Anal Bioanal Chem, 2002 Feb, 372(3), 431 - 5 Epub 2001 Dec 20. Trace cobalt speciation in bacteria and at enzymic active sites using emission Mössbauer spectroscopy; Kamnev AA et al.; 57Co emission Mossbauer spectroscopy (EMS) allows the chemical state of cobalt, as influenced by its coordination environment, to be monitored in biological samples at its physiological (trace) concentrations . To draw attention to EMS as a valuable tool for speciation of cobalt in biocomplexes, the process of cobalt(II) metabolism in cells of the plant growth-promoting rhizobacterium Azospirillum brasilense Sp245 was investigated using EMS of 57CoII-doped bacterial cells . EMS measurements also showed 57CoII-activated glutamine synthetase (GS, a key enzyme of nitrogen metabolism, isolated from this bacterium) to have two different cobalt(II) forms at its active sites, in agreement with data available on other bacterial GSs . Chemical after-effects following electron capture by the nucleus of the parent 57CoII during the 57Co-->57Fe transition, which contribute to the formation of a stabilised daughter 57FeIII component along with the nucleogenic 57FeII forms, are also briefly considered. Mol Microbiol, 2002 Feb, 43(4), 981 - 91 ActP controls copper homeostasis in Rhizobium leguminosarum bv . viciae and Sinorhizobium meliloti preventing low pH-induced copper toxicity; Reeve WG et al.; Two 'calcium-irreparable' acid-sensitive mutants were identified after mutagenizing Rhizobium leguminosarum bv . viciae and Sinorhizobium meliloti with Tn5 . Each mutant contains a single copy of the transposon which, inserted within the actP gene, prevents expression of a P-type ATPase that belongs to the CPx heavy metal-transporting subfamily . Here, we show that both actP-knockout mutants show sensitivity to copper; omission of this heavy metal from low pH-buffered media restores acid tolerance to these strains . Furthermore, complementation of the mutant phenotype requires only the actPgene . An actP-gusA fusion in R . leguminosarum was transcriptionally regulated by copper in a pH-dependent manner.Downstream to actP in both organisms is the hmrR gene that encodes a heavy metal-responsive regulator (HmrR) that belongs to the merR class of regulatory genes . Insertional Inactivation of hmrR abolished transcriptional activation of actP by copper ions and increased the basal level of its expression in their absence . These observations suggest that HmrR can regulate actP transcription positively and negatively.We show that copper homeostasis is an essential mechanism for the acid tolerance of these root nodule bacteria since it prevents this heavy metal from becoming overtly toxic in acidic conditions. Proc R Soc Lond B Biol Sci, 2002 Apr 7, 269(1492), 685 - 94 Sanctions and mutualism stability: why do rhizobia fix nitrogen? West SA, Kiers ET, Simms EL, Denison RF. Why do rhizobia expend resources on fixing N(2) for the benefit of their host plant, when they could use those resources for their own reproduction? We present a series of theoretical models which counter the hypotheses that N(2) fixation is favoured because it (i) increases the exudation of useful resources to related rhizobia in the nearby soil, or (ii) increases plant growth and therefore the resources available for rhizobia growth . Instead, we suggest that appreciable levels of N(2) fixation are only favoured when plants preferentially supply more resources to (or are less likely to senesce) nodules that are fixing more N(2) (termed plant sanctions) . The implications for different agricultural practices and mutualism stability in general are discussed. J Mol Microbiol Biotechnol, 2002 May, 4(3), 187 - 90 Regulation of succinoglycan and galactoglucan biosynthesis in Sinorhizobium meliloti; Becker A et al.; Sinorhizobium meliloti (Rhizobium meliloti) 2011 has the ability to produce the two acidic exopolysaccharides succinoglycan (EPS I) and galactoglucan (EPS II) . EPS I is a branched heteropolysaccharide composed of octasaccharide repeating units, whereas EPS II is a linear heteropolysaccharide consisting of disaccharide subunits . The exo-exs and exp gene clusters are involved in the biosynthesis of EPSI and EPSII, respectively . EPSI and EPSII biosynthesis genes are differentially expressed resulting in a complex regulation of EPS production in S . meliloti . The phosphate concentration was identified as an important factor affecting the expression of exp genes. Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 457 - 62 Identification of isolates from soybean nodules in Xinjiang Region as Sinorhizobium xinjiangense and genetic differentiation of S . xinjiangense from Sinorhizobium fredii; Peng GX et al.; Eight fast-growing rhizobial isolates from Xinjiang soils were identified as Sinorhizobium xinjiangense by analyses of 16S rRNA gene sequences, SDS-PAGE of proteins, intergenic spacer sequences and DNA-DNA hybridization . Based on all of the results, these isolates and the reference strains for S . xinjiangense were a distinct genomic species, although the 16S rRNA genes were closely related to that of Sinorhizobium fredii. Proteomics, 2002 Mar, 2(3), 325 - 37 Characterisation by proteomics of peribacteroid space and peribacteroid membrane preparations from pea (Pisum sativum) symbiosomes; Saalbach G et al.; The legume Rhizobium symbiosis leads to the formation of a new compartment in the plant cell, the symbiosome . This compartment harbours the bacteroids surrounded by a peribacteroid membrane (PBM) originating from the plant plasma membrane . The PBM and the space between the PBM and the bacteroid membrane, called peribacteroid space (PS), mediate the exchange of metabolites between the symbionts . Proteome analysis was used as an approach to characterise the proteins in the PBM and the PS . A standard differential centrifugation procedure including a Percoll gradient was used for symbiosome isolation from pea root nodules . Proteins in the PBM and PS fractions obtained from the symbiosomes were separated by two-dimensional gel electrophoresis, and 89 spots were analysed by tandem mass spectrometry . The proteins of 46 spots could be identified by database search . The results showed that PS and even PBM preparations from pea symbiosomes always contain abundant amounts of bacteroid proteins as a contaminate . Interestingly, in addition to a few PS/PBM proteins a number of endomembrane proteins (less likely representing a contaminate), including V-ATPase, BIP, and an integral membrane protein known from COPI-coated vesicles, were found in the PBM fraction, supporting the role of the endomembrane system in PBM biogenesis. Appl Environ Microbiol, 2002 Apr, 68(4), 1715 - 27 Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli; Vemuri GN et al.; Escherichia coli NZN111, which lacks activities for pyruvate-formate lyase and lactate dehydrogenase, and AFP111, a derivative which contains an additional mutation in ptsG (a gene encoding an enzyme of the glucose phophotransferase system), accumulate significant levels of succinic acid (succinate) under anaerobic conditions . Plasmid pTrc99A-pyc, which expresses the Rhizobium etli pyruvate carboxylase enzyme, was introduced into both strains . We compared growth, substrate consumption, product formation, and activities of seven key enzymes (acetate kinase, fumarate reductase, glucokinase, isocitrate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase) from glucose for NZN111, NZN111/pTrc99A-pyc, AFP111, and AFP111/pTrc99A-pyc under both exclusively anaerobic and dual-phase conditions (an aerobic growth phase followed by an anaerobic production phase) . The highest succinate mass yield was attained with AFP111/pTrc99A-pyc under dual-phase conditions with low pyruvate carboxylase activity . Dual-phase conditions led to significant isocitrate lyase activity in both NZN111 and AFP111, while under exclusively anaerobic conditions, an absence of isocitrate lyase activity resulted in significant pyruvate accumulation . Enzyme assays indicated that under dual-phase conditions, carbon flows not only through the reductive arm of the tricarboxylic acid cycle for succinate generation but also through the glyoxylate shunt and thus provides the cells with metabolic flexibility in the formation of succinate . Significant glucokinase activity in AFP111 compared to NZN111 similarly permits increased metabolic flexibility of AFP111 . The differences between the strains and the benefit of pyruvate carboxylase under both exclusively anaerobic and dual-phase conditions are discussed in light of the cellular constraint for a redox balance. J Bacteriol, 2002 Apr, 184(8), 2296 - 9 Effect of aniA (carbon flux regulator) and PhaC (poly-beta-hydroxybutyrate synthase) mutations on pyruvate metabolism in Rhizobium etli; Dunn MF et al.; The Rhizobium etli poly-beta-hydroxybutyrate synthase (PhaC) mutant SAM100 grows poorly with pyruvate as the carbon source . The inactivation of aniA, encoding a global carbon flux regulator, in SAM100 restores growth of the resulting double mutant (VEM58) on pyruvate . Pyruvate carboxylase (PYC) activity, pyc gene transcription, and holoenzyme content, which were low in SAM100, were restored in strain VEM58 . The genetically engineered overexpression of PYC in SAM100 also allowed its growth on pyruvate . The possible relation between AniA, pyc transcription, and reduced-nucleotide levels is discussed. J Bacteriol, 2002 Apr, 184(8), 2287 - 95 AniA regulates reserve polymer accumulation and global protein expression in Rhizobium etli; Encarnacion S et al.; Previously, it was reported that the oxidative capacity and ability to grow on carbon sources such as pyruvate and glucose were severely diminished in the Rhizobium etli phaC::OmegaSm(r)/Sp(r) mutant CAR1, which is unable to synthesize poly-beta-hydroxybutyric acid (PHB) (M . A . Cevallos, S . Encarnacion, A . Leija, Y . Mora, and J . Mora, J . Bacteriol . 178:1646-1654, 1996) . By random Tn5 mutagenesis of the phaC strain, we isolated the mutants VEM57 and VEM58, both of which contained single Tn5 insertions and had recovered the ability to grow on pyruvate or glucose . Nucleotide sequencing of the region surrounding the Tn5 insertions showed that they had interrupted an open reading frame designated aniA based on its high deduced amino acid sequence identity to the aniA gene product of Sinorhizobium meliloti . R . etli aniA was located adjacent to and divergently transcribed from genes encoding the PHB biosynthetic enzymes beta-ketothiolase (PhaA) and acetoacetyl coenzyme A reductase (PhaB) . An aniA::Tn5 mutant (VEM5854) was constructed and found to synthesize only 40% of the wild type level of PHB . Both VEM58 and VEM5854 produced significantly more extracellular polysaccharide than the wild type . Organic acid excretion and levels of intracellular reduced nucleotides were lowered to wild-type levels in VEM58 and VEM5854, in contrast to those of strain CAR1, which were significantly elevated . Proteome analysis of VEM58 showed a drastic alteration of protein expression, including the absence of a protein identified as PhaB . We propose that the aniA gene product plays an important role in directing carbon flow in R . etli. Mol Microbiol, 1997 Jul, 25(1), 27 - 37 Negative autoregulation of the Rhizobium meliloti fixK gene is indirect and requires a newly identified regulator, FixT; Foussard M et al.; fixK genes are crp/fnr homologues that have been discovered in diverse Rhizobium spp., in which they are usually essential for symbiotic nitrogen fixation . One recurrent function of fixK genes in rhizobia is to activate the transcription of operons required for respiration in the microoxic environment of the nodule . In a similar manner to its Escherichia coli crp and fnr homologues, R . meliloti fixK regulates its own expression negatively . However, we demonstrate here that fixK negative autoregulation is not direct and, instead, involves a newly identified gene, fixT, the expression of which depends on fixK . Inactivation of fixT resulted in derepression of fixK expression under free-living microoxic conditions . Furthermore, constitutively expressed fixT strongly repressed fixK-lacZ expression in the absence of a functional fixK gene . Several lines of evidence indicate that fixT is active via its protein product FixT . FixT does not resemble any protein present in databases so far . Nodules induced by a fixT mutant were Fix+, thus demonstrating that fixT is not essential for symbiotic nitrogen fixation. Mol Microbiol, 1997 Jul, 25(1), 117 - 34 The Rhizobium meliloti exoK gene and prsD/prsE/exsH genes are components of independent degradative pathways which contribute to production of low-molecular-weight succinoglycan; York GM et al.; When grown on medium supplemented with the succinoglycan-binding dye, Calcofluor, and visualized under UV light, colonies of Rhizobium meliloti (Sinorhizobium meliloti) exoK mutants produce a fluorescent halo with a delayed onset relative to wild-type colonies . By conducting transposon mutagenesis of exoK mutants of R . meliloti and screening for colonies with even more severe delays in production of these fluorescent halos, we identified three genes, designated prsD, prsE, and exsH, which are required for the eventual production of fluorescent halos by exoK colonies . Nucleotide sequence indicates that the prsD and prsE genes encode homologues of ABC transporters and membrane fusion proteins of Type I secretion systems, respectively, whereas exsH encodes a homologue of endo-1,3-1,4-beta-glycanases with glycine-rich nonameric repeats typical of proteins secreted by Type I secretion systems . The exoK gene and the prsD/prsE/exsH genes were shown to be components of independent pathways for production of extracellular succinoglycan degrading activities and for production of low-molecular-weight succinoglycan by R . meliloti . Based on these results, we propose that ExsH is a succinoglycan depolymerase secreted by a Type I secretion system composed of PrsD and PrsE, and that the ExsH and ExoK glycanases contribute to production of low-molecular-weight succinoglycan. Yi Chuan Xue Bao, 2002 Feb, 29(2), 181 - 8 {A study of the effects on the symbiotic nitrogen fixation of Sinorhizobium fredii with the introduction of dctABD and nifA genes}; Li YG et al.; A recombinant plasmid pHN307 containing C4-dicarboxylic acid transport genes (dctABD) from Sinorhizobium meliloti, nifA genes from Klebsiella pneumoniae and reporter genes luxAB from pDB30 was constructed by using pTR102 as the vector . The pHN307 was then introduced into the S . fredii HN01, YC4 and GR3 by tri-parental mating, and the stability of pHN307 in the transconjugants under free-living and symbiotic condition was also investigated . The results of plant pot experiment indicated that the introduction of pHN307 in the transconjugants could significantly increase the nodule fresh weight, shoot dry weight (biomass) and total nitrogen content of the symbiotic plants with soybean variety of Heilong 33 . When the transconjugants were in symbiotic with soybean variety of Chuanzao No . 1, HN01 (pHN307) could significantly increase its root nodule number and fresh weight; GR3 (pHN307) could significantly increase its root nodule number, nodule fresh weight, shoot dry weight and total nitrogen content, but YC4(pHN307) demonstrated negative effect under the same condition . The results of this study suggested that the introduction of dctABD and nifA could improve the symbiotic nitrogen fixation efficiency and nodulation ability of the rhizobia tested, respectively, and its effect was relevant to the characteristics of recipient rhizobia and soybean variety. Arch Microbiol, 2002 Apr, 177(4), 290 - 8 Epub 2002 Jan 31. Characterization of the urease gene cluster from Rhizobium leguminosarum bv . viciae; Toffanin A et al.; Moderate levels of urease activity (ca . 300 mU mg(-1)) were detected in Rhizobium leguminosarum bv . viciae UPM791 vegetative cells . This activity did not require urea for induction and was partially repressed by the addition of ammonium into the medium . Lower levels of urease activity (ca . 100 mU mg(-1)) were detected also in pea bacteroids . A DNA region of ca . 9 kb containing the urease structural genes ( ureA, ureB and ureC), accessory genes ( ureD, ureE, ureF, and ureG), and five additional ORFs ( orf83, orf135, orf207, orf223, and orf287) encoding proteins of unknown function was sequenced . Three of these ORFs ( orf83, orf135 and orf207) have a homologous counterpart in a gene cluster from Sinorhizobium meliloti, reported to be involved in urease and hydrogenase activities . R . leguminosarum mutant strains carrying Tn 5 insertions within this region exhibited a urease-negative phenotype, but induced wild-type levels of hydrogenase and nitrogenase activities in bacteroids . orf287 encodes a potential transmembrane protein with a C-terminal GGDEF domain . A mutant affected in orf287 exhibited normal levels of urease activity in culture cells . Experiments aimed at cross-complementing Ni-binding proteins required for urease and hydrogenase synthesis (UreE and HypB, respectively) indicated that these two proteins are not functionally interchangeable in R . leguminosarum. Mol Plant Microbe Interact, 2002 Feb, 15(2), 150 - 9 Sinorhizobium fredii HH103 has a truncated nolO gene due to a -1 frameshift mutation that is conserved among other geographically distant S . fredii strains; Madinabeitia N et al.; Strain SVQ121 is a mutant derivative of Sinorhizobium fredii HH103 carrying a transposon Tn5-lacZ insertion into the nolO-coding region . Sequence analysis of the wild-type gene revealed that it is homologous to that of Rhizobium sp . NGR234, which is involved in the 3 (or 4)-O-carbamoylation of the nonreducing terminus of Nod factors . Downstream of nolO, as in Rhizobium sp . NGR234, the noeI gene responsible for methylation of the fucose moiety of Nod factors was found . SVQ121 Nod factors showed lower levels of methylation into the fucosyl residue than those of HH103-suggesting a polar effect of the transposon insertion into nolO over the noel gene . A noeI HH103 mutant was constructed . This mutant, SVQ503, produced Nod factors devoid of methyl groups, confirming that the S . fredii noeI gene is functional . Neither the nolO nor the noeI mutation affected the ability of HH103 to nodulate several host plants, but both mutations reduced competitiveness to nodulate soybean . The Nod factors produced by strain HH103, like those of other S . fredii isolates, lack carbamoyl residues . By using specific polymerase chain reaction primers, we sequenced the nolO gene of S . fredii strains USDA192, USDA193, USDA257, and 042B(s) . All the analyzed strains showed the same -1 frameshift mutation that is present in the HH103 nolO-coding region . From these results, it is concluded that, regardless of their geographical origin, S . fredii strains carry the nolO-coding region but that it is truncated by the same base-pair deletion. Appl Environ Microbiol, 2002 Mar, 68(3), 1220 - 7 Nucleotide sequence and genetic structure of a novel carbaryl hydrolase gene (cehA) from Rhizobium sp . strain AC100; Hashimoto M et al.; Rhizobium sp . strain AC100, which is capable of degrading carbaryl (1-naphthyl-N-methylcarbamate), was isolated from soil treated with carbaryl . This bacterium hydrolyzed carbaryl to 1-naphthol and methylamine . Carbaryl hydrolase from the strain was purified to homogeneity, and its N-terminal sequence, molecular mass (82 kDa), and enzymatic properties were determined . The purified enzyme hydrolyzed 1-naphthyl acetate and 4-nitrophenyl acetate indicating that the enzyme is an esterase . We then cloned the carbaryl hydrolase gene (cehA) from the plasmid DNA of the strain and determined the nucleotide sequence of the 10-kb region containing cehA . No homologous sequences were found by a database homology search using the nucleotide and deduced amino acid sequences of the cehA gene . Six open reading frames including the cehA gene were found in the 10-kb region, and sequencing analysis shows that the cehA gene is flanked by two copies of insertion sequence-like sequence, suggesting that it makes part of a composite transposon. Appl Environ Microbiol, 2002 Mar, 68(3), 1064 - 70 Morphological changes of rhizobia in peat cultures; Feng L et al.; Morphological changes that take place in peat cultures of several species of rhizobia were examined . These changes seemed to be associated with enhanced survival of cells in peat and after inoculation onto plastic beads, which were used as a model system for seeds . Cell wall changes, in which the periplasmic space appeared to be occluded with electron-dense material, were observed in Rhizobium sp . strain SU343 and Bradyrhizobium lupini WU425 cells after 7 and 14 days in peat, respectively . Nutrient limitation and low O(2) concentration in peat are suggested to be factors involved in the induction of the morphological changes . Polyhydroxybutyrate reserves, which were present in broth-cultured cells of both species of rhizobia, were mobilized after transfer into peat but did not appear to influence survival after inoculation onto beads . Enhanced expression of an iron-manganese superoxide dismutase was also observed after the cells were transferred into peat . We conclude that cell wall thickening in rhizobia after transfer from broth cultures into peat is an adaptive response for long-term survival under nutrient-limited conditions in peat . Cells with thickened walls may also be more resistant to other types of stress, such as that encountered on a seed surface. Mol Genet Genomics, 2002 Feb, 266(6), 1012 - 9 Epub 2002 Jan 23. Genetic mapping of the non-nodulation phenotype of the mutant MN-1008 in tetraploid alfalfa (Medicago sativa); Endre G et al.; Abstract . Roots of the non-nodulating Medicago sativa mutant MN-1008 neither undergo root-hair curling, cortical cell division nor any of the early molecular events that accompany nodule initiation and development following rhizobial infection or treatment with Nod factor . These observations suggested that the mutation(s) impaired a pivotal function in Nod factor perception or in the signal transduction pathway . In this paper we show that the genetic lesion conditioning the recessive non-nodulation phenotype in the tetraploid alfalfa mutant MN-1008 can be localized to a single region on LG5 of the M . sativa genetic map . This conclusion is based on genetic analyses conducted at the tetraploid level, involving both segregation analysis and genetic mapping of the trait with respect to molecular DNA markers . The genetic mapping of the Nod(-) phenotype was performed in a segregating tetraploid F2 population, taking advantage of the availability of an advanced genetic map for diploid alfalfa . Two tightly linked flanking markers have been identified which will facilitate the physical mapping and cloning of the gene(s) that underlie(s) the non-nodulation phenotype. Mol Plant Microbe Interact, 2002 Jan, 15(1), 69 - 74 dpp genes of Rhizobium leguminosarum specify uptake of delta-aminolevulinic acid; Carter RA et al.; An operon with homology to the dppABCDF genes required to transport dipeptides in bacteria was identified in the N2-fixing symbiont, Rhizobium leguminosarum . As in other bacteria, dpp mutants were severely affected in the import of delta-aminolevulinic acid (ALA), a heme precursor . ALA uptake was antagonized by adding dipeptides, indicating that these two classes of molecule share the same transporter . Mutations in dppABCDF did not affect symbiotic N2 fixation on peas, suggesting that the ALA needed for heme synthesis is not supplied by the plant or that another uptake system functions in the bacteroids . The dppABCDF operon of R . leguminosarum resembles that in other bacteria, with a gap between dppA and dppB containing inverted repeats that may stabilize mRNA and may explain why transcription of dppA alone was higher than that of dppBCDF . The dppABCDF promoter was mapped and is most likely recognized by sigma70. Mol Plant Microbe Interact, 2002 Jan, 15(1), 27 - 34 Differential effectiveness of salicylate-dependent and jasmonate/ethylene-dependent induced resistance in Arabidopsis; Ton J et al.; Salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are each involved in the regulation of basal resistance against different pathogens . These three signals play important roles in induced resistance as well . SA is a key regulator of pathogen-induced systemic acquired resistance (SAR), whereas JA and ET are required for rhizobacteria-mediated induced systemic resistance (ISR) . Both types of induced resistance are effective against a broad spectrum of pathogens . In this study, we compared the spectrum of effectiveness of SAR and ISR using an oomycete, a fungal, a bacterial, and a viral pathogen . In noninduced Arabidopsis plants, these pathogens are primarily resisted through either SA-dependent basal resistance (Peronospora parasitica and Turnip crinkle virus {TCV}), JA/ET-dependent basal resistance responses (Alternaria brassicicola), or a combination of SA-, JA-, and ET-dependent defenses (Xanthomonas campestris pv . armoraciae) . Activation of ISR resulted in a significant level of protection against A . brassicicola, whereas SAR was ineffective against this pathogen . Conversely, activation of SAR resulted in a high level of protection against P . parasitica and TCV, whereas ISR conferred only weak and no protection against P . parasitica and TCV, respectively . Induction of SAR and ISR was equally effective against X . campestris pv . armoraciae . These results indicate that SAR is effective against pathogens that in noninduced plants are resisted through SA-dependent defenses, whereas ISR is effective against pathogens that in noninduced plants are resisted through JA/ET-dependent defenses . This suggests that SAR and ISR constitute a reinforcement of extant SA- or JA/ET-dependent basal defense responses, respectively. Prikl Biokhim Mikrobiol, 2002 Jan-Feb, 38(1), 73 - 8 {Survival of Rhizobium in monoculture and binary population with Rhizosphere bacteria}; Sukhovitskaia LA et al.; The survival of pure cultures of Rhizobium leguminosarum bv . pisum and Rhizobium trifolii and their interaction with associative diazotrophic and phosphate-mobilizing bacteria after inoculation of sterile soil were studied . The viable heterotypical diazotrophic and rhizobial phosphate-mobilizing association was shown to be formed whose efficiency was 14% (clover) and 28% (pea) higher compared to monorhizobial inoculates. J Appl Microbiol, 2002, 92(2), 228 - 37 Colonization of the developing rhizosphere of sugar beet seedlings by potential biocontrol agents applied as seed treatments; Walker R et al.; AIMS: Poor colonization of the rhizosphere is a major constraint of seed treatment biological control . The objectives of this study were to; examine the colonization of the rhizosphere of sugar beet seedlings by selected rhizobacteria; determine the influence of the host rhizosphere and percolating water on the distribution of the bacteria; and deliver two biological control agents (BCAs) by co-inoculation . METHODS AND RESULTS: Rifampicin-resistant bacterial strains (Rif +) applied as single treatments to seed sown in columns of field soil produced persistent populations of 5-9 log10 cfu g-1 in the infection court of the damping-off pathogen Aphanomyces cochlioides in a controlled environment . However, isolates varied in their ability to colonize the lower rhizosphere . Percolating water significantly increased the colonization of the upper rhizosphere . Bacterial populations in the soil profiles of "non-rhizosphere" controls declined markedly with time . There was no interaction between the two selected BCAs applied as a seed treatment mixture . CONCLUSIONS: The distribution of the bacteria resulted primarily from root colonization although percolating water may modify the colonization profiles . Co-inoculation of the sugar-beet rhizosphere is a viable proposition . SIGNIFICANCE AND IMAPCT OF THE STUDY: Potential BCAs were successfully delivered to the known infection court of A . cochloides and persisted for the infection period . This bioassay can be used as a tool for the selection of BCAs for field trials. J Appl Microbiol, 2002, 92(1), 13 - 21 Bradyrhizobium sp . nodulating the Mediterranean shrub Spanish broom (Spartium junceum L.); Quatrini P et al.; AIMS: The molecular diversity of 25 strains of rhizobia, isolated in Sicily from root nodules of the Mediterranean shrubby legume Spanish broom (Spartium junceum L.), is presented in relation to the known rhizobial reference strains . METHODS AND RESULTS: Our approach to the study of the S . junceum rhizobial diversity combined the information given by the 16S and the intergenic spacer (IGS) 16S-23S rDNA polymorphic region by obtaining them in a single polymerase chain reaction (PCR) step . The PCR fragment size of the S . junceum isolates was 2400-2500 bp and that of the reference strains varied from 2400 in Bradyrhizobium strains to 2800 in Sinorhizobium strains . Inter- and intrageneric length variability was found among the reference strains . Restriction fragment length polymorphisms (RFLP) analysis allowed us to identify eight genotypes among the S . junceum rhizobia that were clustered into two groups, both related to the Bradyrhizobium lineage . Sequencing of representative strains of the two clusters confirmed these data . The 16S-IGS PCR-RFLP approach, when applied to rhizobial reference strains, allowed very close species (i.e . Rhizobium leguminosarum/R . tropici) to be separated with any of the three enzymes used; however, cluster analysis revealed inconsistencies with the 16S-based phylogenesis of rhizobia . CONCLUSIONS: Rhizobia nodulating S . junceum in the Mediterranean region belong to the Bradyrhizobium lineage . Our results confirm the resolution power of the 16S-23S rDNA in distinguishing among rhizobia genera and species, as well as the usefulness of the PCR-RFLP method applied to the entire 16S-IGS region for a rapid tracking of the known relatives of new isolates . SIGNIFICANCE AND IMPACT OF THE STUDY: The present paper is, to our knowledge, the first report on rhizobia nodulating a Mediterranean wild woody legume. Biotechniques, 2002 Feb, 32(2), 386 - 8, 390, 392-4, passim Genetic tools for pseudomonads, rhizobia, and other gram-negative bacteria; Davison J; Gram-negative bacteria are extraordinarily diverse microorganisms that present a wide variety of characteristics worthy of genetic investigation . For historical reasons, the application of recombinant DNA technology to gram-negative bacteria in general has always lagged behind that of E . coli and its close relatives . However, the past 10 years have seen dramatic advances in the development of new tools and vectors for genetic analysis in non-E . coli hosts . Applications include various kinds of genetic manipulation, conjugation, transposition, site-specific recombination, protein secretion, protein purification, cell suicide, microbial ecology, biodegradation, and plant and animal pathogenicity. Mol Plant Microbe Interact, 2002 Jan, 15(1), 60 - 8 Competitive nodulation blocking of cv . Afghanistan pea is related to high levels of nodulation factors made by some strains of Rhizobium leguminosarum bv . viciae; Hogg B et al.; Cultivar Afghanistan peas are resistant to nodulation by many strains of Rhizobium leguminosarum bv . viciae but are nodulated by strain TOM, which carries the host specificity gene nodX . Some strains that lack nodX can inhibit nodulation of cv . Afghanistan by strain TOM . We present evidence that this "competitive nodulation-blocking" (Cnb) phenotype may result from high levels of Nod factors inhibiting nodulation of cv . Afghanistan peas . The TOM nod gene region (including nodX) is cloned on pIJ1095, and strains (including TOM itself) carrying pIJ1095 nodulate cv . Afghanistan peas very poorly but can nodulate other varieties normally . The presence of pIJ1095, which causes increased levels of Nod factor production, correlates with Cnb . Nodulation of cv . Afghanistan by TOM is also inhibited by a cloned nodD gene that increases nod gene expression and Nod factor production . Nodulation of cv . Afghanistan can be stimulated if nodD on pIJ1095 is mutated, thus severely reducing the level of Nod factor produced . Repression of nod gene expression by nolR eliminates the Cnb phenotype and can stimulate nodulation of cv . Afghanistan . Addition of Nod factors to cv . Afghanistan roots strongly inhibits nodulation . The Cnb+ strains and added Nod factors inhibit infection thread initiation by strain TOM . The sym2A allele determines resistance of cv . Afghanistan to nodulation by strains of R . leguminosarum bv . viciae lacking nodX . We tested whether sym2A is involved in Cnb by using a pea line carrying the sym2A region introgressed from cv . Afghanistan; nodulation in the introgressed line was inhibited by Cnb+ strains . Therefore, the sym2A region has an effect on Cnb, although another locus (or loci) may contribute to the stronger Cnb seen in cv . Afghanistan. Plant Physiol, 2002 Feb, 128(2), 523 - 33 Endogenous Nod-factor-like signal molecules promote early somatic embryo development in Norway spruce; Dyachok JV et al.; Embryogenic cultures of Norway spruce (Picea abies) are composed of pro-embryogenic masses (PEMs) and somatic embryos of various developmental stages . Auxin is important for PEM formation and proliferation . In this report we show that depletion of auxin blocks PEM development and causes large-scale cell death . Extracts of the media conditioned by embryogenic cultures stimulate development of PEM aggregates in auxin-deficient cultures . Partial characterization of the conditioning factor shows that it is a lipophilic, low-molecular-weight molecule, which is sensitive to chitinase and contains GlcNAc residues . On the basis of this information, we propose that the factor is a lipophilic chitin oligosaccharide (LCO) . The amount of LCO correlates to the developmental stages of PEMs and embryos, with the highest level in the media conditioned by developmentally blocked cultures . LCO is not present in nonembryogenic cultures . Cell death, induced by withdrawal of auxin, is suppressed by extra supply of endogenous LCO or Nod factor from Rhizobium sp . NGR234 . The effect can be mimicked by a chitotetraose or chitinase from Streptomyces griseus . Taken together, our data suggest that endogenous LCO acts as a signal molecule stimulating PEM and early embryo development in Norway spruce. Plant Physiol, 2002 Feb, 128(2), 370 - 8 Voltage-dependent cation channels permeable to NH(+)(4), K(+), and Ca(2+) in the symbiosome membrane of the model legume Lotus japonicus; Roberts DM et al.; The symbiosome of nitrogen fixing root nodules mediates metabolite exchange between endosymbiotic rhizobia bacteria and the legume host . In the present study, the ion currents of the symbiosome membrane of the model legume Lotus japonicus were analyzed by patch-clamp recording . Both excised and symbiosome-attached patches exhibited a large inward (toward the cytosolic side of the membrane) current that is activated in a time-dependent manner by negative (on the cytosolic side) potentials . Based on reversal potential determinations and recordings with the impermeant cation N-methyl-glucamine, this current shows a high permeability for monovalent cations with no apparent permeability for anions . The current also showed a finite Ca(2+) permeability . However, the currents were predominantly carried by univalent cations with a slightly greater selectivity for NH(4)(+) over K(+) . Increased Ca(2+) concentration inhibited the current with a K(0.5) for inhibition of 0.317 mM . The current showed strong rectification that is mediated by divalent cations (either Mg(2+) or Ca(2+)) . The influence of divalent cations is symmetrical in nature, because rectification can be exerted in either direction depending upon which side of the membrane has the highest concentration of divalent cations . However, based on observations with symbiosome-attached patches, the direction of the current in vivo is proposed to be toward the cytosol with cytosolic Mg(2+) acting as the putative gating regulator . The findings suggest that L . japonicus possesses a voltage-dependent cation efflux channel that is capable of exporting fixed NH(4)(+), and may also play an additional role in Ca(2+) transport. J Soc Biol, 2001, 195(3), 297 - 302 {Calcium oscillations and Nod signal transduction in Rhizobium-legume symbiosis}; Gough C; Rhizobium nodulation (Nod) factors are lipo-chitooligosaccharides that act as symbiotic signals, eliciting a number of key developmental responses in the roots of legume hosts . One of the earliest responses of root hairs to Nod factors is the induction of sharp oscillations of cytoplasmic calcium ion concentration ("calcium spiking") . This response was first characterised in Medicago sativa and Nod factors were found to be unable to induce calcium spiking in a nodulation-defective mutant of M . sativa . The fact that this mutant lacked any morphological response to Nod factors raised the question of whether calcium spiking could be part of a Nod factor-induced signal transduction pathway leading to nodulation . More recently, calcium spiking has been described in a model legume, Medicago truncatula, and in pea . When nodulation-defective mutants were tested for the induction of calcium spiking in response to Nod factors, three loci of pea and two of M . truncatula were found to be necessary for Nod factor-induced calcium spiking . These loci are also known to be necessary for Nod factor-induction of symbiotic responses such as root hair deformation, nodulin gene expression and cortical cell division . These results therefore constitute strong genetic evidence for the role of calcium spiking in Nod factor transduction . This system provides an opportunity to use genetics to study ligand-stimulated calcium spiking as a signal transduction event. Microbiology, 2002 Feb, 148(Pt 2), 615 - 23 The Rhizobium leguminosarum bv . viciae VF39 gamma-aminobutyrate (GABA) aminotransferase gene (gabT) is induced by GABA and highly expressed in bacteroids; Prell J et al.; A Rhizobium leguminosarum bv . viciae VF39 gene (gabT) encoding a gamma-aminobutyrate (GABA) aminotransferase was identified, cloned and characterized . This gene is thought to be involved in GABA metabolism via the GABA shunt pathway, a theoretical bypass of the 2-oxoglutarate dehydrogenase complex . Mutants in gabT are still able to grow on GABA as a sole carbon and nitrogen source . 2-oxoglutarate-dependent GABA aminotransferase activity is absent in these mutants, while pyruvate-dependent activity remains unaffected . This indicates that at least two enzymes with different substrate specifities are involved in the GABA metabolism of R . leguminosarum bv . viciae VF39 . The gabT promoter was cloned into a newly constructed, stable promoter-probe vector pJP2, suitable for the study of transcriptional GUS fusions in free-living bacteria and during symbiosis . Under free-living conditions the gabT promoter is induced by GABA and repressed by succinate . Transcriptional regulation is mediated by GabR in a repressor-like manner . During symbiosis with the pea host plant gabT is induced and highly expressed in the symbiotic zone . Nodules induced by gabT mutants, however, are still effective in nitrogen fixation. BMC Plant Biol . 2002;2(1):1 . Epub 2002 Jan 02. The molecular genetic linkage map of the model legume Medicago truncatula: an essential tool for comparative legume genomics and the isolation of agronomically important genes; Thoquet P et al.; BACKGROUND: The legume Medicago truncatula has emerged as a model plant for the molecular and genetic dissection of various plant processes involved in rhizobial, mycorrhizal and pathogenic plant-microbe interactions . Aiming to develop essential tools for such genetic approaches, we have established the first genetic map of this species . Two parental homozygous lines were selected from the cultivar Jemalong and from the Algerian natural population (DZA315) on the basis of their molecular and phenotypic polymorphism . RESULTS: An F2 segregating population of 124 individuals between these two lines was obtained using an efficient manual crossing technique established for M . truncatula and was used to construct a genetic map . This map spans 1225 cM (average 470 kb/cM) and comprises 289 markers including RAPD, AFLP, known genes and isoenzymes arranged in 8 linkage groups (2n = 16) . Markers are uniformly distributed throughout the map and segregation distortion is limited to only 3 linkage groups . By mapping a number of common markers, the eight linkage groups are shown to be homologous to those of diploid alfalfa (M . sativa), implying a good level of macrosynteny between the two genomes . Using this M . truncatula map and the derived F3 populations, we were able to map the Mtsym6 symbiotic gene on linkage group 8 and the SPC gene, responsible for the direction of pod coiling, on linkage group 7 . CONCLUSIONS: These results demonstrate that Medicago truncatula is amenable to diploid genetic analysis and they open the way to map-based cloning of symbiotic or other agronomically-important genes using this model plant. Can J Microbiol, 2001 Dec, 47(12), 1068 - 74 Root colonization of faba bean (Vicia faba L.) and pea (Pisum sativum L.) by Rhizobium leguminosarum bv . viciae in the presence of nitrate-nitrogen; Beauchamp CJ et al.; There is a lack of knowledge concerning the effect of nitrate-nitrogen (NO3(-)-N) at levels known to inhibit nodule formation and functioning on root colonization of dinitrogen-fixing legumes . Firstly, this study investigated potential differences between Rhizobium leguminosarum bv . viciae 175F9 and its bioluminescent-labeled strain 175F9.lux on root colonization of faba bean (Vicia faba L.) and pea (Pisum sativum L.) . These two strains similarly colonized the roots of both hosts . Secondly, this study evaluated the effects of 0 and 10 mol x m(-3) NO3(-)-N on root colonization of faba bean and pea by strain 175F9.lux, over time . Averaged over both hosts and harvest dates, the presence of NO3(-)-N increased the rhizobial population and the root length colonized . In addition, our results showed that bioluminescence activity increased from 7 to 14 days after sowing and was not correlated to rhizobial population . Finally, to demonstrate that an increase in bioluminescence activity was not an indirect effect of nitrate on R . leguminosarum bv . viciae 175F9.lux, this study investigated the effects of increasing carbon (mannitol) and nitrogen (NO3(-)-N) concentrations on the rhizobial population and bioluminescence activity . The carbon source was more important than the nitrogen source to increase the rhizobial population and bioluminescence activity, which increased with increasing mannitol concentration, but not with increasing nitrate concentration . Results from this study demonstrated that NO3(-)-N increased rhizobial population, especially for faba bean, and the length of root colonized. Bioorg Med Chem, 2002 Mar, 10(3), 737 - 42 C-terminal truncation of alpha 1,6-fucosyltransferase from Rhizobium sp . does not annul the transferase activity of the enzyme; Bastida A et al.; Recently we have over-expressed the enzyme alpha 1,6-fucosyltransferase from Rhizobium sp . in Escherichia coli . In this heterologous system the enzyme was mainly expressed as inclusion bodies and the one that was expressed soluble showed a short-lasting activity in solution due to precipitation of the protein . A structural analysis of the sequence using the TMpred program predicted a highly hydrophobic region of 19 aa close to the C-terminal of the protein . In order to investigate the influence of this region on the formation of inclusion bodies and the precipitation from solution, we cloned a truncated version of the protein where a C-terminal fragment of 65 aa, including the predicted transmembrane-like region, was removed . The resulting protein was expressed in a soluble form without formation of inclusion bodies . The truncated protein catalyzed the transfer of a fucopyranosyl moiety from GDP-beta-L-Fucose to chitobiose . Comparison of the acceptor specificity between the truncated alpha 1,6-fucosyltransferase and the wild-type enzyme, showed a similar behavior for both enzymes . Our results indicate that the active center is not located in the C-terminal extreme of the protein in contrast to the case of the mammalian glycosyltransferases . Also, these results indicate that the alpha-6-motif III is not directly involved in the catalytic activity of the enzyme. J Theor Biol, 2002 Jan 21, 214(2), 215 - 32 Developmental genetics and evolution of symbiotic structures in nitrogen-fixing nodules and arbuscular mycorrhiza; Provorov NA et al.; Genetic and molecular mechanisms of development are compared for two major plant-microbe endosymbioses: N(2)-fixing nodules (with rhizobia or actinomycetes Frankia) and arbuscular mycorrhiza (with Glomales fungi) . Development from the primordia formed de novo in root tissues is common for all known types of N(2)-fixing nodules . However, their structure varies greatly with respect to: (i) tissue topology (location of vascular bundles is peripherical in legumes or central in non-legumes); (ii) position of nodule primordium (inner or outer cortex in legumes, pericycle in non-legumes); (iii) stability of apical meristem (persistent in the indeterminate nodules, transient in the determinate ones) . In addition, legumes vary in ability to form compartments harboring endosymbiotic rhizobia and located intercellularly (infection threads) and intracellularly (symbiosomes) . Using pea (Pisum sativum) symbiotic mutants, the nodule developmental program is dissected into a range of spatially and temporarily differentiated steps comprising four sub-programs (development of endosymbiotic compartments; nodule histogenesis; autoregulation of nodulation; bacteroid differentiation) . The developmental mutations are suggested in some cases to reverse the endosymbiotic system into the morphologically simpler forms some of which may correspond to the ancestral stages of nodule evolution . The origin of legume-rhizobial and actinorhizal symbioses is suggested to be based on a set of preadaptations many of which had been evolved in angiosperms during coevolution with arbuscular mycorrhizal fungi (e.g., inter- and intracellular maintenance of symbionts, their control via defence-like reactions and recognition of chitin-like molecules) . An analysis of parallel morphological variation in symbiotic mutants and wild-growing legume species enables us to reconstruct the major stages of evolution for N(2)-fixing symbioses . Sheng Wu Gong Cheng Xue Bao, 2001 Sep, 17(5), 598 - 600 {A study on growth of Rhizobium leguminosarum in air pressure oscillating, solid-state fermenter}; Zhao H et al.; Rhizobium leguminosarum L003, a kind of biofertilizers, was cultured in a periodic air pressure oscillating, solid-state fermenter by using straw of wheat as an inert solid support . Effects of air pressure oscillation amplitude, frequency on the viable cells of R . leguminosarum L003 and oxygen transfer rate were investigated . It was found that enhanced oxygen transfer and biochemical reaction occurred in this system . Under the optimized conditions, about a 3-fold increase of the viable cells was obtained in this system compared with that in a static tray fermenter . The overall oxygen transfer rate reached 487.01 mmol/(kg.h) in this system in case of 0.35 MPa, t2 = 0.5 min against 37.46 mmol/(kg.h) in static tray fermenter. Sheng Wu Gong Cheng Xue Bao, 2001 Sep, 17(5), 534 - 8 {Study on intergenera fusion of protoplasts from Rhizobium leguminosorum and Sinorhizobium xinjiangnesis}; Wei GH et al.; Penicillin and chloromycetin were regarded as the sign of resistance to antibodies of R . leguminosorum USDA2370 and S . xinjiangnesis CCBAU110 respectively . Using the protoplast fusion technique, USDA2370 and CCBAU110 were successfully fused . Fusion hybrid can inoculate in the leguminous of parental strains respectively . There were apparent differences between parents and fusion hybrid in cell morphology, colony and pattern of whole-cell protein . The values of DNA homology between fusion hybrid and USDA2370 and CCBAU110 were 56.6% and 10.2% respectively. Mikrobiol Z, 2001 Sep-Oct, 63(5), 59 - 66 {Effect of plant growth stimulators on Rhizobium leguminosarum BV . VICIAE 263b and efficiency of symbiotic nitrogen-fixation in peas}; Kosenko LV et al.; Effect of two plant growth stimulators: bactozol (drug of bacterial origin) and D1 (synthetic analog of phytohormones) on metabolism of pea rhizobia (Rhizobium leguminosarum bv . viciae 2636) and efficiency of their symbiosis with pea plants have been studied . The D1 drug in concentration 0.1% suppressed growth of bacteria . However, bactozol stimulating action on pea rhizobia growth in a pure culture and synthesis of extracellular carbohydrates by them have been established . The trial of three concentrations (0.1%, 0.01%, 0.001%) has shown that bactozol effect has dose-dependent character . The highest effect of stimulation is achieved at concentration 0.1% . Bactozol decreases partially the repressing influence of the mineral nitrogen on growth of rhizobia and their carbohydrate-synthesizing activity under growth of bacteria against a high nitrogen background (20 mM NO3-) . Treatment of pea plants by bactozol (0.1%) increases considerably the efficiency of their symbiosis with pea rhizobia, evoking the growth of the overground and root mass of the plants, quantity of nodules and their nitrogenase activity. Environ Sci Technol, 2001 Sep 15, 35(18), 3676 - 82 Microbial populations associated with the reduction and enhanced mobilization of arsenic in mine tailings; Macur RE et al.; Microbial reduction of arsenate {As(V)} to arsenite {As(III)} and the subsequent effects on As mobilization in contaminated mine tailings were studied under transport conditions . Molecular analysis of bacterial populations and traditional isolation techniques were used in conjunction with column experiments designed to observe relationships among pH (limed vs unlimed treatments), redox potential (Pt electrode), and mobilization of As . Liming increased pH values from approximately 4 to 8, resulting in a 5-fold increase in total As eluted from sterile columns . Elution of As from limed columns was further enhanced by microbial activity . As(III) was the predominant As species eluted from oxic, nonsterile columns . Conversely, in sterile treatments, As(V) was the predominant valence state in column effluent . Denaturing gradient gel electrophoresis coupled with sequence and phylogenetic analysis of 16S rRNA gene segments revealed that liming of the mine tailings stimulated specific Caulobacter-, Sphingomonas-, and Rhizobium-like populations . Pure culture isolates of these bacteria demonstrated the ability to rapidly reduce As(V) in aerated serum bottles . An intracellular As detoxification pathway was implicated in the reduction of As(V) by these isolates . These results indicate that microbial reduction of As(V) in As-contaminated soils may occur under aerobic conditions over relatively short time scales resulting in enhanced As mobilization. Genetika, 2001 Nov, 37(11), 1517 - 21 {Level of phytohormones in various types of symbiotic pea mutants}; Kholodar' AV et al.; The levels of the phytohormones auxin and gibberellin were studied in the original pea (Pisum sativum L.) cultivars Rondo and Ramonskii 77 and in different types of symbiotic mutants (non-nodulating, with single nodules, and supernodulating) induced from them . The results obtained indicated that the levels of the phytohormones in the symbiotic mutants depend on the plant's genotype, developmental phase, and infection with rhizobia . Two mutants were isolated whose phytohormonal statuses markedly differed from the original forms . These mutants may be used for identification of the genes that determine the auxin and gibberellin statuses. Genetika, 2001 Nov, 37(11), 1507 - 12 {Analysis of various types of competition in Tn5-mutants of alfalfa rhizobium bacteria (Sinorhizobium meliloti)}; Onishchuk OP et al.; Nodulation, rhizospheral, and saprophytic types of competitiveness (NC, RC, and SC, respectively) were studied in the highly active strains CXM1-105 and CXM1-188 of the alfalfa rhizobium Sinorhizobium meliloti . The competitiveness was estimated with the use of markers of antibiotic resistance . It was found that the mutant strain T37, which was characterized by a drastically decreased NC, had higher SC and RC than the parental strain . The mutant T107 (with a moderately decreased NC) did not differ from the parental strain with respect to RC but had a higher SC . The mutant T27 (with the lowest NC) did not differ from the parental strain with respect to SC or RC . In the mutant Tb1, the NC and RC were decreased and the SC was the same as in the parental strain . In Tb7, the SC was decreased and RC was increased . In the mutant T795, all of the three types of competitiveness were decreased . The difference between the mutants studied and the parental strain with respect to NC and RC was confirmed using an indirect method (the ability to form effective symbiosis after mixed inoculation together with the an ineffective tester strain CXM1-48) and the X-Gluc staining method (using the S . meliloti RmM4gus tester strain carrying the gene of beta-glucuronidase) . However, the decreased SC that the mutants exhibited when they were cultivated together with parental strains in a plant-growth substrate (vermiculite) was not observed in the case of their cocultivation in liquid media . The independent variation of different types of competitiveness indicate that rhizobia have several separate gene systems determining their survival in in planta and ex planta ecological niches. Microbiol Res, 2001, 156(4), 353 - 8 Chitinolytic and cellulolytic Pseudomonas sp . antagonistic to fungal pathogens enhances nodulation by Mesorhizobium sp . Cicer in chickpea; Sindhu SS et al.; Pseudomonas strains isolated from the rhizosphere of chickpea (Cicer arietinum L.) and green gram (Vigna radiata L.) were screened for the production of chitinases and cellulases . Five Pseudomonas strains were found to produce appreciable amounts of both enzymes in culture-free supernatants and showed growth inhibition of the two fungi Pythium aphanidermatum (Oomycete) and Rhizoctonia solani (Basidiomycete) in plates on potato dextrose agar medium . The fungal growth inhibition was not correlated with cell wall-degrading enzyme activity, which suggested that other antifungal compounds produced by these rhizobacteria were also involved in antagonism . Coinoculation of the Pseudomonas strains with the Mesorhizobium sp . Cicer strain Ca 181 resulted in a significant increase in nodule biomass when grown under sterilized chillum jar conditions . The results suggest that hydrolytic enzymes produced by Pseudomonas sp . contribute to suppression of plant diseases by inhibiting growth of phytopathogenic fungi and also promote nodulation of legumes by rhizobia. Ying Yong Sheng Tai Xue Bao, 2000 Apr, 11(2), 311 - 4 {Research progress on PGPR/AMF interactions}; Long W et al.; As one of the rhizospheric microorganisms PGPR(Plant Growth Promoting Rhizobacteria) and AMF(Arbuscular Mycorrhizal Fungi) play an important role in promoting plant growth . It is of significance to further study and elucidate the interactions between them to utilize and regulate the interactions among rhizospheric microorganisms, and promote and protecte plant growth . Many research results show that on one hand, there exists synergism between PGPR and AMF . AMF can transfer PGPR or act as a media in the process of spread of PGPR along roots, where PGPR create many beneficial conditions for the infection of AMF . Both of them can indirectly enhance the other side's colonization or infection ability through their own promoting role on plant growth . On the other hand, they compete with each other for nutrients and niches, and probably produce some secondary metabolites which cause detrimental effects on the other . However, whether these interactions are synergistic or competitive depends upon the AM fungal or PGPR species involved . So far, the research work is extensive, even in molecular level in some aspects, but not systematic and deep . It is believed however, with the development of techniques in molecular biology and the increasing application of advanced testing methods, the new breakthroughs will be gained in the study and understanding on the interactions. Ying Yong Sheng Tai Xue Bao, 2000 Dec, 11(6), 951 - 3 {Factors affecting colonization of introduced microorganisms on plant roots}; Zhang B et al.; Microorganisms such as biological control agents (BCA), plant growth-promoting rhizobacteria (PGPR) and yield increasing bacteria (YIB) were introduced along growing roots . The colonization process of introduced bacteria was proved that they attached root tipfirst, then distributed along roots, multiplicated there, and survived as certain population size . The colonization location was closely related with root exudates, which was usually at the junction between cortex cells or at the base of lateral roots or root hairs . The variation of colonization by introduced microorganisms in the rhizosphere was caused by biotic and abiotic factors . Biotic factors included the physiological characters of introduced microorganisms and interactions between introduced microorganisms and native microbes . The more important factors were plant genotypes which associated with introduced beneficial microbes and regulated the population and community of those microbes affecting the colonization of introduced microorganisms . Abiotic factors here referred to soil environmental conditions, e.g., soil texture, water content, soil temperature and pH value. Can J Microbiol, 2001 Nov, 47(11), 1058 - 62 Co-inoculation with Bacillus sp . CECT 450 improves nodulation in Phaseolus vulgaris L; Camacho M et al.; The strain Bacillus sp . CECT 450 increased nodulation on bean (Phaseolus vulgaris L.) when co-inoculated with Rhizobium tropici CIAT 899 . This positive effect occured under controlled conditions on perlite-vermiculite, sand, or in a mixture of soil and sand . This increase was also observed in a field assay . Nodulation kinetic studies suggested that the synergistic effect is pronounced during the latter stages of cultivation . In contrast, the same bacteria co-inoculated with Bradyrhizobium japonicum USDA 110 reduced nodulation on soybean (Glycine max (L.) Merr.) . Inoculation with Bacillus sp . CECT 450 alone had no effect on bean plants, but reduced root growth in soybean . The survival of Bacillus sp . CECT 450 on inoculated seeds was high, even when inoculated seeds were maintained for several months at room temperature. Int J Syst Evol Microbiol, 2001 Nov, 51(Pt 6), 1983 - 6 Phylogenetic relationships of three bacterial strains isolated from the pasture legume Biserrula pelecinus L; Nandasena KG et al.; Three bacterial strains (WSM 1283, WSM 1284, WSM 1497) isolated from root nodules of the pasture legume Biserrula pelecinus L . growing in Morocco, Italy and Greece, respectively, were studied in order to determine their phylogenetic relationship to the other members of the family Rhizobiaceae . A polyphasic approach, which included analyses of morphological and physiological characteristics, plasmid profiles, symbiotic performance and 16S rRNA gene sequencing, indicated that these strains belong to the genus Mesorhizobium. Ying Yong Sheng Tai Xue Bao, 2001 Aug, 12(4), 639 - 40 {Effect of pH on nodulation of soybean rhizobia from Weifang and Huayuankou soils}; Yang J et al.; The effect of pH on the nodulation of Sinorhizobium fredii and Bradyrhizobium japonicum was examined by analyzing the indigent soybean rhizobia, predominant indigent rhizobia, and specific rhizobia, respectively . The results showed that very acid and very alkaline environment could retard the nodulation and inhibit the growth of the rhizobia . Sinorhizohium fredii could endure environment more strongly than Bradyrhizobium japonicum, and had a high competitive nodulation capacity . Bradyhizobium japonicum could endure acid environment more strongly than Sinorhizobium fredii . In very acid and very alkaline environment, the nodulation capacity of S . fredii and B . japonicum was mainly determined by their physiological characteristics. Ying Yong Sheng Tai Xue Bao, 2001 Aug, 12(4), 597 - 600 {Diversity of Sinorhizobium fredii strains}; Chen M et al.; Rhizobial strains were isolated from soils growing with soybean cultivar Heilong 33 and Willimas . Fifty Sinorhizobium fredii strains were chosen, and their biological characteristics including growth velocity, acid-alkali endurance, resistance of intrinsic antibiotics, utilization of carbon and nitrogen sources, absorption of congo red, ability of melanin production, and plasmid profiles were comparatively researched . The dendrogram was described using the method of cluster analysis, and the biodiversity of Sinorhizobium fredii from different soils was proved. FEMS Microbiol Lett, 2001 Dec 18, 205(2), 171 - 8 Molecular diversity of the plasmid genotypes among Rhizobium gene pools of sesbanias from different habitats of a semi-arid region (Delhi); Mohmmed A et al.; Plasmid genotypes of root nodulating rhizobial isolates of Sesbania, sampled from six ecologically distinct habitats, were characterized . Plasmid profile analysis revealed nine different plasmid types having molecular masses ranging from 30 to 300 MDa, distributed among six profile types that grouped the isolates into six plasmid classes . The six plasmid profiles were diverged from each other and lack many common plasmid types among them . Variation in number and types of symbiotic (Sym) plasmid was assessed by hybridization of plasmid profiles with sym gene probes . Relatedness among different plasmid types was assessed by hybridization of total DNAs as well as plasmid profiles of different isolates with labelled intact plasmid . Plasticity of plasmid genotype and possible recombination between different plasmid types is suggested from the results obtained . Structural diversity among sym plasmids was assessed by PCR amplified product profiles using primer corresponding to the reiterated nif promoter consensus element (NPC-PCR) . A total of 26 NPC-PCR profile types were recognized . Genetic diversity among sym plasmids of isolates belonging to the same plasmid class and having similar sym plasmid suggested recombinations and rearrangements of sequences within the sym plasmids . Cluster analysis based upon similarity among profile types sorted the isolates across the ecological gradient . We suggest that habitat heterogeneity and plasticity of plasmid genotype together contribute for the generation of genetic diversity leading to strainal differentiation in rhizobia. J Bacteriol, 2002 Jan, 184(1), 177 - 82 Site-specific integrative elements of rhizobiophage 16-3 can integrate into proline tRNA (CGG) genes in different bacterial genera; Semsey S et al.; The integrase protein of the Rhizobium meliloti 41 phage 16-3 has been classified as a member of the Int family of tyrosine recombinases . The site-specific recombination system of the phage belongs to the group in which the target site of integration (attB) is within a tRNA gene . Since tRNA genes are conserved, we expected that the target sequence of the site-specific recombination system of the 16-3 phage could occur in other species and integration could take place if the required putative host factors were also provided by the targeted cells . Here we report that a plasmid (pSEM167) carrying the attP element and the integrase gene (int) of the phage can integrate into the chromosomes of R . meliloti 1021 and eight other species . In all cases integration occurred at so-far-unidentified, putative proline tRNA (CGG) genes, indicating the possibility of their common origin . Multiple alignment of the sequences suggested that the location of the att core was different from that expected previously . The minimal attB was identified as a 23-bp sequence corresponding to the anticodon arm of the tRNA. J Bacteriol, 2002 Jan, 184(1), 171 - 6 Dynamics of genome architecture in Rhizobium sp . strain NGR234; Mavingui P et al.; Bacterial genomes are usually partitioned in several replicons, which are dynamic structures prone to mutation and genomic rearrangements, thus contributing to genome evolution . Nevertheless, much remains to be learned about the origins and dynamics of the formation of bacterial alternative genomic states and their possible biological consequences . To address these issues, we have studied the dynamics of the genome architecture in Rhizobium sp . strain NGR234 and analyzed its biological significance . NGR234 genome consists of three replicons: the symbiotic plasmid pNGR234a (536,165 bp), the megaplasmid pNGR234b (>2,000 kb), and the chromosome (>3,700 kb) . Here we report that genome analyses of cell siblings showed the occurrence of large-scale DNA rearrangements consisting of cointegrations and excisions between the three replicons . As a result, four new genomic architectures have emerged . Three consisted of the cointegrates between two replicons: chromosome-pNGR234a, chromosome-pNGR234b, and pNGR234a-pNGR234b . The other consisted of a cointegrate of the three replicons (chromosome-pNGR234a-pNGR234b) . Cointegration and excision of pNGR234a with either the chromosome or pNGR234b were studied and found to proceed via a Campbell-type mechanism, mediated by insertion sequence elements . We provide evidence showing that changes in the genome architecture did not alter the growth and symbiotic proficiency of Rhizobium derivatives. Arch Microbiol, 2001 Dec, 176(6), 421 - 6 Epub 2001 Sep 21. Purification and properties of two chitinolytic enzymes of Serratia plymuthica HRO-C48; Frankowski J et al.; The chitinolytic rhizobacterium Serratia plymuthica HRO-C48 was previously selected as a biocontrol agent of phytopathogenic fungi . One endochitinase (E.C . 3.2.1.14), CHIT60, and one N-acetyl-beta-1,4- D-hexosaminidase (E.C . 3.2.1.52), CHIT100, were purified and characterized . The endochitinase CHIT60, with an apparent molecular mass of 60.5 kDa, had a N-terminal amino acid sequence highly similar to that of chitinases A from Serratia liquefaciens and Serratia marcescens . The enzyme activity had its peak at 55 degrees C and pH 5.4, and increased by more than 20% in the presence of 10 mM Ca(2+), Co(2+) or Mn(2+) . Activity was inhibited by 80% in the presence of 10 mM Cu(2+) . CHIT100 appeared to be a monomeric enzyme with a molecular mass of 95.6 kDa and a pI of 6.8 . Optimal activity was obtained at 43 degrees C and pH 6.6, and decreased by more than 90 % in the presence of 10 mM Co(2+) or Cu(2+) . CHIT100 (100 microg ml(-1)) inhibited spore germination and germ tube elongation of the phytopathogenic fungus Botrytis cinerea by 28 % and 31.6 %, respectively . With CHIT60 (100 microg ml(-1)), the effect was more pronounced: 78 % inhibition of of germination and 63.9 % inhibition of germ tube elongation. Int J Biochem Cell Biol, 2002 Jan, 34(1), 33 - 42 Dimerization of Rhizobium meliloti NifH protein in Saccharomyces cerevisiae cells requires simultaneous expression of NifM protein; Petrova N et al.; Compared to free living diazotrophs, the nitrogenase system of symbiotic microorganisms, like Rhizobium (Synorhizobium) meliloti, was poorly studied . The aim of our research was to investigate whether (by analogy with Klebsiella pneumoniae) the NifM product is required and sufficient to obtain active R . meliloti Fe-protein . We cloned nifH gene of R . meliloti and nifM gene of K . pneumoniae in suitable yeast vectors . When introduced into Saccharomyces cerevisiae cells, both genes were effectively expressed to proteins similar to the native products in its immunoreactivity and apparent molecular mass . The association of R . meliloti NifH protein into dimer structure required co-expression of NifM that also conferred stability of NifH polypeptide . However, the NifH protein synthesized in yeast did not show enzyme activity, suggesting that the NifM of K . pneumoniae is incapable of activating the NifH protein of R . meliloti. Acta Biochim Pol, 2001, 48(2), 359 - 65 Nod genes and Nod signals and the evolution of the Rhizobium legume symbiosis; Debelle F et al.; The establishment of the nitrogen-fixing symbiosis between rhizobia and legumes requires an exchange of signals between the two partners . In response to flavonoids excreted by the host plant, rhizobia synthesize Nod factors (NFs) which elicit, at very low concentrations and in a specific manner, various symbiotic responses on the roots of the legume hosts . NFs from several rhizobial species have been characterized . They all are lipo-chitooligosaccharides, consisting of a backbone of generally four or five glucosamine residues N-acylated at the non-reducing end, and carrying various O-substituents . The N-acyl chain and the other substituents are important determinants of the rhizobial host specificity . A number of nodulation genes which specify the synthesis of NFs have been identified . All rhizobia, in spite of their diversity, possess conserved nodABC genes responsible for the synthesis of the N-acylated oligosaccharide core of NFs, which suggests that these genes are of a monophyletic origin . Other genes, the host specific nod genes, specify the substitutions of NFs . The central role of NFs and nod genes in the Rhizobium-legume symbiosis suggests that these factors could be used as molecular markers to study the evolution of this symbiosis . We have studied a number of NFs which are N-acylated by alpha,beta-unsaturated fatty acids . We found that the ability to synthesize such NFs does not correlate with taxonomic position of the rhizobia . However, all rhizobia that produce NFs such nodulate plants belonging to related tribes of legumes, the Trifolieae, Vicieae, and Galegeae, all of them being members of the so-called galegoid group . This suggests that the ability to recognize the NFs with alpha-beta-unsaturated fatty acids is limited to this group of legumes, and thus might have appeared only once in the course of legume evolution, in the galegoid phylum. Nucleic Acids Res, 2001 Dec 1, 29(23), 4800 - 7 A mRNA-based thermosensor controls expression of rhizobial heat shock genes; Nocker A et al.; Expression of several heat shock operons, mainly coding for small heat shock proteins, is under the control of ROSE (repression of heat shock gene expression) in various rhizobial species . This negatively cis-acting element confers temperature control by preventing expression at physiological temperatures . We provide evidence that ROSE-mediated regulation occurs at the post-transcriptional level . A detailed mutational analysis of ROSE(1)-hspA translationally fused to lacZ revealed that its highly conserved 3'-half is required for repression at normal temperatures (30 degrees C) . The mRNA in this region is predicted to form an extended secondary structure that looks very similar in all 15 known ROSE elements . Nucleotides involved in base pairing are strongly conserved, whereas nucleotides in loop regions are more divergent . Base substitutions leading to derepression of the lacZ fusion at 30 degrees C exclusively resided in potential stem structures . Optimised base pairing by elimination of a bulged residue and by introduction of complementary nucleotides in internal loops resulted in ROSE elements that were tightly repressed not only at normal but also at heat shock temperatures . We propose a model in which the temperature-regulated secondary structure of ROSE mRNA influences heat shock gene expression by controlling ribosome access to the ribosome-binding site. Plant J, 2001 Oct, 28(2), 191 - 9 Evidence for structurally specific negative feedback in the Nod factor signal transduction pathway; Oldroyd GE et al.; Nod factor is a critical signalling molecule in the establishment of the legume/rhizobial symbiosis . The Nod factor of Sinorhizobium meliloti carries O-sulphate, O-acetate and C16:2 N-acyl attachments that define its activity and host specificity . Here we assess the relative importance of these modifications for the induction of calcium spiking in Medicago truncatula . We find that Nod factor structures lacking the O-sulphate, structures lacking the O-acetate and N-acyl groups, and structures lacking the O-acetate combined with a C18:1 N-acyl group all show calcium spiking when applied at high concentrations . These calcium responses are blocked in dmi1 and dmi2 mutants, suggesting that they function through the Nod factor signal transduction pathway . The dmi3 mutant, which is proposed to function in the Nod factor signal transduction pathway downstream of calcium spiking, shows increased sensitivity to Nod factor . This increased sensitivity is only active with wild-type Nod factor and was not present when the plants were treated with mutant Nod factor structures . We propose that the Nod factor signal transduction pathway is under negative feedback regulation that is activated at or downstream of DMI3 and requires structural components of the Nod factor molecule for activity. Can J Microbiol, 2001 Oct, 47(10), 889 - 94 Rhizobitoxine production and symbiotic compatibility of Bradyrhizobium from Asian and North American lineages of amphicarpaea; Parker MA et al.; Reciprocal inoculations with Bradyrhizobium sp . isolates from the North American legume Amphicarpaea bracteata (L.) Fern . (Phaseoleae-Glycininae) and from a Japanese population of its close relative Amphicarpaea edgeworthii (Benth.) var . japonica were performed to analyze relative symbiotic compatibility . Amphicarpaea edgeworthii plants formed few or no nodules with any North American bradyrhizobial strains isolated from A . bracteata, but all A . bracteata lineages formed effective nitrogen-fixing nodules with Japanese Bradyrhizobium isolates from A . edgeworthii . However, one group of A . bracteata plants (lineage Ia) when inoculated with Japanese bradyrhizobia developed a striking leaf chlorosis similar to that known to be caused by rhizobitoxine . The beta-cystathionase inhibition assay demonstrated that significant amounts of rhizobitoxine were present in nodules formed by these Japanese bradyrhizobia . No North American bradyrhizobial isolate from A . bracteata induced chlorosis on any plants, and the beta-cystathionase assay failed to detect rhizobitoxine in nodules formed by these isolates . The role of rhizobitoxine in A . edgeworthii nodulation development was tested by inoculating plants with a Bradyrhizobium elkanii rhizobitoxine-producing strain, USDA 61, and two mutant derivatives, RX17E and RX18E, which are unable to synthesize rhizobitoxine . Amphicarpaea edgeworthii inoculated with wild-type USDA 61 developed >150 nodules per plant, while plants inoculated with RX17E and RX18E developed fewer than 10 nodules per plant . Thus, efficient nodule development in A . edgeworthii appears to be highly dependent on rhizobitoxine production by Bradyrhizobium strains. J Bacteriol, 2001 Dec, 183(24), 7241 - 52 Improved soybean root association of N-starved Bradyrhizobium japonicum; Lopez-Garcia SL et al.; In this study, we addressed the effects of N limitation in Bradyrhizobium japonicum for its association with soybean roots . The wild-type strain LP 3001 grew for six generations with a growth rate of 1.2 day(-1) in a minimal medium with 28 mM mannitol as the carbon source and with the N source {(NH(4))(2)SO(4)} limited to only 20 microM . Under these conditions, the glutamine synthetase (GS) activity was five to six times higher than in similar cultures grown with 1 or 0.1 mM (NH(4))(2)SO(4) . The NtrBC-inducible GSII form of this enzyme accounted for 60% of the specific activity in N-starved rhizobia, being negligible in the other two cultures . The exopolysaccharide (EPS) and capsular polysaccharide (CPS) contents relative to cell protein were significantly higher in the N-starved cultures, but on the other hand, the poly-3-hydroxybutyrate level did not rise in comparison with N-sufficient cultures . In agreement with the accumulation of CPS in N-starved cultures, soybean lectin (SBL) binding as well as stimulation of rhizobial adsorption to soybean roots by SBL pretreatment were higher . The last effect was evident only in cultures that had not entered stationary phase . We also studied nodC gene induction in relation to N starvation . In the chromosomal nodC::lacZ fusion Bj110-573, nodC gene expression was induced by genistein 2.7-fold more in N-starved young cultures than in nonstarved ones . In stationary-phase cultures, nodC gene expression was similarly induced in N-limited cultures, but induction was negligible in cultures limited by another nutrient . Nodulation profiles obtained with strain LP 3001 grown under N starvation indicated that these cultures nodulated faster . In addition, as culture age increased, the nodulation efficiency decreased for two reasons: fewer nodules were formed, and nodulation was delayed . However, their relative importance was different according to the nutrient condition: in older cultures the overall decrease in the number of nodules was the main effect in N-starved cultures, whereas a delay in nodulation was more responsible for a loss in efficiency of N-sufficient cultures . Competition for nodulation was studied with young cultures of two wild-type strains differing only in their antibiotic resistance, the N-starved cultures being the most competitive. J Bacteriol, 2001 Dec, 183(24), 7067 - 75 Identification of essential amino acids in the Azorhizobium caulinodans fucosyltransferase NodZ; Chazalet V et al.; The nodZ gene, which is present in various rhizobial species, is involved in the addition of a fucose residue in an alpha 1-6 linkage to the reducing N-acetylglucosamine residue of lipo-chitin oligosaccharide signal molecules, the so-called Nod factors . Fucosylation of Nod factors is known to affect nodulation efficiency and host specificity . Despite a lack of overall sequence identity, NodZ proteins share conserved peptide motifs with mammalian and plant fucosyltransferases that participate in the biosynthesis of complex glycans and polysaccharides . These peptide motifs are thought to play important roles in catalysis . NodZ was expressed as an active and soluble form in Escherichia coli and was subjected to site-directed mutagenesis to investigate the role of the most conserved residues . Enzyme assays demonstrate that the replacement of the invariant Arg-182 by either alanine, lysine, or aspartate results in products with no detectable activity . A similar result is obtained with the replacement of the conserved acidic position (Asp-275) into its corresponding amide form . The residues His-183 and Asn-185 appear to fulfill functions that are more specific to the NodZ subfamily . Secondary structure predictions and threading analyses suggest the presence of a "Rossmann-type" nucleotide binding domain in the half C-terminal part of the catalytic domain of fucosyltransferases . Site-directed mutagenesis combined with theoretical approaches have shed light on the possible nucleotide donor recognition mode for NodZ and related fucosyltransferases. J Bacteriol, 2001 Dec, 183(24), 6999 - 7006 Regulation of gene expression in response to oxygen in Rhizobium etli: role of FnrN in fixNOQP expression and in symbiotic nitrogen fixation; Lopez O et al.; Previously, we reported finding duplicated fixNOQP operons in Rhizobium etli CFN42 . One of these duplicated operons is located in the symbiotic plasmid (fixNOQPd), while the other is located in a cryptic plasmid (fixNOQPf) . Although a novel FixL-FixKf regulatory cascade participates in microaerobic expression of both fixNOQP duplicated operons, we found that a mutation in fixL eliminates fixNOQPf expression but has only a moderate effect on expression of fixNOQPd . This suggests that there are differential regulatory controls . Interestingly, only the fixNOQPd operon was essential for symbiotic nitrogen fixation (L . Girard, S . Brom, A . Davalos, O . Lopez, M . Soberon, and D . Romero, Mol . Plant-Microbe Interact . 13:1283-1292, 2000) . Searching for potential candidates responsible for the differential expression, we characterized two fnrN homologs (encoding transcriptional activators of the cyclic AMP receptor protein {CRP}-Fnr family) in R . etli CFN42 . One of these genes (fnrNd) is located on the symbiotic plasmid, while the other (fnrNchr) is located on the chromosome . Analysis of the expression of the fnrN genes using transcriptional fusions with lacZ showed that the two fnrN genes are differentially regulated, since only fnrNd is expressed in microaerobic cultures of the wild-type strain while fnrNchr is negatively controlled by FixL . Mutagenesis of the two fnrN genes showed that both genes participate, in conjunction with FixL-FixKf, in the microaerobic induction of the fixNOQPd operon . Participation of these genes is also seen during the symbiotic process, in which mutations in fnrNd and fnrNchr, either singly or in combination, lead to reductions in nitrogen fixation . Therefore, R . etli employs a regulatory circuit for induction of the fixNOQPd operon that involves at least three transcriptional regulators of the CRP-Fnr family . This regulatory circuit may be important for ensuring optimal production of the cbb(3), terminal oxidase during symbiosis. Arch Biochem Biophys, 2001 Dec 1, 396(1), 99 - 105 Purification of Cajanus cajan root lectin and its interaction with rhizobial lipopolysaccharide as studied by different spectroscopic techniques; Naeem A et al.; A lectin present in roots of Cajanus cajan seedlings was isolated and purified by affinity chromatography . Sugar specificity assayed by hemagglutination-inhibition activity indicated that lectin belongs to glucose/mannose-specific group . The root lectin was found to be mannose-specific from the second day onwards as it was reconfirmed by specific elution of different days' sample from mannose agarose matrix . The maximum interaction of lectin with goat IgM was obtained in 10-day-old sample, indicating the highest crude lectin content . Lectin (total amount of eluted protein) from different days soil sample showed a maximum amount in 10-day-old sample . For further studies, the lectin has been isolated from the roots of 10-day C . cajan seedlings and purified on mannose-CL agarose column by affinity chromatography . Lectin was found to be a dimer of 18.5-kDa subunit as revealed by SDS-PAGE . Tryptophan quenching fluorescence was studied for C . cajan root lectin . Secondary structure of C . cajan root lectin as studied by circular dichroism was found to be a typical beta-pleated sheet structure . The interaction of purified root lectin with C . cajan-specific rhizobial lipopolysaccharide and its inhibition by specific and nonspecific sugars was demonstrated by fluorescence and circular dichroism . Results discussed in this paper were studied for the first time by different spectroscopic methods, suggesting that C . cajan root lectin-lipopolysaccharide interaction is specific . (c)2001 Elsevier Science. Microbiol Res, 2001, 156(3), 279 - 84 Cold stress induced high molecular weight membrane polypeptides are responsible for cold tolerance in Rhizobium DDSS69; Sardesai N et al.; Cold stress induces a lag phase in the growth cycle of Rhizobium DDSS69 . Two cold sensitive mutants of DDSS69 were generated through Tn5 tagged mutagenesis . These mutants do not grow below 15 degrees C but show a growth curve comparable with the wild type grown at 5 degrees C . There is a rapid induction of two high molecular weight membrane polypeptides of 135 and 119 kDa within 15 min of exposure to 5 degrees C in DDSS69 . PAGE membrane protein profiles of stressed and non-stressed cells reveal differential regulation of genes . At 15 degrees C both mutants lack the high molecular weight polypeptides, suggesting a role in alleviation of cold stress. Microbiol Res, 2001, 156(3), 209 - 23 Suppression of maize root diseases caused by Macrophomina phaseolina, Fusarium moniliforme and Fusarium graminearum by plant growth promoting rhizobacteria; Pal KK et al.; A plant growth-promoting isolate of a fluorescent Pseudomonas sp . EM85 and two bacilli isolates MR-11(2) and MRF, isolated from maize rhizosphere, were found strongly antagonistic to Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina, causal agents of foot rots and wilting, collar rots/stalk rots and root rots and wilting, and charcoal rots of maize, respectively . Pseudomonas sp . EM85 produced antifungal antibiotics (Afa+), siderophore (Sid+), HCN (HCN+) and fluorescent pigments (Flu+) besides exhibiting plant growth promoting traits like nitrogen fixation, phosphate solubilization, and production of organic acids and IAA . While MR-11(2) produced siderophore (Sid+), antibiotics (Afa+) and antifungal volatiles (Afv+), MRF exhibited the production of antifungal antibiotics (Afa+) and siderophores (Sid+) . Bacillus spp . MRF was also found to produce organic acids and IAA, solubilized tri-calcium phosphate and fixed nitrogen from the atmosphere . All three isolates suppressed the diseases caused by Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina in vitro . A Tn5:: lacZ induced isogenic mutant of the fluorescent Pseudomonas EM85, M23, along with the two bacilli were evaluated for in situ disease suppression of maize . Results indicated that combined application of the two bacilli significantly (P = 0.05) reduced the Macrophomina-induced charcoal rots of maize by 56.04% . Treatments with the MRF isolate of Bacillus spp . and Tn5:: lacZ mutant (M23) of fluorescent Pseudomonas sp . EM85 significantly reduced collar rots, root and foot rots, and wilting of maize caused by Fusarium moniliforme and F . graminearum (P = 0.05) compared to all other treatments . All these isolates were found very efficient in colonizing the rhizotic zones of maize after inoculation . Evaluation of the population dynamics of the fluorescent Pseudomonas sp . EM85 using the Tn5:: lacZ marker and of the Bacillus spp . MRF and MR-11(2) using an antibiotic resistance marker revealed that all the three isolates could proliferate successfully in the rhizosphere, rhizoplane and endorhizosphere of maize, both at 30 and 60 days after seeding . Four antifungal compounds from fluorescent Pseudomonas sp . EM85, one from Bacillus sp . MR-11(2) and three from Bacillus sp . MRF were isolated, purified and tested in vitro and in thin layer chromatography bioassays . All these compounds inhibited R . solani, M . phaseolina, F . moniliforme, F . graminearum and F . solani strongly . Results indicated that antifungal antibiotics and/or fluorescent pigment of fluorescent Pseudomonas sp . EM85, and antifungal antibiotics of the bacilli along with the successful colonization of all the isolates might be involved in the biological suppression of the maize root diseases. Phytochem Anal, 2001 Sep-Oct, 12(5), 305 - 11 Low molecular weight organic acids and fatty acids in root exudates of two Lupinus cultivars at flowering and fruiting stages; Lucas Garcia JA et al.; Low molecular weight organic acids (LOAs) and fatty acids in root exudates of two lupin cultivars, Lupinus albus cv . Multolupa and L . luteus cv . Tremosilla, were determined at flowering and fruiting stages . LOAs were analysed by capillary electrophoresis . Acetic and citric acids were the most abundant, especially the latter in L . luteus at the flowering stage (5922.79 micrograms/g dry root) . The significant decrease in acid content of both cultivars from flowering to fruiting stages was also striking . The highest levels of acetic acid were detected in L . luteus at fruiting stage (1542.03 micrograms/g dry root) . The significant citrate production in L . luteus could be related to the low phosphorus concentration in the studied soils but not to proteoid roots, which were detected only in L . albus . The source of the LOAs detected in these exudates is also discussed, since they may be produced either by the plant or by the associated rhizobacteria . The profile of phospholipid fatty acids was determined by high-resolution GC . A high level of 18:2 omega 6 (a fatty-acid specific to fungi) was found in exudates of L . luteus (a mycorrhizal plant) in contrast to L . albus (a non-mycorrhizal plant). Arch Microbiol, 2001 Nov, 176(5), 355 - 63 Cloning and characterization of the pyruvate carboxylase from Sinorhizobium meliloti Rm1021; Dunn MF et al.; The gene encoding pyruvate carboxylase (pyc) was isolated from a Sinorhizobium meliloti Rm1021 cosmid bank by complementation of a Rhizobium tropici pyc mutant . PYC-negative mutants of S . meliloti Rm1021 were isolated by transposon mutagenesis and were unable to grow with glucose or pyruvate as sole carbon sources, but were symbiotically competent in combination with alfalfa plants . PYC activity assays, pyc::lacZ gene fusion studies and an in vivo biotinylation assay showed that PYC activity in S . meliloti was dependent mainly on biotin availability and not on changes in gene transcription . The subunit and holo-enzyme molecular masses of the S . meliloti PYC indicated that the enzyme was an alpha4 homotetramer . The S . meliloti PYC had a high apparent Ka (0.23 mM) for the allosteric activator acetyl-CoA and was product-inhibited by sub-millimolar concentrations of oxaloacetate . In contrast to other bacterial alpha4-PYCs which have been characterized, the S . meliloti enzyme was not strongly inhibited by L-aspartate. Annu Rev Phytopathol, 1999, 37, 473 - 491 HOST VARIATION FOR INTERACTIONS WITH BENEFICIAL PLANT-ASSOCIATED MICROBES; Smith KP et al.; Beneficial plant-associated microbes can profoundly influence plant health by suppressing disease, enhancing nutrient uptake, fixing atmospheric nitrogen, and promoting plant growth . Host variation, among cultivars or plant genotypes, for response to beneficial microorganisms suggests that plant genes play a role in supporting these interactions . Such host variation can be found among diverse groups of microorganisms including rhizobia, mycorrhizal fungi, and microbial biocontrol agents . Discrete variation among plant genotypes for interaction with beneficial microbes has led to the discovery of single genes that specify compatible interactions . Continuous variation for interaction phenotypes such as disease suppression, plant growth, or nutrient uptake have led to hypotheses, and in some cases genetic descriptions, of multigenic control of these interactions . Future research into the role of plant genes involved in hosting beneficial plant-associated microbes will provide greater insight into this relatively unexplored area of biology and should provide new tools to improve plant health in agriculture. Microbiology, 2001 Nov, 147(Pt 11), 3113 - 9 Altered expression of two light-dependent genes in a microcystin-lacking mutant of Microcystis aeruginosa PCC 7806; Dittmann E et al.; Microcystin is a potent inhibitor of eukaryotic protein phosphatases and has been implicated in causing hepatotoxicity to humans and animals worldwide . It is produced primarily by the bloom-forming cyanobacterium Microcystis aeruginosa, although the function of the peptide in this micro-organism is unknown . In this study, a microcystin-related protein, MrpA, was identified using a microcystin-lacking mutant of M . aeruginosa, PCC 7806 . Comparative two-dimensional protein electrophoresis showed that MrpA was strongly expressed in wild-type PCC 7806, but was not detectable in the mcyB mutant . MrpA showed similarity to the RhiA protein from Rhizobium leguminosarum, which is encoded by the rhiABC operon and controlled by quorum-sensing mediators . Sequencing of mrpA flanking regions in M . aeruginosa PCC 7806 revealed the presence of a rhiB homologue, mrpB, directly downstream of mrpA . Northern blot analyses of mrpA expression in cells exposed to different light conditions revealed a rapid decline of transcription under high light conditions . Most striking was a strong increase in transcript levels from cultures irradiated with blue light . The mrpA transcription level was strongly reduced in two independent microcystin-lacking mutants under all light conditions investigated. J Bacteriol, 2001 Dec, 183(23), 6771 - 7 Multiple N-acyl homoserine lactone signals of Rhizobium leguminosarum are synthesized in a distinct temporal pattern; Blosser-Middleton RS et al.; A common form of bacterial quorum sensing involves the production and release of acyl homoserine lactone (AHL) signal metabolites . The nitrogen-fixing symbiont Rhizobium leguminosarum reportedly produces at least six different AHLs, but little is known about the regulation of biosynthesis of these molecules . We used a radiolabeling protocol to quantify the relative amounts of AHLs synthesized over time by R . leguminosarum cells with and without the symbiosis plasmid pRL1JI . Cells containing pRL1JI were found to produce three predominant signals . In decreasing order of abundance, these were N-(3-oxo)octanoyl homoserine lactone {(3-O)C(8)HSL}, N-octanoyl homoserine lactone, and N-hexanoyl homoserine lactone . Cells without pRL1JI produced only two major signals, N-(3-hydroxy-7-cis)tetradecanoyl homoserine lactone {(3-OH)C(14:1)HSL} and (3-O)C(8)HSL . Each AHL exhibited a distinct temporal pattern of synthesis, suggesting that each AHL is subject to unique regulatory mechanisms . While (3-O)C(8)HSL was produced in both cultures, the patterns of synthesis were different in cells with and without pRL1JI, possibly as a result of redundant gene functions that are present on both the chromosome and the symbiosis plasmid . None of the AHLs appeared to regulate its own biosynthesis, although exogenous (3-OH)C(14:1)HSL did activate synthesis of the three AHLs made by cells containing pRL1JI . These results indicate that the synthesis of multiple AHLs in R . leguminosarum is regulated by complex mechanisms that operate independently of quorum sensing itself but that (3-OH)C(14:1)HSL can supersede these controls in pRL1JI-containing cells . This work provides an important global perspective for AHL regulation that both complements and contrasts with the results of previous studies performed with isolated gene systems. DNA Seq, 2001 Jul, 12(1), 1 - 12 Isolation and sequencing of Rhizobium leguminosarum Bv . Trifolii PssN, PssO and PssP genes encoding the proteins involved in polymerization and translocation of exopolysaccharide; Mazur A et al.; Rhizobium leguminosarum bv . trifolii produces an acidic exopolysaccharide (EPS) that plays an important role in symbiotic interaction with clover plants . The sequence of 6.0-kb DNA fragment located upstream of the previously described prsDEorf3 and pssCDE genes involved in exopolysaccharide biosynthesis revealed three new genes designated pssN, pssO and pssP . The predicted protein product of pssP gene shares a significant homology to members of the membrane-periplasmic auxiliary (MPA1) family, that are involved in polymerization of the repeating subunits of EPS . The putative pssN protein product is highly homologous to the family of the outer membrane auxiliary (OMA) proteins engaged in translocation of polysaccharides in bacteria . The PssO did not reveal homology to the known bacterial proteins, but showed characteristic features of outer membrane proteins, and with PssN and PssP, it might be a part of the system involved in polymerization and translocation of EPS across the bacterial membranes. Yi Chuan Xue Bao, 2001, 28(10), 964 - 70 {Cloning and functional analysis of glnB from Azospirillum brasilense Yu62}; Li ZH et al.; The glnB gene of A . brasilense Yu62 was determined in a 3.7 kb EcoRI + PstI fragment . The glnA is located downstream of glnB and an ORF for hypothetical protein is on upstream of glnB . The deduced amino acid sequence of PII encoded by glnB is 71%, 77%, 79% and 69% identical to that of K . pneumoniae, Bradyrhizobium japonicum, Rhizobium leguninosarum and E . coli, respectively . A Km-casette was inserted into BglII site of glnB coding region and GlnB- mutant was obtained by homologous recombination . The GlnB- mutant has lost the nitrogenase activity, i.e.: Nif- . For the functional confirmation of glnB gene, a complementary test was carried out and it was shown that C-glnB(glnB::Km/glnB) can restore the nitrogenase activity . When the recombinant plasmid pVK-II which containined the coding region of glnB was introduced into A . brasilense Yu62 and A . brasilense Yu62 DraT-, respectively, the Yu62-II (containing pVK-II) and draT-II(containing pVK-II) showed higher nitrogenase activity than wild type . These results confirmed that glnB plays an important role in the regulation of nitrogen in A . brasilense. Biochem Biophys Res Commun, 2001 Nov 9, 288(4), 757 - 64 Mammalian PASKIN, a PAS-serine/threonine kinase related to bacterial oxygen sensors; Hofer T et al.; The PAS domain is a versatile protein fold found in many archaeal, bacterial, and plant proteins capable of sensing environmental changes in light intensity, oxygen concentration, and redox potentials . The oxygen sensor FixL from Rhizobium species contains a heme-bearing PAS domain and a histidine kinase domain that couples sensing to signaling . We identified a novel mammalian PAS protein (PASKIN) containing a domain architecture resembling FixL . PASKIN is encoded by an evolutionarily conserved single-copy gene which is ubiquitously expressed . The human PASKIN and mouse Paskin genes show a conserved intron-exon structure and share their promoter regions with another ubiquitously expressed gene that encodes a regulator of protein phosphatase-1 . The 144-kDa PASKIN protein contains a PAS region homologous to the FixL PAS domain and a serine/threonine kinase domain which might be involved in signaling . Thus, PASKIN is likely to function as a mammalian PAS sensor protein . Can J Microbiol, 2001 Sep, 47(9), 807 - 12 Effects of some salts and sodicity on the growth of a Rhizobium leguminosarum bv . viceae strain isolated from a salt-affected soil; Faituri MY et al.; The effects of sodium (Na+), calcium (Ca2+), magnesium (Mg2+), and boron (B) concentrations and sodicity, as measured by the sodium adsorption ratio (SAR), on the growth of a Rhizobium leguminosarum bv . viceae strain isolated from a salt-affected soil were studied . The rate of growth was measured in a yeast extract-mannitol broth, amended with salts having electrical conductivity (EC) of 4, 8, and 16 dS x m(-1) . Each salinity level was prepared to achieve SAR values of 10, 20, and 30 with or without graded B concentrations of 0.5, 1, 3, and 5 mg x L(-1) . We found that salinity levels equal to or more than 8 dS x m(-1) had negative effects on Rhizobium growth during the first days of incubation, but the effects became less pronounced after 1 week . Na+ concentrations of more than 1.1 g x L(-1) retarded growth, especially at high SAR values (i.e., at low Ca2+ concentrations) . The retardation of growth increased with increases in EC up to 16 dS x m(-1), at all sodicity levels . Mg2+ added together with Na+ or with Ca2+ + Na+ affected growth more negatively than Ca2+ + Na+ alone . The effect of Mg2+ became more pronounced with increased salinities and sodicities . It was concluded that EC of more than 4 dS x m(-1) retarded growth of Rhizobium, but only at high sodicity levels . The relative specific ion effect on growth was in the order Na+ < Ca2+ < Mg2+ . The harmful effect of Mg2+ on this strain was accentuated by adding Ca2+ to the cultural medium . When SAR increased from 10 to 30, Na+ had no clear effect on growth, irrespective of the accompanied cations, i.e, Ca2+, Mg2+, or Ca2+ + Mg2+ . Growth was reduced by B concentrations as low as 0.5 mg x L(-1), and the B effect was enhanced by increased salinity. Appl Environ Microbiol, 2001 Nov, 67(11), 4999 - 5009 DNA sequence and mutational analysis of rhizobitoxine biosynthesis genes in Bradyrhizobium elkanii; Yasuta T et al.; We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii . The nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxA showed that several open reading frames (ORFs) were located downstream of rtxA . A large-deletion mutant of B . elkanii, USDA94 Delta rtx::Omega 1, which lacks rtxA, ORF1 (rtxC), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol . The broad-host-range cosmid pLAFR1, which contains rtxA and these ORFs, complemented rhizobitoxine production in USDA94 Delta rtx::Omega 1 . Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxA and rtxC are necessary for rhizobitoxine production . Insertional mutagenesis of the N-terminal and C-terminal regions of rtxA indicated that rtxA is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis . An insertional mutant of rtxC produced serinol and dihydrorhizobitoxine but no rhizobitoxine . Moreover, the rtxC product was highly homologous to the fatty acid desaturase of Pseudomonas syringae and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase . This result suggested that rtxC encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production . In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia. Mol Microbiol, 2001 Oct, 42(1), 195 - 204 RepA negatively autoregulates the transcription of the repABC operon of the Rhizobium etli symbiotic plasmid basic replicon; Ramirez-Romero MA et al.; The basic replicon of Rhizobium etli CE3, like other members of the repABC plasmid family, is constituted by the repABC operon . RepC is essential for replication, and RepA and RepB play a role in plasmid segregation . It has been shown that deletion derivatives lacking the repAB genes have an increased copy number, indicating that these genes participate in the control of plasmid copy number . RepA is also a trans-incompatibility factor . To understand the regulation of the repABC operon, in this paper: (i) the transcription start site of the repABC operon was determined; (ii) the promoter region was identified by site-directed mutagenesis of the putative -35 and -10 hexameric elements; and (iii) RepA was recognized as a negative regulator of the transcription of the repABC operon. Biochemistry, 2001 Oct 30, 40(43), 12932 - 42 Insights into the signal transduction mechanism of RmFixL provided by carbon monoxide recombination kinetics; Rodgers KR et al.; This report presents evidence for interdomain steps of the ligand-coupled signal transduction mechanism of the oxygen receptor from Rhizobium meliloti, RmFixL . Photolysis of the CO adducts of heme domain (RmFixLN) and heme kinase (RmFixL*) proteins allowed tracking of second-order heme CO recombination reactions by transient absorbance . Whereas CO rebinding to RmFixLN is characterized by a single kinetic phase, rebinding to RmFixL* is characterized by two kinetic phases . Evidence indicates that CO rebinds to two interconvertible deoxyRmFixL* conformers that are produced sequentially after photolysis . Since the second conformer is only observed when the kinase domain is present, its production is concluded to be an interdomain signal transmission event that is coupled to heme ligand release . Because receptor clustering is a recurring theme in signal transduction mechanisms, the dependence of molecular weight upon heme ligation was investigated at equilibrium . Gel permeation chromatography and native gel electrophoresis showed that the molecular weight distribution for both RmFixLN and RmFixL* depends on heme ligation . At equilibrium, oxyRmFixLN and oxyRmFixL* exist as monomers and dimers, respectively . Their deoxy analogues, metRmFixLN and metRmFixL*, exist as dimers and as a mixture of tetramers and 9-mers, respectively . Assembly of these oligomers is reversible . The physiological relevance of these ligand-coupled assemblies and the kinetic factors controlling CO recombination are discussed. Proc Natl Acad Sci U S A, 1995 May 9, 92(10), 4197 - 201 Molecular mechanisms of defense by rhizobacteria against root disease; Cook RJ et al.; Genetic resistance in plants to root diseases is rare, and agriculture depends instead on practices such as crop rotation and soil fumigation to control these diseases . "Induced suppression" is a natural phenomenon whereby a soil due to microbiological changes converts from conducive to suppressive to a soilborne pathogen during prolonged monoculture of the susceptible host . Our studies have focused on the wheat root disease "take-all," caused by the fungus Gaeumannomyces graminis var . tritici, and the role of bacteria in the wheat rhizosphere (rhizobacteria) in a well-documented induced suppression (take-all decline) that occurs in response to the disease and continued monoculture of wheat . The results summarized herein show that antibiotic production plays a significant role in both plant defense by and ecological competence of rhizobacteria . Production of phenazine and phloroglucinol antibiotics, as examples, account for most of the natural defense provided by fluorescent Pseudomonas strains isolated from among the diversity of rhizobacteria associated with take-all decline . There appear to be at least three levels of regulation of genes for antibiotic biosynthesis: environmental sensing, global regulation that ties antibiotic production to cellular metabolism, and regulatory loci linked to genes for pathway enzymes . Plant defense by rhizobacteria producing antibiotics on roots and as cohabitants with pathogens in infected tissues is analogous to defense by the plant's production of phytoalexins, even to the extent that an enzyme of the same chalcone/stilbene synthase family used to produce phytoalexins is used to produce 2,4-diacetylphloroglucinol . The defense strategy favored by selection pressure imposed on plants by soilborne pathogens may well be the ability of plants to support and respond to rhizosphere microorganisms antagonistic to these pathogens. Mol Plant Microbe Interact, 2001 Oct, 14(10), 1189 - 96 The antioxidants of legume nodule mitochondria; Iturbe-Ormaetxe I et al.; The mitochondria of legume root nodules are critical to sustain the energy-intensive process of nitrogen fixation . They also generate reactive oxygen species at high rates and thus require the protection of antioxidant enzymes and metabolites . We show here that highly purified mitochondria from bean nodules (Phaseolus vulgaris L . cv . Contender x Rhizobium leguminosarum bv . phaseoli strain 3622) contain ascorbate peroxidase primarily in the inner membrane (with lesser amounts detected occasionally in the matrix), guaiacol peroxidases in the outer membrane and matrix, and manganese superoxide dismutase (MnSOD) and an ascorbate-regenerating system in the matrix . This regenerating system relies on homoglutathione (instead of glutathione) and pyridine nucleotides as electron donors and involves the enzymes monodehydroascorbate reductase, dehydroascorbate reductase, and homoglutathione reductase . Homoglutathione is synthesized in the cytosol and taken up by the mitochondria and bacteroids . Although bacteroids synthesize glutathione, it is not exported to the plant in significant amounts . We propose a model for the detoxification of peroxides in nodule mitochondria in which membrane-bound ascorbate peroxidase scavenges the peroxide formed by the electron transport chain using ascorbate provided by L-galactono-1,4-lactone dehydrogenase in the inner membrane . The resulting monodehydroascorbate and dehydroascorbate can be recycled in the matrix or cytosol . In the matrix, the peroxides formed by oxidative reactions and by MnSOD may be scavenged by specific isozymes of guaiacol peroxidase, ascorbate peroxidase, and catalase. Mol Plant Microbe Interact, 2001 Oct, 14(10), 1168 - 77 Overlapping plant signal transduction pathways induced by a parasitic nematode and a rhizobial endosymbiont; Koltai H et al.; Root-knot nematodes and rhizobia establish interactions with roots characterized by the de novo induction of host structures, termed giant cells and nodules, respectively . Two transcription regulators, PHAN and KNOX, required for the establishment of meristems were previously shown to be expressed in tomato giant cells . We isolated the orthologues of PHAN and KNOX (Mt-phan and Mt-knox-1) from the model legume Medicago truncatula, and established the spatial distribution of their expression in situ . We confirmed that Mt-phan and Mt-knox-1 are expressed in lateral root initials and in nematode-induced giant cells and showed that they are expressed in nodules induced by Sinorhizobium meliloti . Expression of both genes becomes spatially restricted as the nodules develop . We further examined nematode feeding sites for the expression of two genes involved in nodule formation, ccs52 (encodes a mitotic inhibitor) and ENOD40 (encodes an early, nodulation mitogen), and found transcripts of both genes to be present in and around giant cells induced in Medicago . Collectively, these results reveal common elements of host responses to mutualistic and parasitic plant endosymbionts and imply that overlapping regulatory pathways lead to giant cells and nodules . We discuss these pathways in the context of phytohormones and parallels between beneficial symbiosis and disease. J Exp Bot, 2001 Nov, 52(364), 2181 - 6 Redifferentiation of bacteria isolated from Lotus japonicus root nodules colonized by Rhizobium sp . NGR234; Muller J et al.; In most studies concerning legume root nodules, the question to what extent the nodule-borne bacteroids survive nodule senescence has not been properly addressed . At present, there is no "model system" to study these aspects in detail . Such a system with Lotus japonicus and the broad host range Rhizobium sp . NGR234 has been developed . L . japonicus L . cv . Gifu was inoculated with Rhizobium sp . NGR234 and grown over a 12 week time period . The first nodules could be harvested after 3 weeks . Nodulation reached a plateau after 11 weeks with a mean of 64 nodules having a biomass of nearly 100 mg FW per plant . Nodules were harvested and homogenized at different stages of plant development . Microscopic inspection of the extracts revealed that, typically, nodules contained c . 15x10(9) bacteroids g(-1) FW, and that about 60% of the bacteroids were viable as judged by vital staining . When aliquots of the extracts were plated on selective media, a substantial number of "colony-forming units" was observed in all cases, indicating that a considerable fraction of the bacteroids had the potential to redifferentiate into growing bacteria . In nodules from the early developmental stages, the fraction of total bacteroids yielding CFUs amounted to about 20%, or one-third of the bacteroids judged to be viable after extraction, and it increased slightly when the plants started to flower . In order to see how nodule senescence affected the survival and redifferentiation potential of bacteroids, some plants were placed in the dark for 1 week . This led to typical symptoms of senescence in the nodules such as an almost complete loss of nitrogenase activity and a considerable decrease in soluble proteins . However, surprisingly, the number of total and viable bacteroids g(-1) nodule FW remained virtually constant, and the fraction of total bacteroids yielding CFUs did not decrease but significantly increased up to 75% of the bacteroids judged to be viable after extraction . This result indicates that during nodule senescence bacteroids might be induced to redifferentiate into the state of free-living, growing bacteria. Mol Microbiol, 2001 Sep, 41(6), 1357 - 64 Feedback regulation of the Bradyrhizobium japonicum nodulation genes; Loh JT et al.; Lipochitin Nod signals are produced by rhizobia and are required for the establishment of a nitrogen-fixing symbiosis with a legume host . The nodulation genes encode products required for the synthesis of this signal and are induced in response to plant-produced flavonoid compounds . The addition of chitin and lipo-chitin oligomers to Bradyrhizobium japonicum cultures resulted in a significant reduction in the expression of a nod-lacZ fusion . Intracellular expression of NodC, encoding a chitin synthase, also reduced nod gene expression . In contrast, expression of the ChiB chitinase increased nod gene expression . The chain length of the oligosaccharide was important in feedback regulation, with chitotetraose molecules the best modulators of nod gene expression . Feedback regulation is mediated by the induction of nolA by chitin, resulting in elevated levels of the repressor protein, NodD2. Genome Biol . 2001;2(9):RESEARCH0037 . Epub 2001 Aug 23. On the species of origin: diagnosing the source of symbiotic transcripts; Hraber PT et al.; BACKGROUND: Most organisms have developed ways to recognize and interact with other species . Symbiotic interactions range from pathogenic to mutualistic . Some molecular mechanisms of interspecific interaction are well understood, but many remain to be discovered . Expressed sequence tags (ESTs) from cultures of interacting symbionts can help identify transcripts that regulate symbiosis, but present a unique challenge for functional analysis . Given a sequence expressed in an interaction between two symbionts, the challenge is to determine from which organism the transcript originated . For high-throughput sequencing from interaction cultures, a reliable computational approach is needed . Previous investigations into GC nucleotide content and comparative similarity searching provide provisional solutions, but a comparative lexical analysis, which uses a likelihood-ratio test of hexamer counts, is more powerful . RESULTS: Validation with genes whose origin and function are known yielded 94% accuracy . Microbial (non-plant) transcripts comprised 75% of a Phytophthora sojae-infected soybean (Glycine max cv Harasoy) library, contrasted with 15% or less in root tissue libraries of Medicago truncatula from axenic, Phytophthora medicaginis-infected, mycorrhizal, and rhizobacterial treatments . Mycorrhizal libraries contained about 23% microbial transcripts; an axenic plant library contained a similar proportion of putative microbial transcri