc); Prithiviraj B et al.; Lipo-chito-oligosaccharides (LCOs) are bacteria-to-plant signal molecules essential for the establishment of rhizobia-legume symbioses . LCOs invoke a number of physiological changes in the host plants, such as root hair deformation, cortical cell division and ontogeny of complete nodule structures . The responses of five soybean cultivars to Nod BJ: V (C(18:1) MeFuc) isolated from Bradyrhizobium japonicum strain 532C were studied with a new technique . Two distinct types of root hair deformation were evident (i) bulging, in which root hairs were swollen at the tip or at the base depending on the cultivars and (ii) curling . The nodulating capacity of B . japonicum 532C varied among cultivars . Cultivars that produced a bulging reaction when treated with LCO had fewer nodules and the roots had low phenol contents . Cultivars that produced curling had higher numbers of nodules and the roots had higher amounts of phenol . Further, the roots of cultivars that showed root hair bulging were able to degrade LCO much faster than cultivars that manifested curling . The results of the present study establish relationships among the type of LCO-induced root hair deformation, root system LCO-degrading ability and nodulation capacity of soybean cultivars. Phytochemistry, 2000 Nov, 55(5), 429 - 38 Peptides isolated from cell walls of Medicago truncatula nodules and uninfected root; Frueauf JB et al.; The hydroxyproline-rich root nodules of legumes provide a microaerobic niche for symbiotic nitrogen-fixing Rhizobacteria . The contributions of the cell wall and associated structural proteins, particularly the hydroxyproline-rich glycoproteins (HRGPs), are therefore of interest . Our approach involved identification of the protein components by direct chemical analysis of the insoluble wall . Chymotryptic peptide mapping showed a "P3-type" extensin containing the highly arabinosylated Ser-Hyp4-Ser-Hyp-Ser-Hyp4-Tyr3-Lys motif as a major component . Cell wall amino acid analyses and quantitative hydroxyproline arabinoside profiles, predominantly of tri- and tetraarabinosides, confirmed this extensin as the major structural protein in the cell walls of both root nodules and uninfected roots . On the other hand, judging from the Pro, Glu and non-glycosylated Hyp content, the nodule-specific proline-rich glycoproteins, such as the early nodulins (ENOD-PRPs), are present in much lesser amounts . Although we isolated no PRP peptides from nodule cell walls, a single PRP peptide from root cell walls confirmed the presence of a PRP in roots and represented the first direct evidence for a crosslinked PRP in muro . Compared with root cell walls (approximately 7% protein dry weight) nodule cell walls contained significantly more protein (approximately 13% dry weight) with an overall amino acid and peptide composition indicating the presence of structural protein unrelated to the HRGPs. Chem Biol, 2000 Dec, 7(12), 919 - 30 Structural control of polyketide formation in plant-specific polyketide synthases; Jez JM et al.; BACKGROUND: Polyketide synthases (PKSs) generate molecular diversity by utilizing different starter molecules and by controlling the final length of the polyketide . Although exploitation of this mechanistic variability has produced novel polyketides, the structural foundation of this versatility is unclear . Plant-specific PKSs are essential for the biosynthesis of anti-microbial phytoalexins, anthocyanin floral pigments, and inducers of Rhizobium nodulation genes . 2-Pyrone synthase (2-PS) and chalcone synthase (CHS) are plant-specific PKSs that share 74% amino acid sequence identity . 2-PS forms the triketide methylpyrone from an acetyl-CoA starter molecule and two malonyl-CoAs . CHS uses a p-coumaroyl-CoA starter molecule and three malonyl-CoAs to produce the tetraketide chalcone . Our goal was to elucidate the molecular basis of starter molecule selectivity and control of polyketide length in this class of PKS.Results: The 2.05 A resolution crystal structure of 2-PS complexed with the reaction intermediate acetoacetyl-CoA was determined by molecular replacement . 2-PS and CHS share a common three-dimensional fold, a set of conserved catalytic residues, and similar CoA binding sites . However, the active site cavity of 2-PS is smaller than the cavity in CHS . Of the 28 residues lining the 2-PS initiation/elongation cavity, four positions vary in CHS . Point mutations at three of these positions in CHS (T197L, G256L, and S338I) altered product formation . Combining these mutations in a CHS triple mutant (T197L/G256L/S338I) yielded an enzyme that was functionally identical to 2-PS.Conclusions: Structural and functional characterization of 2-PS together with generation of a CHS mutant with an initiation/elongation cavity analogous to 2-PS demonstrates that cavity volume influences the choice of starter molecule and controls the final length of the polyketide . These results provide a structural basis for control of polyketide length in other PKSs, and suggest strategies for further increasing the scope of polyketide biosynthetic diversity. J Clin Microbiol, 2001 Jan, 39(1), 235 - 40 Limited genetic diversity of Brucella spp; Gandara B et al.; Multilocus enzyme electrophoresis (MLEE) of 99 Brucella isolates, including the type strains from all recognized species, revealed a very limited genetic diversity and supports the proposal of a monospecific genus . In MLEE-derived dendrograms, Brucella abortus and a marine Brucella sp . grouped into a single electrophoretic type related to Brucella neotomae and Brucella ovis . Brucella suis and Brucella canis formed another cluster linked to Brucella melitensis and related to Rhizobium tropici . The Brucella strains tested that were representatives of the six electrophoretic types had the same rRNA gene restriction fragment length polymorphism patterns and identical ribotypes . All 99 isolates had similar chromosome profiles as revealed by the Eckhardt procedure. FEMS Microbiol Ecol, 2001 Jan, 34(3), 267 - 278 Effect of heat stress on cell activity and cell morphology of the tropical rhizobium, Sinorhizobium arboris; Rasanen LA et al.; The effect of heat stress on the growth, physiological state, cell activity and cell morphology of the tropical Sinorhizobium arboris strain HAMBI 2190 was studied . The cells were chromosomally tagged with the firefly luciferase gene, luc . Since the bioluminescence phenotype is dependent on cellular energy reserves it was used as an indicator of the metabolic status of the cell population under various heat conditions . Variations in the numbers and lengths of growth phases between individual cultures indicated that the growth pattern at 40 degrees C was disturbed compared to growth at 37 or 28 degrees C . In addition, the cell morphology was changed radically . The number of culturable cells and the luciferase activity declined when the cultures were incubated at 40 degrees C . By contrast, under all conditions studied, the cells could be stained with 5-(and 6-)sulfofluorescein diacetate, indicating esterase activity . This demonstrated that although the culturability and cellular energy reserves decreased considerably during heat stress, a majority of the of S . arboris cell population maintained basal enzyme activity. Mol Microbiol, 2001 Jan, 39(2), 379 - 91 Lipid A and O-chain modifications cause Rhizobium lipopolysaccharides to become hydrophobic during bacteroid development; Kannenberg EL et al.; Modifications to the lipopolysaccharide (LPS) structure caused by three different growth conditions were investigated in the pea-nodulating strain Rhizobium leguminosarum 3841 . The LPSs extracted by hot phenol-water from cultured cells fractionated into hydrophilic water and/or hydrophobic phenol phases . Most of the LPSs from cells grown under standard conditions extracted into the water phase, but a greater proportion of LPSs were extracted into the phenol phase from cells grown under acidic or reduced-oxygen conditions, or when isolated from root nodules as bacteroids . Compared with the water-extracted LPSs, the phenol-extracted LPSs contained greater degrees of glycosyl methylation and O-acetylation, increased levels of xylose, glucose and mannose and increased amounts of long-chain fatty acids attached to the lipid A moiety . The water- and phenol-phase LPSs also differed in their reactivity with monoclonal antibodies and in their polyacrylamide gel electrophoretic banding patterns . Phenol-extracted LPSs from rhizobia grown under reduced-oxygen conditions closely resembled the bulk of LPSs isolated from pea nodule bacteria (i.e . mainly bacteroids) in their chemical properties, reactivities with monoclonal antibodies and extraction behaviour . This finding suggests that, during symbiotic bacteroid development, reduced oxygen tension induces structural modifications in LPSs that cause a switch from predominantly hydrophilic to predominantly hydrophobic molecular forms . Increased hydrophobicity of LPSs was also positively correlated with an increase in the surface hydrophobicity of whole cells, as shown by the high degree of adhesion to hydrocarbons of bacterial cells isolated from nodules or from cultures grown under low-oxygen conditions . The implications of these LPS modifications are discussed for rhizobial survival and function in different soil and in planta habitats. Appl Environ Microbiol, 2001 Jan, 67(1), 396 - 402 Small-subunit rRNA genotyping of rhizobia nodulating Australian Acacia spp; Lafay B et al.; The structure of rhizobial communities nodulating Acacia in southeastern Australia from south Queensland to Tasmania was investigated by a molecular approach . A total of 118 isolates from nodule samples from 13 different Acacia species collected at 44 sites were characterized by small-subunit (SSU) ribosomal DNA (rDNA) PCR-restriction fragment length polymorphism analysis . Nine rhizobial genomospecies were identified, and these taxa corresponded to previously described genomospecies (B . Lafay and J . J . Burdon, Appl . Environ . Microbiol . 64:3989-3997, 1998) . Eight of these genomospecies belonged to the Bradyrhizobium lineage and accounted for 96.6% of the isolates . The remaining genomospecies corresponded to Rhizobium tropici . For analysis of geographic patterns, results were grouped into five latitudinal regions regardless of host origin . In each region, as observed previously for rhizobial isolates taken from non-Acacia legumes (Lafay and Burdon, Appl . Environ . Microbiol . 64:3989-3997, 1998), rhizobial communities were dominated by one or two genomospecies, the identities of which varied from place to place . Despite this similarity in patterns, the most abundant genomospecies for Acacia isolates differed from the genomospecies found in the non-Acacia-derived rhizobial collection, suggesting that there is a difference in nodulation patterns of the Mimosoideae and the Papilionoideae . Only two genomospecies were both widespread and relatively abundant across the range of sites sampled . Genomospecies A was found in all regions except the most northern sites located in Queensland, whereas genomospecies B was not detected in Tasmania . This suggests that genomospecies A might be restricted to the more temperate regions of Australia, whereas in contrast, genomospecies B occurs in different climatic and edaphic conditions across the whole continent . The latter hypothesis is supported by the presence of genomospecies B in southwestern Australia, based on partial SSU rDNA sequence data (N . D . S . Marsudi, A . R . Glenn, and M . J . Dilworth, Soil Biol . Biochem . 31:1229-1238, 1998). Heredity, 2000 Oct, 85 Pt 4, 366 - 72 Evolution of nitrogen fixation in spatially structured populations of Rhizobium; Bever JD et al.; Symbiosis between legumes and nitrogen-fixing bacteria is thought to bring mutual benefit to each participant . However, it is not known how rhizobia benefit from nodulation of legume hosts because they fix nitrogen only after differentiating into bacteroids, terminally differentiated cells that cannot reproduce . Because free-living rhizobia can reproduce, and may benefit from the increase of plant root exudates stimulated by nodulation, evolution of symbiotic nitrogen fixation may depend upon kin selection . However, unrelated nonmutualists may also benefit from increased plant exudates and nitrogen-fixing populations are therefore vulnerable to invasion by nonfixing, saprophytic Rhizobium . The access of nonfixing Rhizobium to the plant exudates associated with nodules depends upon the spatial structure of the Rhizobium populations within the soil . We investigate the influence of spatial structure on the evolution of N-fixation within a Rhizobium population using a mathematical model . Our model demonstrates that spatial structure is necessary for the evolution of N-fixation and that N-fixation is more likely to evolve with increasing degrees of spatial structure . In fact, we identify three dynamic outcomes that depend upon the relative strength of the costs of N-fixation relative to the degree of spatial structure and benefits resulting from nodulations . If the costs are relatively high, N-fixation will not evolve; if the costs are relatively low, N-fixing genes will fix in the population, but at intermediate conditions, a stable mixture of N-fixing bacteria and nonfixing bacteria will be maintained . The conditions for coexistence of N-fixing bacteria and nonfixing bacteria expand under a saturating relationship between nodule numbers and N-fixing genotype frequency. Curr Opin Microbiol, 2000 Dec, 3(6), 613 - 7 Hanging by a thread: invasion of legume plants by rhizobia; Gage DJ et al.; Nitrogen-fixing nodules on plants such as alfalfa, pea and vetch arise from the root inner cortex and grow via a persistent meristem . Thus, these nodules are defined as indeterminate . The formation of functional indeterminate nodules requires that symbiotic bacteria, collectively called rhizobia, gain access to the interior of roots and root nodules via infection threads . Recent work has begun to elucidate the important functions of the root cell cytoskeleton in infection thread formation . It has also recently become apparent that rhizobial Nod factors and rhizobial exopolysaccharides play key roles in the initiation and elongation of infection threads. Biochim Biophys Acta, 2000 Dec 15, 1517(1), 139 - 42 A Phaseolus vulgaris lipoxygenase gene expressed in nodules and in Rhizobium tropici inoculated roots; Porta H et al.; A genomic clone encoding a common bean lipoxygenase (PvLOX5) was isolated from a Phaseolus vulgaris library . Reverse transcription-polymerase chain reaction analysis revealed that PvLOX5 is expressed during nodule development and in Rhizobium tropici inoculated roots . There was no detectable expression of PvLOX5 in non-inoculated roots, healthy leaves, leaves after Pseudomonas syringae pv . tabaci infection, floral buds or dry seeds. Curr Microbiol, 2001 Jan, 42(1), 59 - 64 Survival and nodulating ability of indigenous and inoculated Rhizobium leguminosarum biovar trifolii in sterilized and unsterilized soil treated with sewage sludge; Purchase D et al.; Rhizobium leguminosarum biovar trifolii was detected in soil from 41 of 47 plots, within nine sewage sludge-treated sites with different soil characteristics and heavy metal contents . However, although population size varied widely, there was no consistent correlation with soil heavy metal concentration . Indigenous populations in 20 plots within four selected sites retained their ability to induce effective nodule formation after incubation of soil in the dark for 165 days . In sterilized (gamma-irradiated) soil, Rhizobium survival varied from 0.01% to 95% depending on the soil sample and strain used . Metal-resistant strains with non-mucoid colonies survived less well than mucoid metal-sensitive strains. Curr Microbiol, 2001 Jan, 42(1), 53 - 8 Poly-beta-hydroxybutyrate metabolism is affected by changes in respiratory enzymatic activities due to cold stress in two psychrotrophic strains of Rhizobium; Sardesai N et al.; Cold stress resulted in a decrease in the poly-beta-hydroxybutyrate (PHB) content of non-cold-acclimated Rhizobium DDSS69 cultures . Analysis of the specific activity of beta-ketothiolase and beta-hydroxybutyrate dehydrogenase revealed that decrease in PHB levels was a result of the inhibition of synthesis of PHB rather than an increase in its breakdown . Rhizobium ATR1, a cold-acclimated strain, revealed the presence of a stable PHB metabolism that did not show any significant differences either in PHB levels or in the activity of enzymes of the PHB metabolism under cold stress, suggesting that PHB is not involved in cold tolerance . Analysis of specific activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of the pentose phosphate pathway showed the upward regulation of alternate pathways of carbohydrate metabolism under cold stress to rapidly generate energy to overcome the stress . There is diversity in the switching mechanisms of carbon metabolism among cold-acclimated and non-cold-acclimated Rhizobium isolates . Upward regulation of malate dehydrogenase in both isolates suggests that it is a critical input for cold tolerance. Curr Microbiol, 2001 Jan, 42(1), 26 - 31 Numerical taxonomy of Sarothamnus scoparius rhizobia; Sajnaga E et al.; Twenty nodule isolates from Sarothamnus scoparius (broom) growing in Poland and nine strains from plants growing in Japan were studied for phenotypic properties, plasmid presence, phage sensitivity, and host plant specificity . By numerical analysis of phenotypic properties, it was found that the studied nodule bacteria, originating from geographically different countries, constitute two separate groups affiliated to the bradyrhizobium cluster . The membership of S . scoparius rhizobia in the Bradyrhizobium genus was also supported by their long generation time, alkaline reaction in YEM medium with mannitol, lack of plasmids, and wide host plant range. Mol Microbiol, 2000 Nov, 38(4), 828 - 38 HrpB2 and HrpF from Xanthomonas are type III-secreted proteins and essential for pathogenicity and recognition by the host plant; Rossier O et al.; The interaction between the plant pathogen Xanthomonas campestris pv . vesicatoria and its host plants is controlled by hrp genes (hypersensitive reaction and pathogenicity), which encode a type III protein secretion system . Among type III-secreted proteins are avirulence proteins, effectors involved in the induction of plant defence reactions . Using non-polar mutants, we investigated the role of 12 hrp genes in the secretion of the avirulence protein AvrBs3 from X . c . pv . vesicatoria and a heterologous protein, YopE, from Yersinia pseudotuberculosis . Genes conserved among type III secretion systems (hrcQ, hrcR, hrcS and hrcT) as well as non-conserved genes (hrpB1, hrpB2, hrpB4, hrpB5, hrpD5 and hrpD6) were shown to be required for secretion . Protein localization studies using specific antibodies showed that HrpB1 and HrpB4, as well as the putative ATPase HrcN, were mainly found in the soluble fraction of the bacterial cell . In contrast, HrpB2 and HrpF, which is related to NolX of Rhizobium fredii, are secreted into the culture medium in an hrp-dependent manner . As HrpB2, but not HrpF, is essential for type III protein secretion, there might be a hierarchy in the secretion process . We propose that HrpF, which is dispensable for protein secretion but required for AvrBs3 recognition in planta, functions as a translocator of effector proteins into the host cell. Mol Microbiol, 2000 Nov, 38(4), 750 - 9 Critical protective role of bacterial superoxide dismutase in rhizobium-legume symbiosis; Santos R et al.; In nitrogen-poor soils, rhizobia elicit nodule formation on legume roots, within which they differentiate into bacteroids that fix atmospheric nitrogen . Protection against reactive oxygen species (ROS) was anticipated to play an important role in Rhizobium-legume symbiosis because nitrogenase is extremely oxygen sensitive . We deleted the sodA gene encoding the sole cytoplasmic superoxide dismutase (SOD) of Sinorhizobium meliloti . The resulting mutant, deficient in superoxide dismutase, grew almost normally and was only moderately sensitive to oxidative stress when free living . In contrast, its symbiotic properties in alfalfa were drastically affected . Nitrogen-fixing ability was severely impaired . More strikingly, most SOD-deficient bacteria did not reach the differentiation stage of nitrogen-fixing bacteroids . The SOD-deficient mutant nodulated poorly and displayed abnormal infection . After release into plant cells, a large number of bacteria failed to differentiate into bacteroids and rapidly underwent senescence . Thus, bacterial SOD plays a key protective role in the symbiotic process. J Bacteriol, 2001 Jan, 183(1), 214 - 20 Methylotrophic Methylobacterium bacteria nodulate and fix nitrogen in symbiosis with legumes; Sy A et al.; Rhizobia described so far belong to three distinct phylogenetic branches within the alpha-2 subclass of Proteobacteria . Here we report the discovery of a fourth rhizobial branch involving bacteria of the Methylobacterium genus . Rhizobia isolated from Crotalaria legumes were assigned to a new species, "Methylobacterium nodulans," within the Methylobacterium genus on the basis of 16S ribosomal DNA analyses . We demonstrated that these rhizobia facultatively grow on methanol, which is a characteristic of Methylobacterium spp . but a unique feature among rhizobia . Genes encoding two key enzymes of methylotrophy and nodulation, the mxaF gene, encoding the alpha subunit of the methanol dehydrogenase, and the nodA gene, encoding an acyltransferase involved in Nod factor biosynthesis, were sequenced for the type strain, ORS2060 . Plant tests and nodA amplification assays showed that "M . nodulans" is the only nodulating Methylobacterium sp . identified so far . Phylogenetic sequence analysis showed that "M . nodulans" NodA is closely related to Bradyrhizobium NodA, suggesting that this gene was acquired by horizontal gene transfer. Int Microbiol, 1998 Sep, 1(3), 225 - 30 Partial characterization and photolabeling of a Rhizobium meliloti polysaccharide methyltransferase with S-adenosylmethionine; Ruiz OA et al.; S-Adenosylmethionine (SAM) has been used to directly cross-link a polysaccharide specific methyltransferase isolated from Rhizobium meliloti HA . This peculiar enzyme transfers a methyl group to the 2-O-galacturonosyl residue of a teichuronic type polysaccharide and was very unstable . The apparent Km for SAM was 0.46 mM . The Hill coefficient, n, was 1 . The enzyme had an optimum pH of 8.2 and requires Mn2+ at concentration of 2 mM . The enzyme was inactivated by saline concentrations of 120 mM or greater and was eluted from Superose columns with an apparent molecular weight of 28 kDa . The isoelectric point was close to 7.0 . To elucidate the relationship between chemical structure and catalytic function, (3H)SAM was cross-linked to the enzyme and the enzymatic activity was assayed in presence and in absence of commercial substrate analogs . Cross-linking was performed by direct irradiation of enzyme and (3H)SAM . The uptake of radioactivity was linear up to about 20 min and then reached a plateau . This irreversible junction is specific, as shown by a number of different criteria . Several competitive inhibitors were able to affect this photoactivated cross-linkage . As the concentration of inhibitors increased, both, the level of photolabeling and enzyme activity always decreased . The SAM-enzyme adduct was shown to be a single protein band by SDS polyacrylamide gel electrophoresis. Syst Appl Microbiol, 2000 Oct, 23(3), 418 - 25 Restriction fragment length polymorphism analysis of 16S rDNA and low molecular weight RNA profiling of rhizobial isolates from shrubby legumes endemic to the Canary islands; Jarabo-Lorenzo A et al.; Thirty-six strains of slow-growing rhizobia isolated from nodules of four woody legumes endemic to the Canary islands were characterised by 16S rDNA PCR-RFLP analyses (ARDRA) and LMW RNA profiling, and compared with reference strains representing Bradyrhizobium japonicum, B . elkanii, B . liaoningense, and two unclassified Bradyrhizobium sp . (Lupinus) strains . Both techniques showed similar results, indicating the existence of three genotypes among the Canarian isolates . Analysis of the combined RFLP patterns obtained with four endonucleases, showed the existence of predominant genotype comprising 75% of the Canarian isolates (BTA-1 group) and the Bradyrhizobium sp . (Lupinus) strains . A second genotype was shared by nine Canarian isolates (BGA-1 group) and the B . japonicum and B . liaoningense reference strains . The BES-5 strain formed an independent group, as also did the B . elkanii reference strains . LMW RNA profile analysis consistently resolved the same three genotypes detected by 16S ARDRA among the Canarian isolates, and suggested that all these isolates are genotypically more related to B . japonicum than to B . elkanii or B . liaoningense . Cluster analysis of the combined 16S ARDRA and LMW RNA profiles resolved the BTA-1 group with the Bradyrhizobium sp . (Lupinus) strains, and the BES-5 isolate, as a well separated sub-branch of the B . japonicum cluster . Thus, the two types of analyses indicated that the isolates related to BTA-1 conform a group of bradyrhizobial strains that can be clearly distinguishable from representatives of the tree currently described Bradyrhizobium species . No correlation between genotypes, host legumes, and geographic location was found. Eur J Biochem, 2000 Dec, 267(24), 7224 - 30 Identification and characterization of a novel transcriptional regulator, MatR, for malonate metabolism in Rhizobium leguminosarum bv . trifolii; Lee HY et al.; A novel gene, matR, located upstream of matABC, transcribed in the opposite direction, and encoding a putative regulatory protein by sequence analysis was discovered from Rhizobium leguminosarum bv . trifolii . The matA, matB, and matC genes encode malonyl-CoA decarboxylase, malonyl-CoA synthetase, and a presumed malonate transporter, respectively . Together, these enzymes catalyze the uptake and conversion of malonate to acetyl-CoA . The deduced amino-acid sequence of matR showed sequence similarity with GntR from Bacillus subtilis in the N-terminal region encoding a helix-turn-helix domain . Electrophoretic mobility shift assay indicated that MatR bound to a fragment of DNA corresponding to the mat promoter region . The addition of malonate or methylmalonate increased the association of MatR and DNA fragment . DNase I footprinting assays identified a MatR binding site encompassing 66 nucleotides near the mat promoter . The mat operator region included an inverted repeat (TCTTGTA/TACACGA) centered -46.5 relative to the transcription start site . Transcriptional assays, using the luciferase gene, revealed that MatR represses transcription from the mat promoter and malonate alleviates MatR-mediated repression effect on the expression of Pmat-luc+ reporter fusion. Mol Plant Microbe Interact, 2000 Dec, 13(12), 1283 - 92 Differential regulation of fixN-reiterated genes in Rhizobium etli by a novel fixL-fixK cascade; Girard L et al.; Among the complexities in the regulation of nitrogen fixation in the Rhizobiaceae are reiteration of regulatory components as well as variant roles for each component between species . For Rhizobium etli CFN42, we reported that the symbiotic plasmid (pCFN42d) contains a key regulatory gene (fixKd) and genes for a symbiotic cytochrome oxidase (fixNOQPd) . Here we discuss the occurrence of reiteration of these genes (fixKf and fixNOQPf) and the finding of an unusual fixL homolog on a plasmid previously considered cryptic (pCFN42f) . The structure of the deduced FixL polypeptide is suggestive of a fusion of the receiver and transmitter modules of a two-component regulatory system as described in R . leguminosarum bv . viciae VF39 . Gene fusion analysis, coupled with mutation of each regulatory element, revealed that free-living expression of FixKf was dependent fully on FixL . In contrast, synthesis of FixKd was not detected under the conditions tested . The FixKf protein is needed for microaerobic expression of both fixN reiterations, whereas the FixKd protein appears to be dispensable . Interestingly, expression of the fixN reiterations exhibits a differential dependence for FixL, where transcription of fixNf was suppressed in the absence of FixL but expression of fixNd still showed significant levels . This suggests the existence of a FixL-independent mechanism for expression of the fixNd reiteration . Surprisingly, mutations in fixL, fixKd, or fixKf (either singly or in combination) did not alter symbiotic effectiveness . A mutation in fixNd (but not in fixNf) was, however, severely affected, indicating a differential role for these reiterations in nitrogen fixation. Appl Environ Microbiol, 2000 Dec, 66(12), 5469 - 71 The Bradyrhizobium japonicum proline biosynthesis gene proC is essential for symbiosis; King ND et al.; Plant host-derived proline is proposed to serve as an energy source for rhizobia in the rhizosphere and in symbiotic root nodules . The Bradyrhizobium japonicum proC gene was isolated, and a proC mutant strain that behaved as a strict proline auxotroph in culture was constructed . The proC strain elicited undeveloped nodules on soybeans that lacked nitrogen fixation activity and plant hemoglobin . We conclude that the proC gene is essential for symbiosis and suggest that the mutant does not obtain an exogenous supply of proline in association with soybeans sufficient to satisfy its auxotrophy. Appl Environ Microbiol, 2000 Dec, 66(12), 5437 - 47 Photosynthetic bradyrhizobia are natural endophytes of the African wild rice Oryza breviligulata; Chaintreuil C et al.; We investigated the presence of endophytic rhizobia within the roots of the wetland wild rice Oryza breviligulata, which is the ancestor of the African cultivated rice Oryza glaberrima . This primitive rice species grows in the same wetland sites as Aeschynomene sensitiva, an aquatic stem-nodulated legume associated with photosynthetic strains of Bradyrhizobium . Twenty endophytic and aquatic isolates were obtained at three different sites in West Africa (Senegal and Guinea) from nodal roots of O . breviligulata and surrounding water by using A . sensitiva as a trap legume . Most endophytic and aquatic isolates were photosynthetic and belonged to the same phylogenetic Bradyrhizobium/Blastobacter subgroup as the typical photosynthetic Bradyrhizobium strains previously isolated from Aeschynomene stem nodules . Nitrogen-fixing activity, measured by acetylene reduction, was detected in rice plants inoculated with endophytic isolates . A 20% increase in the shoot growth and grain yield of O . breviligulata grown in a greenhouse was also observed upon inoculation with one endophytic strain and one Aeschynomene photosynthetic strain . The photosynthetic Bradyrhizobium sp . strain ORS278 extensively colonized the root surface, followed by intercellular, and rarely intracellular, bacterial invasion of the rice roots, which was determined with a lacZ-tagged mutant of ORS278 . The discovery that photosynthetic Bradyrhizobium strains, which are usually known to induce nitrogen-fixing nodules on stems of the legume Aeschynomene, are also natural true endophytes of the primitive rice O . breviligulata could significantly enhance cultivated rice production. J Bacteriol, 2000 Dec, 182(24), 7088 - 91 Characterization of a major cluster of nif, fix, and associated genes in a sugarcane endophyte, Acetobacter diazotrophicus; Lee S et al.; A major 30.5-kb cluster of nif and associated genes of Acetobacter diazotrophicus (syn . Gluconacetobacter diazotrophicus), a nitrogen-fixing endophyte of sugarcane, was sequenced and analyzed . This cluster represents the largest assembly of contiguous nif-fix and associated genes so far characterized in any diazotrophic bacterial species . Northern blots and promoter sequence analysis indicated that the genes are organized into eight transcriptional units . The overall arrangement of genes is most like that of the nif-fix cluster in Azospirillum brasilense, while the individual gene products are more similar to those in species of Rhizobiaceae or in Rhodobacter capsulatus. J Mol Biol, 2000 Dec 1, 304(3), 447 - 60 Rapid determination of protein folds using residual dipolar couplings; Fowler CA et al.; Over the next few years, various genome projects will sequence many new genes and yield many new gene products . Many of these products will have no known function and little, if any, sequence homology to existing proteins . There is reason to believe that a rapid determination of a protein fold, even at low resolution, can aid in the identification of function and expedite the determination of structure at higher resolution . Recently devised NMR methods of measuring residual dipolar couplings provide one route to the determination of a fold . They do this by allowing the alignment of previously identified secondary structural elements with respect to each other . When combined with constraints involving loops connecting elements or other short-range experimental distance information, a fold is produced . We illustrate this approach to protein fold determination on (15)N-labeled Eschericia coli acyl carrier protein using a limited set of (15)N-(1)H and (1)H-(1)H dipolar couplings . We also illustrate an approach using a more extended set of heteronuclear couplings on a related protein, (13)C, (15)N-labeled NodF protein from Rhizobium leguminosarum . Plant Mol Biol, 2000 Aug, 43(5-6), 773 - 86 Cell cycle regulation in the course of nodule organogenesis in Medicago; Foucher F et al.; The molecular mechanisms of de novo meristem formation, cell differentiation and the integration of the cell cycle machinery into appropriate stages of the developmental programmes are still largely unknown in plants . Legume root nodules, which house nitrogen-fixing rhizobia, are unique plant organs and their development may serve as a model for organogenetic processes in plants . Nodules form and are essential for the plant only under limitation of combined nitrogen in the soil . Moreover, their development is triggered by external mitogenic signals produced by their symbiotic partners, the rhizobia . These signals, the lipochitooligosaccharide Nod factors, act as host-specific morphogens and induce the re-entry of root cortical cells into mitotic cycles . Maintenance of cell division activity leads to the formation of a persistent nodule meristem from which cells exit continuously and enter the nodule differentiation programme, involving multiple cycles of endoreduplication and enlargement of nuclear and cell volumes . While the small diploid 2C cells remain uninfected, the large polyploid cells can be invaded and, after completing the differentiation programme, host the nitrogen-fixing bacteroids . This review summarizes the present knowledge on cell cycle reactivation and meristem formation in response to Nod factors and reports on a novel plant cell cycle regulator that can switch mitotic cycles to differentiation programmes. J Biol Inorg Chem, 2000 Oct, 5(5), 642 - 54 Nitrosyl adducts of FixL as probes of heme environment; Rodgers KR et al.; This report presents a spectroscopic investigation of the nitrosyl adducts of FixL, the sensor in the signal transduction system responsible for regulating nitrogen fixation in Rhizobium meliloti . Variable-temperature resonance Raman (RR), electron spin resonance (ESR), and variable-temperature UV-visible absorption data are presented for the ferrous NO adducts of two FixL deletion derivatives, FixLN (the heme-containing domain) and FixL* (a functional heme-kinase) . A temperature-dependent equilibrium is observed between the five-coordinate (5-c) and six-coordinate (6-c) ferrous nitrosyl adducts, with lower temperatures favoring formation of the 6-c nitrosyl adduct . This equilibrium is perturbed as the solution freezes, and the amount of 5-c FixL-NO increases sharply until a nearly constant ratio of 6-c to 5-c adducts is obtained . Complexation between the heme domain of FixL and its response regulator, FixJ, is revealed through specfic FixJ-induced increase in the energy separation between 5-c and 6-c FixL-NO . Ferric nitrosyl adducts of FixL* and FixLN autoreduce to their corresponding ferrous nitrosyl adducts . The kinetic behavior of this reduction is monophasic for FixL*-NO, while the reaction for ferric FixLN-NO is biphasic . These results suggest conformational inhomogeneity in the heme pocket of FixLN and conformational homogeneity in that of FixL* . Hence the kinase domain plays a role in distal pocket conformational stability . Implications for the signal transduction mechanism are discussed. Curr Microbiol, 2000 Dec, 41(6), 402 - 9 Crossing the limits of Rhizobium existence in extreme conditions; Kulkarni S et al.; An ecological survey was conducted to characterize 5000 Rhizobium sp . sesbania strains of diverse geographical origin, isolated from the root nodules of Sesbania aculeata growing in neutral (pH 7) and alkaline (pH 8.5 and above) soils . The rhizobia from the alkaline soil showed significantly higher salt tolerance than those isolated from neutral soil . Upper limits of stress survival of rhizobial isolates, Rhizobium sp . NBRI0102 sesbania selected from neutral soil, and Rhizobium sp . NBRI2505 sesbania selected from alkaline soil, were studied under free living conditions . Rhizobium sp . NBRI0102 sesbania and Rhizobium sp . NBRI2505 sesbania tolerated yeast extract mannitol broth (YEB) containing 10% and 28% salt (NaCl, wt/vol) for up to 18 h of incubation at 30 degrees C . Growth of Rhizobium sp . NBRI0102 sesbania and Rhizobium sp . NBRI2505 sesbania at pH 7, 11, and 12 was identical, except for a lag period of about 10 h in the growth of Rhizobium sp . NBRI0102 sesbania at pH 11 and 12, as compared with pH 7 . Rhizobium sp . NBRI0102 sesbania and Rhizobium sp . NBRI2505 sesbania survived at 50 degrees C and 65 degrees C, in YEB at pH 7 for up to 4 and 2 h, respectively . To our knowledge, this is the first report of rhizobia demonstrating survival of Rhizobium sp . NBRI2505 sesbania, estimated by counting viable cells, to such extreme conditions of salt and temperature, individually . In contrast to Rhizobium sp . NBRI0102 sesbania, high temperature was tolerated efficiently by Rhizobium sp . NBRI2505 sesbania, in the presence of salt at higher pH . Our results suggest that the possession of the trait of high salt tolerance might be of some evolutionary significance for the survival of rhizobia in alkaline soils, at high pH and temperature. Plant Physiol, 2000 Nov, 124(3), 1039 - 48 Localization of a Nod factor-binding protein in legume roots and factors influencing its distribution and expression; Kalsi G et al.; The roots of the legume Dolichos biflorus contain a lectin/nucleotide phosphohydrolase (Db-LNP) that binds to the Nod factor signals produced by rhizobia that nodulate this plant . In this study we show that Db-LNP is differentially distributed along the surface of the root axis in a pattern that correlates with the zone of nodulation of the root . Db-LNP is present on the surface of young and emerging root hairs and redistributes to the tips of the root hairs in response to treatment of the roots with a rhizobial symbiont or with a carbohydrate ligand . This redistribution does not occur in response to a non-symbiotic rhizobial strain or a root pathogen . Db-LNP is also present in the root pericycle where its level decreases upon initiation of nodule formation . Maximum levels of Db-LNP are found in 2-d-old roots, and the expression of this root protein is increased when the plants are grown in the absence of NO(3)(-) and NH(4)(+) . These results support the possibility that Db-LNP is involved in the initiation of the Rhizobium legume symbiosis. Proc Natl Acad Sci U S A, 2000 Nov 21, 97(24), 13413 - 8 Dissection of nodulation signaling using pea mutants defective for calcium spiking induced by nod factors and chitin oligomers; Walker SA et al.; Changes in intracellular calcium in pea root hairs responding to Rhizobium leguminosarum bv . viciae nodulation (Nod) factors were analyzed by using a microinjected calcium-sensitive fluorescent dye (dextran-linked Oregon Green) . Within 1-2 min after Nod-factor addition, there was usually an increase in fluorescence, followed about 10 min later by spikes in fluorescence occurring at a rate of about one spike per minute . These spikes, corresponding to an increase in calcium of approximately 200 nM, were localized around the nuclear region, and they were similar in terms of lag and period to those induced by Nod factors in alfalfa . Calcium responses were analyzed in nonnodulating pea mutants, representing seven loci that affect early stages of the symbiosis . Mutations affecting three loci (sym8, sym10, and sym19) abolished Nod-factor-induced calcium spiking, whereas a normal response was seen in peas carrying alleles of sym2(A), sym7, sym9, and sym30 . Chitin oligomers of four or five N-acetylglucosamine residues could also induce calcium spiking, although the response was qualitatively different from that induced by Nod factors; a rapid increase in intracellular calcium was not observed, the period between spikes was lower, and the response was not as sustained . The chitin-oligomer-induced calcium spiking did not occur in nodulation mutants (sym8, sym10, and sym19) that were defective for Nod-factor-induced spiking, suggesting that this response is related to nodulation signaling . From our data and previous observations on the lack of mycorrhizal infection in some of the sym mutants, we propose a model for the potential order of pea nodulation genes in nodulation and mycorrhizal signaling. Biochemistry, 2000 Nov 14, 39(45), 13810 - 6 Roles of Ile209 and Ile210 on the heme pocket structure and regulation of histidine kinase activity of oxygen sensor FixL from Rhizobium meliloti; Mukai M et al.; FixL is a sensor histidine kinase having a heme-containing domain as an O(2) sensing site . In the study presented here, Ile209 and Ile210 located near the heme iron of the heme domain of Rhizobium meliloti FixL (RmFixL) were mutated, and the mutational effects on the regulation of the kinase activity and the heme pocket structure were examined by the autophosphorylation assay and UV-visible absorption and resonance Raman (RR) spectroscopies . The mutation of these residues disrupted the regulation of the kinase activity by the sensor (heme) domain, indicating that Ile209 and Ile210 play important roles in the signal transduction between the heme and the kinase domains . By measurement of the resonance Raman and optical absorption spectra of Ile209 and Ile210 mutants in several oxidation, spin, and ligation states, it was found that both residues are highly flexible, and their side chains sterically interact with the O(2) ligand, when it binds to the heme iron . On the basis of the results, we propose an O(2) sensing mechanism of RmFixL; the kinase activity is regulated via conformational changes of Ile209 and Ile210 induced by the O(2) binding to the sensory center. Microbiology, 2000 Nov, 146 ( Pt 11), 2997 - 3005 Phylogenetic diversity of rhizobial strains nodulating Robinia pseudoacacia L; Ulrich A et al.; Lack of knowledge exists regarding the diversity of rhizobial strains nodulating black locust (Robinia pseudoacacia L.), which is a neophytic tree species widely distributed in Europe . Seventeen rhizobial strains isolated from nodules of black locust at a German location were examined by phenotypic characterization and 16S rDNA analysis . The isolates were classified in nine 16S rDNA genotypes using a set of seven endonucleases . Based on RFLP analysis and sequencing, the strains were shown to belong to the genera Mesorhizobium (76%) and Rhizobium (24%) . Five genotypes were identical to the species Mesorhizobium amorphae, Mesorhizobium loti, Mesorhizobium huakuii, Rhizobium leguminosarum and Rhizobium tropici . A strong similarity between the 16S rDNA sequence of another two genotypes and M . amorphae (99.9%) as well as the Mesorhizobium strain R88b (99.8%) was found . The two remaining genotypes were classified in the genus Rhizobium, without a significant relationship at the species level . Comparing isolates nodulating Rob . pseudoacacia and Amorpha fruticosa, a parallel picture of phylogenetic diversity was detected with a range of phylogenetically different rhizobia and M . amorphae dominating . For this study, 18 rhizobial strains which had originally been isolated from a forest in Maryland where black locust is native were additionally analysed . Results revealed seven genotypes all belonging to the genus Mesorhizobium, with four genotypes identical to the isolates from the German sampling location . Whereas the genotype identical to M . amorphae dominated within the strains obtained from the German location, the dominance of a genotype identical to M . huakuii was found among the strains from the native location . Summarizing data from both locations, Rob . pseudoacacia was nodulated with various genomic species, most of which belonged to the genus Mesorhizobium . Concerning phenotypic features such as growth rate, pH tolerance or use of certain carbohydrates, most isolates corresponded to described species and genera . However, there were differences in salt tolerance between these isolates and the corresponding reference strains . Overall, the results demonstrated a high phenotypic and phylogenetic diversity of rhizobial strains nodulating Rob . pseudoacacia . This may be a characteristic of neophytic and other widely spread legumes and may contribute to the success of black locust as a pioneer tree species for the temperate zone. Microbiology, 2000 Nov, 146 ( Pt 11), 2987 - 96 The Rhizobium leguminosarum bv . viciae glnD gene, encoding a uridylyltransferase/uridylyl-removing enzyme, is expressed in the root nodule but is not essential for nitrogen fixation; Schluter A et al.; A Rhizobium leguminosarum bv . viciae VF39 gene (glnD) encoding the uridylyltransferase/uridylyl-removing enzyme, which constitutes the sensory component of the nitrogen regulation (ntr) system, was identified, cloned and characterized . The deduced amino acid sequence contains the conserved active site motif of the nucleotidyltransferase superfamily and is highly homologous to the glnD gene products of other bacterial species . Downstream of the VF39 glnD resides an open reading frame with similarity to the Salmonella typhimurium virulence factor gene mviN . Mutation of the glnD gene abolished the ability to use nitrate as a sole nitrogen source but not glutamine . In addition, neither uridylylation of P(II) nor induction of the ntr-regulated glnII gene (encoding glutamine synthetase II) under ammonium deficiency could be observed in mutant strains . This strongly suggests that glnD mutants harbour a permanently deuridylylated P(II) protein and as a consequence are unable to activate transcription from NtrC-dependent promoters . The glnD gene itself is expressed constitutively, irrespective of the nitrogen content of the medium . A functional GlnD protein is not essential for nitrogen fixation in R . leguminosarum bv . viciae, but in situ detection of glnD expression in the symbiotic and infection zone of the root nodule and quantitative measurements suggest that at least part of the ntr system functions in symbiosis . The results also indicate that the N-terminal part of GlnD is essential for the cell, as deletions in the 5'-region of the gene appear to be lethal and mutations possibly affecting the expression of the first half of the protein have a significant effect on the vitality of the mutant strain. Mol Plant Microbe Interact, 2000 Nov, 13(11), 1271 - 4 The dnaJ (hsp40) locus in Rhizobium leguminosarum bv . phaseoli is required for the establishment of an effective symbiosis with Phaseolus vulgaris; Labidi M et al.; P121R25 is a Tn5-induced mutant of the effective Rhizobium leguminosarum bv . phaseoli strain P121R that is unable to use glutamate as the sole carbon and nitrogen source and is defective in symbiotic nitrogen fixation . Enzymatic analysis showed that three enzymes implicated in glutamate metabolism (glutamate dehydrogenase, 2-oxoglutarate dehydrogenase, and glutamate synthase) were affected by this mutation . Sequencing of the chromosomal locus bordering the Tn5 in P121R25 indicated the presence of the dnaK and dnaJ genes in an arrangement similar to that described in R . leguminosarum bv . viciae (GenBank accession number Y14649) . The mutation was located in the dnaJ (hsp40) gene. Mol Plant Microbe Interact, 2000 Nov, 13(11), 1228 - 36 A guaB mutant strain of Rhizobium tropici CIAT899 pleiotropically defective in thermal tolerance and symbiosis; Riccillo PM et al.; Rhizobium tropici strain CIAT899 displays a high intrinsic thermal tolerance, and had been used in this work to study the molecular basis of bacterial responses to high temperature . We generated a collection of R . tropici CIAT899 mutants affected in thermal tolerance using TnS-luxAB mutagenesis and described the characterization of a mutant strain, CIAT899-10T, that fails to grow under conditions of high temperature . Strain CIAT899-10T carries a single transposon insertion in a gene showing a high degree of similarity with the guaB gene of Escherichia coli and other organisms, encoding the enzyme inosine monophosphate dehydrogenase . The guaB strain CIAT899-10T does not require guanine for growth due to an alternative pathway via xanthine dehydrogenase and, phenotypically, in addition to the thermal sensitivity, the mutant is also defective in symbiosis with beans, forming nodules that lack rhizobial content . Guanine and its precursors restore wild-type tolerance to grow at high temperature . Our data show that, in R . tropici, the production of guanine via inosine monophosphate dehydrogenase is essential for growth at extreme temperatures and for effective nodulation. Mol Plant Microbe Interact, 2000 Nov, 13(11), 1204 - 13 Saprophytic intracellular rhizobia in alfalfa nodules; Timmers AC et al.; In indeterminate alfalfa nodules, the establishment of the senescent zone IV, in which both symbionts undergo simultaneous degeneration, has been considered, until now, as the end point of the symbiotic interaction . However, we now describe an additional zone, zone V, proximal to the senescent zone IV and present in alfalfa nodules more than 6 weeks old . In zone V, a new round of bacterial release occurs from remaining infection threads, leading to the reinvasion of plant cells that have completely senesced . These intracellular rhizobia are rod shaped and do not display the ultrastructural differentiation features of bacteroids observed in the more distal zones of the nodule . Interestingly, we have found that oxygen is available in zone V at a concentration compatible with both bacterial development and nitrogen fixation gene expression in newly released rhizobia . However, this expression is not correlated with acetylene reduction . Moreover, the pattern of nifH expression in this zone, as well as new data relating to expression in zone II, strongly suggest that nifH transcription in the nodule is under the control of a negative regulator in addition to oxygen . Our results support the conclusion that zone V is an ecological niche where intracellular rhizobia take advantage of the interaction for their exclusive benefit and live as parallel saprophytic partners . The demonstration of such an advantage for rhizobia in nodules was the missing evidence that Rhizobium-legume interactions are indeed symbiotic and, in particular, suggests that benefits to the two partners are associated with different developmental stages within the nodule. Mol Plant Microbe Interact, 2000 Nov, 13(11), 1163 - 9 Use of green fluorescent protein color variants expressed on stable broad-host-range vectors to visualize rhizobia interacting with plants; Stuurman N et al.; We developed two sets of broad-host-range vectors that drive expression of the green fluorescent protein (GFP) or color variants thereof (henceforth collectively called autofluorescent proteins {AFPs}) from the lac promoter . These two sets are based on different replicons that are maintained in a stable fashion in Escherichia coli and rhizobia . Using specific filter sets or a dedicated confocal laser scanning microscope setup in which emitted light is split into its color components through a prism, we were able to unambiguously identify bacteria expressing enhanced cyan fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) in mixtures of the two . Clearly, these vectors will be valuable tools for competition, cohabitation, and rescue studies and will also allow the visualization of interactions between genetically marked bacteria in vivo . Here, we used these vectors to visualize the interaction between rhizobia and plants . Specifically, we found that progeny from different rhizobia can be found in the same nodule or even in the same infection thread . We also visualized movements of bacteroids within plant nodule cells. Extremophiles, 2000 Oct, 4(5), 253 - 8 The Na+/H+ antiporter of alkaliphilic Bacillus sp; Kitada M et al.; The Na+/H+ antiporter, which appears to predominantly contribute to the alkaliphily of Bacillus halodurans C-125, was studied in an alkali-sensitive mutant of this strain and a transformant with restored alkaliphily . The alkali-sensitive mutant, strain 38154, which has lost the ability to grow above pH 9.5, was found to lack electrogenic Na+/H+ antiport activity driven by deltapsi (membrane potential, interior negative), and it showed defective regulation of intracellular pH under alkaline conditions . On the other hand, a transformant carrying a 2.0-kb DNA fragment from the parental genome that complemented this defect was able to maintain an intracellular pH lower than that of the external milieu, and it was found to have recovered the Na+/H+ antiport activity driven by deltapsi . Sequence analyses found that a 5.1-kb DNA region contained four open reading frames (ORF-1 to ORF-4) . Direct sequencing of the corresponding region in mutant 38154 revealed a G-to-A substitution, which resulted in an amino acid substitution from Gly-393 to Arg in the putative ORF-1 product . It has been recently found that a region homologous to the DNA fragment responsible for the alkaliphily of strain C-125 exists in the genomes of Bacillus subtilis, Sinorhizobium (Rhizobium) meliloti, and Staphylococcus aureus . These homologues are present as a cluster of seven ORFs in each case . The shaA gene product of B . subtilis shows significant similarity to the ORF-1 product of strain C-125 . Disruption of the shaA gene resulted in a decrease in Na+/H+ antiport activity, and growth of the shaA-disrupted strain was impaired when the external Na+ concentration was increased . We conclude that the shaA gene encodes a Na+/H+ antiporter, which plays an important role in extrusion of cytotoxic Na+. Appl Environ Microbiol, 2000 Nov, 66(11), 5078 - 82 Bradyrhizobium sp . Strains that nodulate the leguminous tree Acacia albida produce fucosylated and partially sulfated nod factors; Ferro M et al.; We determined the structures of Nod factors produced by six different Bradyrhizobium sp . strains nodulating the legume tree Acacia albida (syn . Faidherbia albida) . Compounds from all strains were found to be similar, i.e., O-carbamoylated and substituted by an often sulfated methyl fucose and different from compounds produced by Rhizobium-Mesorhizobium-Sinorhizobium strains nodulating other species of the Acaciae tribe. Appl Environ Microbiol, 2000 Nov, 66(11), 4785 - 9 Genetic diversity and dynamics of Sinorhizobium meliloti populations nodulating different alfalfa cultivars in Italian soils; Carelli M et al.; We analyzed the genetic diversity of 531 Sinorhizobium meliloti strains isolated from nodules of Medicago sativa cultivars in two different Italian soils during 4 years of plant growth . The isolates were analyzed for DNA polymorphism with the random amplified polymorphic DNA method . The populations showed a high level of genetic polymorphism distributed throughout all the isolates, with 440 different haplotypes . Analysis of molecular variance allowed us to relate the genetic structure of the symbiotic population to various factors, including soil type, alfalfa cultivar, individual plants within a cultivar, and time . Some of these factors significantly affected the genetic structure of the population, and their relative influence changed with time . At the beginning of the experiment, the soil of origin and, even more, the cultivar significantly influenced the distribution of genetic variability of S . meliloti . After 3 years, the rhizobium population was altered; it showed a genetic structure based mainly on differences among plants, while the effects of soil and cultivar were not significant. FEMS Microbiol Ecol, 2000 Oct 1, 34(1), 9 - 16 Characterization of halotolerant rhizobia isolated from root nodules of Canavalia rosea from seaside areas; Chen W et al.; Twelve nodule isolates from Canavalia rosea, an indigenous leguminous halophyte growing in the seaside areas of southern Taiwan, were effective symbionts for the original host and able to grow at NaCl concentrations up to 3-3.5% (w/v) . The taxonomy of these isolates was investigated using a polyphasic approach, including phenotypic characteristics, banding patterns of total proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), genomic fingerprint patterns from random amplified polymorphic DNA (RAPD) analysis, pulsed-field gel electrophoresis (PFGE) analysis, amplified 16S rDNA restriction analysis (ARDRA), 16S rRNA gene sequencing, and nifH gene sequencing . Based on the SDS-PAGE, RAPD, PFGE and ARDRA results, the 12 isolates are highly diverse . The 16S rRNA and nifH gene sequences were determined for isolates with distinct ARDRA patterns and compared with other members of the rhizobial species . We propose these isolates should be classified into the genus Sinorhizobium and distinguished from the current species of this genus. Mol Plant Microbe Interact, 2000 Oct, 13(10), 1109 - 20 The Lotus japonicus LjSym4 gene is required for the successful symbiotic infection of root epidermal cells; Bonfante P et al.; The role of the Lotus japonicus LjSym4 gene during the symbiotic interaction with Mesorhizobium loti and arbuscular mycorrhizal (AM) fungi was analyzed with two mutant alleles conferring phenotypes of different strength . Ljsym4-1 and Ljsym4-2 mutants do not form nodules with M . loti . Normal root hair curling and infection threads are not observed, while a nodC-dependent deformation of root hair tips indicates that nodulation factors are still perceived by Ljsym4 mutants . Fungal infection attempts on the mutants generally abort within the epidermis, but Ljsym4-1 mutants allow rare, successful, infection events, leading to delayed arbuscule formation . On roots of mutants homozygous for the Ljsym4-2 allele, arbuscule formation was never observed upon inoculation with either of the two AM fungi, Glomus intraradices or Gigaspora margarita . The strategy of epidermal penetration by G . margarita was identical for Ljsym4-2 mutants and the parental line, with appressoria, hyphae growing between two epidermal cells, penetration of epidermal cells through their anticlinal wall . These observations define a novel, genetically controlled step in AM colonization . Although rhizobia penetrate the tip of root hairs and AM fungi access an entry site near the base of epidermal cells, the LjSym4 gene is necessary for the appropriate response of this cell type to both microsymbionts . We propose that LjSym4 is required for the initiation or coordinated expression of the host plant cell's accommodation program, allowing the passage of both microsymbionts through the epidermis layer. Genetika, 2000 Sep, 36(9), 1173 - 88 {Nodulation as a model for studying differentiation in higher plants}; Pavlova ZB et al.; The stages of the legume-rhizobial symbiosis and nodule structure in various legume plants are briefly reviewed . Modern data on the mechanisms involved in the control of nodule initiation and morphogenesis are considered. Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8901 - 8906 enod40 induces dedifferentiation and division of root cortical cells in legumes; Charon C et al.; Under nitrogen-limiting conditions Rhizobium meliloti can establish symbiosis with Medicago plants to form nitrogen-fixing root nodules . Nodule organogenesis starts with the dedifferentiation and division of root cortical cells . In these cells the early nodulin gene enod40, which encodes an unusually small peptide (12 or 13 amino acids), is induced from the beginning of this process . Herein we show that enod40 expression evokes root nodule initiation . (i) Nitrogen-deprived transgenic Medicago truncatula plants overexpressing enod40 exhibit extensive cortical cell division in their roots in the absence of Rhizobium . (ii) Bombardment of Medicago roots with an enod40-expressing DNA cassette induces dedifferentiation and division of cortical cells and the expression of another early nodulin gene, Msenod12A . Moreover, transient expression of either the enod40 region spanning the oligopeptide sequence or only the downstream region without this sequence induces these responses . Our results suggest that the cell-specific growth response elicited by enod40 is involved in the initiation of root nodule organogenesis. Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 5467 - 5472 Expression of early nodulin genes in alfalfa mycorrhizae indicates that signal transduction pathways used in forming arbuscular mycorrhizae and Rhizobium-induced nodules may be conserved; van Rhijn P et al.; Transcripts for two genes expressed early in alfalfa nodule development (MsENOD40 and MsENOD2) are found in mycorrhizal roots, but not in noncolonized roots or in roots infected with the fungal pathogen Rhizoctonia solani . These same two early nodulin genes are expressed in uninoculated roots upon application of the cytokinin 6-benzylaminopurine . Correlated with the expression of the two early nodulin genes, we found that mycorrhizal roots contain higher levels of trans-zeatin riboside than nonmycorrhizal roots . These data suggest that there may be conservation of signal transduction pathways between the two symbioses-nitrogen-fixing nodules and phosphate-acquiring mycorrhizae. J Plant Growth Regul, 2000 Jun, 19(2), 155 - 166 Molecular Mechanisms in Root Nodule Development; Crespi M et al.; Under nitrogen-limiting conditions, bacteria from the family Rhizobiaceae establish a symbiosis with leguminous plants to form nitrogen-fixing root nodules . These organs require a coordinated control of the spatiotemporal expression of plant and bacterial genes during morphogenesis . Both plant and bacterial signals are involved in this regulation in the plant host . Plant genes induced during nodule development, the so-called nodulin genes, have been extensively characterized . Products of several of these genes show homologies to known regulators of signal transduction pathways in other plant or animal systems . Initial functional analysis of the molecular mechanisms implicated in nodulation have been undertaken using model legumes . Insertion mutagenesis and transgenic technologies to modify nodulin gene expression, as well as pharmacologic approaches, have been used to analyze molecular mechanisms involved in morphologic responses induced by the bacterial symbiont in the plant . G protein-mediated transduction mechanisms have been implicated, and the nin transcription factor appears to be required for early steps in nodule development . ENOD40, a gene coding for an RNA that contains only short ORFs, seems to be closely tied to nodule primordium formation . In addition, a vascular-associated Kruppel-like transcription factor and small Rab type G-proteins affect bacteroid differentiation and the function of the nitrogen-fixing zone . These initial results presage a wealth of information that will be obtained from the application of genomic approaches to legumes. J Plant Growth Regul, 2000 Jun, 19(2), 113 - 130 Fundamental Concepts in Symbiotic Interactions: Light and Dark, Day and Night, Squid and Legume; Hirsch AM et al.; The legume-Rhizobium symbiosis and that between Euprymna scolopes and Vibrio fischeri show some surprising physiological similarities as well as differences . Both interactions rely on exchange of signal molecules, some of which are derived from bacterial cell surface molecules . Although the legume-Rhizobium symbiosis is nutritionally based as are many animal-microbe symbioses, it is not obligate because the plant initiates nodule formation only when the soil is deficient in nitrogen . In contrast, the squid-Vibrio symbiosis is obligate for the squid but is not nutritionally based . Rather, the bacteria produce light, which enables the animal to evade predators . These similarities and differences are described and discussed in term of the overall question of whether or not these two symbiotic relationships have evolved from commensal or pathogenic/parasitic interactions between prokaryotes and eukaryotes. Int J Syst Evol Microbiol, 2000 Sep, 50 Pt 5, 1887 - 91 Sinorhizobium meliloti associated with Medicago sativa and Melilotus spp . in arid saline soils in Xinjiang, China; Yan AM et al.; Of 42 rhizobial isolates from Medicago sativa and Melilotus spp . growing in arid saline fields in Xinjiang, China, 40 were identified as Sinorhizobium meliloti by a polyphasic approach . However, diverse groups were obtained from these isolates in numerical taxonomy and SDS-PAGE of proteins . They could grow at pH 10.5 and were tolerant to 2.5-4.0% (w/v) NaCl. Glycobiology, 2000 Oct, 10(10), 1013 - 23 The type and yield of lipopolysaccharide from symbiotically deficient rhizobium lipopolysaccharide mutants vary depending on the extraction method; Ridley BL et al.; At least 18 lipopolysaccharide (LPS) extraction methods are available, and no single method is universally applicable . Here, the LPSs from four R.etli, one R.leguminosarum bv . trifolii mutant, 24AR, and the R.etli parent strain, CE3, were isolated by hot phenol/water (phi;/W), and phenol/EDTA/triethylamine (phi/EDTA/TEA) extraction . The LPS in various preparations was quantified, analyzed by deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE), and by immunoblotting . These rhizobia normally have two prominent LPS forms: LPS I, which has O-polysaccharide, and LPS II, which has none . The LPS forms obtained depend on the method of extraction and vary depending on the mutant that is extracted . Both methods extract LPS I and LPS II from CE3 . The phi/EDTA/TEA, but not the phi/W, method extracts LPS I from mutants CE358 and CE359 . Conversely, the phi;/W but not the phi;/EDTA/TEA method extracts CE359 LPS V, an LPS form with a truncated O-polysaccharide . phi/EDTA/TEA extraction of mutant CE406 gives good yields of LPS I and II, while phi/W extraction gives very small amounts of LPS I . The LPS yield from all the strains using phi/EDTA/TEA extraction is fairly consistent (3-fold range), while the yields from phi/W extraction are highly variable (850-fold range) . The phi/EDTA/TEA method extracts LPS I and LPS II from mutant 24AR, but the phi/W method partitions LPS II exclusively into the phenol phase, making its recovery difficult . Overall, phi/EDTA/TEA extraction yields more forms of LPS from the mutants and provides a simpler, faster, and less hazardous alternative to phi/W extraction . Nevertheless, it is concluded that careful analysis of any LPS mutant requires the use of more than one extraction method. Plant Physiol, 2000 Oct, 124(2), 781 - 94 Production of the isoflavones genistein and daidzein in non-legume dicot and monocot tissues; Yu O et al.; Metabolic engineering for production of isoflavones in non-legume plants may provide the health benefits of these phytoestrogens from consumption of more widely used grains . In legumes, isoflavones function in both the symbiotic relationship with rhizobial bacteria and the plant defense response . Expression of a soybean isoflavone synthase (IFS) gene in Arabidopsis plants was previously shown to result in the synthesis and accumulation of the isoflavone genistein in leaf and stem tissue (Jung et al., 2000) . Here we further investigate the ability of the heterologous IFS enzyme to interact with the endogenous phenylpropanoid pathway, which provides the substrate for IFS, and produces genistein in several plant tissue systems . In tobacco (Nicotiana tabacum) floral tissue that synthesizes anthocyanins, genistein production was increased relative to leaves . Induction of the flavonoid/anthocyanin branch of the phenylpropanoid pathway through UV-B treatment also enhanced genistein production in Arabidopsis . In a monocot cell system, introduced expression of a transcription factor regulating genes of the anthocyanin pathway was effective in conferring the ability to produce genistein in the presence of the IFS gene . Introduction of a third gene, chalcone reductase, provided the ability to synthesize an additional substrate of IFS resulting in production of the isoflavone daidzein in this system . The genistein produced in tobacco, Arabidopsis, and maize (Zea mays) cells was present in conjugated forms, indicating that endogenous enzymes were capable of recognizing genistein as a substrate . This study provides insight into requirements for metabolic engineering for isoflavone production in non-legume dicot and monocot tissues. Plant Physiol, 2000 Oct, 124(2), 733 - 40 Nod factors and chitooligomers elicit an increase in cytosolic calcium in aequorin-expressing soybean cells; Muller J et al.; Rhizobial Nod factors (NFs) function as nodulation signals that trigger symbiotic responses of leguminous host plants . NFs consist of a chitin oligomer backbone carrying a fatty acid at the non-reducing end . Depending on the rhizobial strain, NFs carry additional substituents, which may determine host specificity . Transgenic suspension-cultured soybean (Glycine max {L.} Merr.) cells expressing aequorin have been used to record cytosolic {Ca(2+)} changes upon treatment with purified NFs and chitin fragments . Both compounds elicited an increase of cytosolic {Ca(2+)} at nanomolar concentrations . The shape and amplitude of cytosolic {Ca(2+)} changes was similar to the response elicited by un-derivatized chitin oligomers . Cells challenged first with NFs did not respond to a subsequent treatment with chitin oligomers and vice versa . Dose-response experiments showed that un-derivatized chitin oligomers were more active compared with NFs . The capacity of NFs to elicit the calcium response depended on their structure . The presence of reducing end substituents in methylfucosylated NFs from Rhizobium sp . NGR234 and the O-acetyl group at the non-reducing end in NFs from Sinorhizobium meliloti attenuated the activity to cause the calcium changes . The sulfate group in NFs from Rhizobium tropici did not affect the elicitor activity . Pentameric S . meliloti NFs were more active than tetrameric molecules, whereas trimeric or dimeric degradation products were inactive . Substituents in NFs may have the function to avoid stimulation of defense reactions mediated by the perception system for chitin oligomers. J Appl Microbiol, 2000 Sep, 89(3), 463 - 71 Genotypic and phenotypic differentiation of an antifungal biocontrol strain belonging to Bacillus subtilis; Marten P et al.; Physiological and molecular fingerprints of biotechnologically relevant rhizobacteria are necessary for registration, patenting, recognition and quality checking of the strains . To characterize the biological control agent, Bacillus subtilis B2g, the strain was compared with other plant-associated B . subtilis isolates . Phenotypic characterization included biochemical and nutritional properties, in vitro activity and analysis of potential antagonistic mechanisms towards several plant pathogenic fungi . According to the phenotypic characteristics, it was not possible to differentiate the biocontrol agent from the other strains, although the enzymatic fingerprint was unique . Genotypic diversity among the isolates was characterized by molecular fingerprinting methods using REP-PCR (repetitive extragenomic palindromic PCR), and macrorestriction of genomic DNA and electrophoretic separation of DNA fragments by pulsed-field gel electrophoresis (PFGE) . A protocol for PFGE analysis using restriction enzyme SfiI for B . subtilis was developed . PFGE typing of B . subtilis B2g resulted in a unique fingerprint . Therefore, it was possible to differentiate B . subtilis B2g, the biocontrol agent of Phytovit, from other antifungal B . subtilis isolates. Annu Rev Microbiol, 2000, 54, 257 - 88 Root nodulation and infection factors produced by rhizobial bacteria; Spaink HP; Rhizobia are soil bacteria that can engage in a symbiosis with leguminous plants that produces nitrogen-fixing root nodules . This symbiosis is based on specific recognition of signal molecules, which are produced by both the bacterial and plant partners . In this review, recognition factors from the bacterial endosymbionts are discussed, with particular attention to secreted and cell surface glycans . Glycans that are discussed include the Nod factors, the extracellular polysaccharides, the lipopolysaccharides, the K-antigens, and the cyclic glucans . Recent advances in the understanding of the biosynthesis, secretion, and regulation of production of these glycans are reviewed, and their functions are compared with glycans produced by other bacteria, such as plant pathogens. Mol Gen Genet, 2000 Sep, 264(1-2), 75 - 81 Functional analysis of chimeras derived from the Sinorhizobium meliloti and Mesorhizobium loti nodC genes identifies regions controlling chitin oligosaccharide chain length; Kamst E et al.; The rhizobial nodulation gene nodC encodes an N-acetylglucosaminyltransferase that is responsible for the synthesis of chitin oligosaccharides . These oligosaccharides are precursors for the synthesis of the lipo-chitin oligosaccharides that induce cell division and differentiation during the development of nitrogen-fixing root nodules in leguminous plants . The NodC proteins of Mesorhizobium loti and Sinorizobium meliloti yield chitinpentaose and chitintetraose as their main products, respectively . In order to localize regions in these enzymes that are responsible for this difference in product chain length, a set of six chimeric enzymes, comprising different combinations of regions of the NodC proteins from these two bacteria, was expressed in Escherichia coli . The oligosaccharides produced were analyzed using thin-layer chromatography . The major conclusion from this work is that a central 91-amino acid segment does not play any obvious role in determining the difference in the chain length of the major product . Furthermore, the characteristically predominant synthesis of chitintetraose by S . meliloti NodC is mainly dependent on a C-terminal region of maximally 164 amino acids; exchange of only this C-terminal region is sufficient to completely convert the M . loti chitinpentaose synthase into an S . meliloti-like chitintetraose synthase . The N-terminal region of 170 amino acids also plays a role in restricting the length of the major product to a tetramer . However, the role of the C-terminal region is clearly dominant, since exchanging the N-terminal region has no effect on the relative amounts of chitintetraose and -pentaose produced when the C-terminal region of S . meliloti NodC is present . The length of a predicted beta-strand around residue 300 in the C-terminal region of various NodC proteins is the only structural element that seems to be related to the length of the chitin oligosaccharides produced by these enzymes; the higher the amount of chitintetraose relative to chitinpentaose, the shorter the predicted beta-strand . This element may therefore be important for the effect of the C-terminal 164 amino acids on chitin oligosaccharide chain length. Antonie Van Leeuwenhoek, 2000 Jul, 78(1), 63 - 71 Symbiosis of Astragalus cicer with its microsymbionts: partial nodC gene sequence, host plant specificity, and root nodule structure; Wdowiak S et al.; Astragalus cicer (cicer milkvetch) nodule bacteria were investigated for host plant specificity and partial nodC gene sequences, whilst their native host was studied for the microscopic structure of root nodules . The strains under investigation formed nodules not only on the original host but also on Astragalus glycyphyllos, Astragalus sinicus, Lotus corniculatus, and Phaseolus vulgaris . The nodules induced on the cicer milkvetch were classified as indeterminate and characterized by apical, persistent meristem, a large bacteroid region with infected and uninfected cells, and elongated bacteroids singly located inside peribacteroid membranes . By comparison of the partial nodC gene sequences of a representative strain of astragali rhizobia to those contained in the GenBank database, a close symbiotic relationship of A . cicer microsymbionts to Rhizobium sp . (Oxytropis) was found. Appl Environ Microbiol, 2000 Oct, 66(10), 4292 - 9 Generation of new hydrogen-recycling Rhizobiaceae strains by introduction of a novel hup minitransposon; Bascones E et al.; Hydrogen evolution by nitrogenase is a source of inefficiency for the nitrogen fixation process by the Rhizobium-legume symbiosis . To develop a strategy to generate rhizobial strains with H(2)-recycling ability, we have constructed a Tn5 derivative minitransposon (TnHB100) that contains the ca . 18-kb H(2) uptake (hup) gene cluster from Rhizobium leguminosarum bv . viciae UPM791 . Bacteroids from TnHB100-containing strains of R . leguminosarum bv . viciae PRE, Bradyrhizobium japonicum, R . etli, and Mesorhizobium loti expressed high levels of hydrogenase activity that resulted in full recycling of the hydrogen evolved by nitrogenase in nodules . Efficient processing of the hydrogenase large subunit (HupL) in these strains was shown by immunoblot analysis of bacteroid extracts . In contrast, Sinorhizobium meliloti, M . ciceri, and R . leguminosarum bv . viciae UML2 strains showed poor expression of the hup system that resulted in H(2)-evolving nodules . For the latter group of strains, no immunoreactive material was detected in bacteroid extracts using anti-HupL antiserum, suggesting a low level of transcription of hup genes or HupL instability . A general procedure for the characterization of the minitransposon insertion site and removal of antibiotic resistance gene included in TnHB100 has been developed and used to generate engineered strains suitable for field release. Plant Sci, 2000 Oct 16, 159(1), 57 - 63 Growth promoting effect of two Sinorhizobium meliloti strains (a wild type and its genetically modified derivative) on a non-legume plant species in specific interaction with two arbuscular mycorrhizal fungi; Galleguillos C et al.; In the present study, we have investigated whether the ubiquitous rhizosphere soil organism Sinorhizobium meliloti has a plant growth promoting (PGP) effect on non-leguminous plant species . Such PGP activity was investigated for both a wild type strain and its genetically modified (GM) derivative, which had an enhanced biofertilizer capability . The PGP effect of these rhizobial strains was tested in interaction with two arbuscular-mycorrhizal (AM) fungi: G . mosseae or G . intraradices on lettuce (Lactuca sativa L.) plants . Both rhizobial strains were efficient in increasing lettuce biomass and also induced modifications on root morphology, particularly in mycorrhizal plants; thus these strains behave as plant growth promoting rhizobacteria . In non-mycorrhizal plants, both strains exhibited a similar growth promoting effect on lettuce . However, both rhizobial strains differed in mycorrhizal plants with regard to (i) biomass production, (ii) the length of axis and lateral roots, and (iii) the number of lateral roots formed; effects which were, in turn, affected by the AM fungus involved . Microbial treatments were more effective on root growth and morphology at earlier developmental stages (20 days of plant growth) but, in a later stage (after 40 days), the microbial effects were more relevant at increasing plant biomass . The interaction between the GM rhizobial strain and G . mosseae produced the highest growth promoting effect (476% over control), in spite of the fact that G . intraradices showed a quicker and higher colonization ability than G . mosseae . Microbial interactions inducing PGP effects did not benefit AM colonization nor the succinate dehydrogenase activity in the AM fungal mycelium . Irrespective of the underlying mechanisms, which are being now investigated, the interactions between rhizobial strains, as free-living saprophs, and AM fungi are noteworthy, and depend on the microbial genotype involved. Plant Cell, 2000 Sep, 12(9), 1647 - 66 Four genes of Medicago truncatula controlling components of a nod factor transduction pathway; Catoira R et al.; Rhizobium nodulation (Nod) factors are lipo-chitooligosaccharides that act as symbiotic signals, eliciting several key developmental responses in the roots of legume hosts . Using nodulation-defective mutants of Medicago truncatula, we have started to dissect the genetic control of Nod factor transduction . Mutants in four genes (DMI1, DMI2, DMI3, and NSP) were pleiotropically affected in Nod factor responses, indicating that these genes are required for a Nod factor-activated signal transduction pathway that leads to symbiotic responses such as root hair deformations, expressions of nodulin genes, and cortical cell divisions . Mutant analysis also provides evidence that Nod factors have a dual effect on the growth of root hair: inhibition of endogenous (plant) tip growth, and elicitation of a novel tip growth dependent on (bacterial) Nod factors . dmi1, dmi2, and dmi3 mutants are also unable to establish a symbiotic association with endomycorrhizal fungi, indicating that there are at least three common steps to nodulation and endomycorrhization in M . truncatula and providing further evidence for a common signaling pathway between nodulation and mycorrhization. Biochim Biophys Acta, 2000 Jun 15, 1479(1-2), 203 - 13 Kinetic and hydrodynamic studies of the NodL O-acetyl transferase of Rhizobium leguminosarum: a random-order ternary complex mechanism for acetyl transfer by a roughly spherical trimeric protein; Hindson VJ et al.; The nodL gene product of Rhizobium leguminosarum is required for O-acetylation of diffusible lipo-oligosaccharide signalling factors which are involved in the host-specific nodulation of legume roots . Kinetic studies of the forward reaction, using the substrate analogues chitosan pentaose and chitosan tetraose and the acyl donors acetyl-CoA and propionyl-CoA, and the dead-end inhibitor EtCoA are consistent with a steady-state random-order ternary complex mechanism in which the off rate of the O-acetyl chitosan oligomer appears to be partially rate-determining . Moreover, the linearity of primary double-reciprocal plots favours the view that the interconversion of the ternary complex of NodL and its substrates with that of enzyme and bound products is not significantly faster than k(cat) . Dissociation constants for coenzyme A and acetyl-CoA were determined by titration microcalorimetry to be 16.5 and 7.2 microM respectively, the latter in agreement with the kinetically derived value of 7.0 microM . The physical state of purified NodL, as determined by equilibrium centrifugation, velocity sedimentation and quasi-elastic light scattering, is that of a roughly spherical, trimeric protein with little tendency to self-associate. Proc Natl Acad Sci U S A, 2000 Sep 26, 97(20), 11114 - 9 Symbiosis-specific expression of Rhizobium etli casA encoding a secreted calmodulin-related protein; Xi C et al.; Symbiosis between Rhizobium and its leguminous host requires elaborate communication between the partners throughout the interaction process . A calmodulin-like protein, termed calsymin, was identified in Rhizobium etli; a calmodulin-related protein in a Gram-negative bacterium had not been described previously . Calsymin possesses three repeated homologous domains . Each domain contains two predicted EF-hand Ca(2+)-binding motifs . Ca(2+)-binding activity of calsymin was demonstrated on purified protein . R . etli efficiently secretes calsymin without N-terminal cleavage of the protein . The gene encoding calsymin, casA, is exclusively expressed during colonization and infection