Rhizobacterium

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 transcripts . CONCLUSIONS: Comparative lexical analysis offers numerous advantages over alternative approaches . Many of the transcripts isolated from mixed cultures were of unknown function, suggesting specificity to symbiotic metabolism and therefore candidates likely to be interesting for further functional investigation . Future investigations will determine whether the abundance of non-plant transcripts in a pure plant library indicates procedural artifacts, horizontally transferred genes, or other phenomena.

Microbiol Res, 2001, 156(2), 145 - 9
Improvement in bioavailability of tricalcium phosphate to Cymbopogon martinii var . motia by rhizobacteria, AMF and Azospirillum inoculation; Ratti N et al.; The interactive effects of phosphate solubilizing bacteria, N2 fixing bacteria and arbuscular mycorrhizal fungi (AMF) were studied in a low phosphate alkaline soil amended with tricalcium insoluble source of inorganic phosphate on the growth of an aromatic grass palmarosa (Cymbopogon martinii) . The microbial inocula consisted of the AM fungus Glomus aggregatum, phosphate solubilizing rhizobacteria Bacillus polymyxa and N2 fixing bacteria Azospirillum brasilense . These rhizobacteria behaved as "mycorrhiza helper" and enhanced root colonization by G . aggregatum in presence of tricalcium phosphate at the rate of 200 mg kg(-1) soil (P1 level) . Dual inoculation of G . aggregatum and B . polymyxa yielded 21.5 g plant dry weight (biomass), while it was 21.7 g in B . polymyxa and A . brasilense inoculated plants as compared to 14.9 g of control at the same level . Phosphate content was maximum (0.167%) in the combined treatment of G . aggregatum, B . polymyxa and A . brasilense at P1 level, however acid phosphatase activity was recorded to be 4.75 pmol mg(-1) min(-1) in G . aggregatum, B . polymyxa and A . brasilense treatment at P0 level . This study indicates that all microbes inoculated together help in the uptake of tricalcium phosphate which is otherwise not used by the plants and their addition at 200 mg kg(-1) of soil gave higher productivity to palmarosa plants.

J Bacteriol, 2001 Oct, 183(20), 6054 - 64
Genetic locus required for antigenic maturation of Rhizobium etli CE3 lipopolysaccharide; Duelli DM et al.; Rhizobium etli modifies lipopolysaccharide (LPS) structure in response to environmental signals, such as low pH and anthocyanins . These LPS modifications result in the loss of reactivity with certain monoclonal antibodies . The same antibodies fail to recognize previously isolated R . etli mutant strain CE367, even in the absence of such environmental cues . Chemical analysis of the LPS in strain CE367 demonstrated that it lacked the terminal sugar of the wild-type O antigen, 2,3,4-tri-O-methylfucose . A 3-kb stretch of DNA, designated as lpe3, restored wild-type antigenicity when transferred into CE367 . From the sequence of this DNA, five open reading frames were postulated . Site-directed mutagenesis and complementation analysis suggested that the genes were organized in at least two transcriptional units, both of which were required for the production of LPS reactive with the diagnostic antibodies . Growth in anthocyanins or at low pH did not alter the specific expression of gusA from the transposon insertion of mutant CE367, nor did the presence of multiple copies of lpe3 situated behind a strong, constitutive promoter prevent epitope changes induced by these environmental cues . Mutations of the lpe genes did not prevent normal nodule development on Phaseolus vulgaris and had very little effect on the occupation of nodules in competition with the wild-type strain.

J Biotechnol, 2001 Oct 4, 91(2-3), 269 - 82
Improvement of forage production in Calliandra calothyrsus: methodology for the identification of an effective inoculum containing Rhizobium strains and arbuscular mycorrhizal isolates; Lesueur D et al.; The overall aim of this paper is to describe the selection of effective rhizobia and arbuscular mycorrhizas (AM), which after inoculation, will significantly improve the forage production of Calliandra calothyrsus under field conditions . To achieve this objective, the following activities were carried out: (i) establishment from both nodules and soil samples of a collection of microsymbionts (rhizobium and AM) of C . calothyrsus from Central America (Mexico, Honduras, Guatemala, Nicaragua and Costa Rica), also from outside its native range in Cameroon, Kenya and New Caledonia; (ii) identification under glasshouse conditions of the most effective rhizobia and AM isolates; (iii) production of a solid selected inoculum for field trials; (iv) examination of the impact of the inoculation on the growth of C . calothyrsus monitored under nursery conditions . We have screened 446 rhizobia strains in the nursery and identified six as being very effective at nodulating the host plant . They originated from Costa Rica (CCCR15 and CCCR1), from New Caledonia (CCNC26), from Cameroon (CCC22) and from Kenya (KWN35 and KCC6) . In relation to AM, five isolates have been selected for the ability to infect and promote growth of the host plant--two isolates of Gigaspora albida isolated from Kenya (GA1b and GA2); one isolate of Scutellospora verrucosa isolated from Kenya (SV2c); one isolate of Scutellospora calospora isolated from Guatemala (SC2) and one isolate of Glomus etunicatum isolated from Honduras (GE1) . Further experiments will test these selected inocula, singly and in mixtures, in order to obtain an inoculant which significantly improves the growth of C . calothyrsus and to enable its distribution to farmers who use this woody legume for forage production on their farms.

J Biotechnol, 2001 Oct 4, 91(2-3), 257 - 68
An interdisciplinary research strategy to improve symbiotic nitrogen fixation and yield of common bean (Phaseolus vulgaris) in salinised areas of the Mediterranean basin; Drevon JJ et al.; The main findings of a cooperative research group of agronomists, plant breeders, microbiologists, physiologists and molecularists to improve the symbiotic nitrogen fixation (SNF) and N2-dependent yield of common bean under moderate salinity in the Mediterranean basin are summarised . Agronomic surveys in reference production areas show large spatial and temporal variations in plant nodulation and growth, and in efficiency of utilisation of the rhizobial symbiosis . The latter was associated with a large rhizobial diversity, including new bean nodulating species . Macrosymbiont diversity in SNF and adaptation to NaCl was found . However, contrasts between plant genotypes could be altered by specific interactions with some native rhizobia . Therefore, variations in soil rhizobial population, in addition to agronomic practices and environmental constraints, may have contributed to erratic results observed in field inoculations . At the mechanistic level, nodule C and N metabolisms, and abcissic acid content, were related to SNF potential and tolerance to NaCl . Their relation with nodule conductance to O2 diffusion was addressed by in situ hybridisation of candidate carbonic anhydrase and aquaporin genes in nodule cortex . The limits and prospects of the cooperative strategy are discussed.

J Biotechnol, 2001 Oct 4, 91(2-3), 243 - 55
Effect of pH and soybean cultivars on the quantitative analyses of soybean rhizobia populations; Yang SS et al.; Quantitative analyses of fast- and slow-growing soybean rhizobia populations in soils of four different provinces of China (Hubei, Shan Dong, Henan, and Xinjiang) have been carried out using the most probable number technique (MPN) . All soils contained fast- (FSR) and slow-growing (SSR) soybean rhizobia . Asiatic and American soybean cultivars grown at acid, neutral and alkaline pH were used as trapping hosts for FSR and SSR strains . The estimated total indigenous soybean-rhizobia populations of the Xinjiang and Shan Dong soil samples greatly varied with the different soybean cultivars used . The soybean cultivar and the pH at which plants were grown also showed clear effects on the FSR/SSR rations isolated from nodules . Results of competition experiments between FSR and SSR strains supported the importance of the soybean cultivar and the pH on the outcome of competition for nodulation between FSR and SSR strains . In general, nodule occupancy by FSRs significantly increased at alkaline pH . Bacterial isolates from soybean cultivar Jing Dou 19 inoculated with Xinjiang soil nodulate cultivars Heinong 33 and Williams very poorly . Plasmid and lipopolysaccharide (LPS) profiles and PCR-RAPD analyses showed that cultivar Jing Dou 19 had trapped a diversity of FSR strains . Most of the isolates from soybean cultivar Heinong 33 inoculated with Xinjiang soil were able to nodulate Heinong 33 and Williams showed very similar, or identical, plasmid, LPS and PCR-RAPD profiles . All the strains isolated from Xinjiang province, regardless of the soybean cultivar used for trapping, showed similar nodulation factor (LCO) profiles as judged by thin layer chromatographic analyses . These results indicate that the existence of soybean rhizobia sub-populations showing marked cultivar specificity, can affect the estimation of total soybean rhizobia populations indigenous to the soil, and can also affect the diversity of soybean rhizobial strains isolated from soybean nodules.

J Biotechnol, 2001 Oct 4, 91(2-3), 211 - 21
Novel pathway for phosphatidylcholine biosynthesis in bacteria associated with eukaryotes; Lopez-Lara IM et al.; Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesised by either of two pathways, the CDP-choline pathway or the methylation pathway . Many prokaryotes lack PC, but it can be found in significant amounts in membranes of distantly related bacteria such as Rhizobacteria and Spirochetes . Enzymatic methylation of phosphatidylethanolamine via the methylation pathway was thought to be the only biosynthetic pathway to yield PC in bacteria . However, a novel choline-dependent pathway for PC biosynthesis has been discovered in Sinorhizobium meliloti . In this pathway, a novel enzymatic activity, PC synthase, condenses choline directly with CDP-diacylglyceride to form PC in one step . Surprisingly, genomes of some pathogens (Pseudomonas aeruginosa, Borrelia burgdorferi and Legionella pneumophila) contain genes similar to the sinorhizobial gene for phosphatidylcholine synthase . We, therefore, suggest that the new PC synthase pathway is present in a number of bacteria displaying symbiotic or pathogenic associations with eukaryotes and that the eukaryotic host functions as the provider of choline for this pathway.

J Biotechnol, 2001 Oct 4, 91(2-3), 189 - 95
Recent developments in the structural organization and regulation of nitrogen fixation genes in Herbaspirillum seropedicae; Pedrosa FO et al.; Herbaspirillum seropedicae is a nitrogen-fixing bacterium found in association with economically important gramineae . Regulation of nitrogen fixation involves the transcriptional activator NifA protein . The regulation of NifA protein and its truncated mutant proteins is described and compared with that of other nitrogen fixation bacteria . Nitrogen fixation control in H . seropedicae, of the beta-subgroup of Proteobacteria, has regulatory features in common with Klebsiella pneumoniae, of the gamma-subgroup, at the level of nifA expression and with rhizobia and Azospirillum brasilense, of the alpha-subgroup, at the level of control of NifA by oxygen.

J Biotechnol, 2001 Oct 4, 91(2-3), 181 - 8
The diversity of rhizobia nodulating beans in Northwest Argentina as a source of more efficient inoculant strains; Aguilar OM et al.; The common bean (Phaseolus vulgaris L.) is cultivated widely in Central and South America and particularly in the Northwest of Argentina . In order to describe the diversity of the common bean nodulating rhizobial population from the bean producing area in Northwest Argentina (NWA), a collection of about 400 isolates of common beans recovered from nodules and soil samples from NWA were characterized by using nifH-PCR, analysis of genes coding for 16S rRNA and nodC, and REP-fingerprinting, respectively . It was found that species Rhizobium etli is predominant in common bean nodules although a high degree of diversity was found within the species . Other bean nodulating genotypes recovered from soils by using Leucaena sp . as the trapping host was found to have the 16S rDNA alleles of species such as Sinorhizobium fredii, Sinorhizobium saheli, Sinorhizobium teranga, Mesorhizobium loti, and Rhizobium tropici . Some of the bean genotypes that were found to be more efficient in green house experiments were selected and assayed in two successive bean-cropping seasons in the field environment in NWA, and an increase in yields with inoculation was found . The performance of strains isolated from the region indicates potential for exploiting the diversity.

J Biotechnol, 2001 Oct 4, 91(2-3), 169 - 80
AFLP fingerprinting as a tool to study the genetic diversity of Rhizobium galegae isolated from Galega orientalis and Galega officinalis; Terefework Z et al.; AFLP fingerprints of Rhizobium galegae strains that infect Galega orientalis and Galega officinalis obtained from different geographical sources, and of taxonomically diverse rhizobia representing the recognized species, were generated . Comparisons of the fingerprints from fluorescent labeled AFLP products using capillary electrophoresis on ABI prism 310, slab gel electrophoresis on ABI prism 377 genetic analyzers and silver staining were in good agreement . All methods delineated the G . orientalis strains from G . officinalis strains, the G . orientalis strains formed a tight cluster whereas the G . officinalis strains seem to show a greater level of genetic diversity . Comparison of fluorescent AFLP with other detection methods revealed that fluorescent labeling is more sensitive and practical, in addition, the deleterious effect of radioactivity associated with 32P-labeling, the delicate process of blotting polyacrylamide gels or the tedious procedure of silver staining can be avoided . The automated system facilitated a large number of runs at a time and the subsequent analysis of the data by generating exportable raw data . The congruency of the experiments was analyzed using the Bionumerics software.

J Biotechnol, 2001 Oct 4, 91(2-3), 143 - 53
Rhizobia from wild legumes: diversity, taxonomy, ecology, nitrogen fixation and biotechnology; Zahran HH; Wild legumes (herb or tree) are widely distributed in arid regions and actively contribute to soil fertility in these environments . The N2-fixing activity and tolerance to drastic conditions may be higher in wild legumes than in crop legumes . The wild legumes in arid zones harbor diverse and promiscuous rhizobia in their root-nodules . Specificity existed only in few rhizobia from wild legumes, however, the majority of them are with wide host range . Based on phenotypic characteristics and molecular techniques (protein profiles, polysaccharides, plasmids, DNA-DNA hybridization, 16SrRNA, etc.), the root-nodule bacteria that was isolated from wild legumes had been classified into four genera (Rhizobium, Bradyrhizobium, Mesorhizobium and Sinorhizobium) . The rhizobia of wild legumes in arid zones, exhibit higher tolerance to the prevailing adverse conditions, e.g . salt stress, elevated temperatures and desiccation . These rhizobia may be used to inoculate wild, as well as, crop legumes, cultivated in reclaimed desert lands . Recent reports indicated that the wild-legume rhizobia formed successful symbioses with some grain legumes . Moreover, intercropping of some N2-fixing tree legumes (e.g . Lablab, Leucaena, Sesbania, etc.) to pasture grasses improved biomass yield and herb quality . In recent years, the rhizobia of wild legumes turn the attention of biotechnologists . These bacteria may have specific traits that can be transferred to other rhizobia through genetic engineering tools or used to produce industrially important compounds . Therefore, these bacteria are very important from both economic and environmental points of view.

J Biotechnol, 2001 Oct 4, 91(2-3), 117 - 26
Natural endophytic association between Rhizobium etli and maize (Zea mays L.); Gutierrez-Zamora ML et al.; Maize (Zea mays) and bean (Phaseolus vulgaris) have been traditionally grown in association for thousands of years in Mesoamerica . From surface sterilized maize roots, we have isolated over 60 Rhizobium strains that correspond to Rhizobium etli bv . phaseoli (the main symbiont of bean) on the basis of 16S rRNA gene restriction patterns, metabolic enzyme electropherotypes, organization of nif genes, and the ability to nodulate beans . The colonization capacity of some of the isolates was tested with an unimproved maize cultivar and with 30 maize land races . Increases in plant dry weight upon R . etli inoculation were recorded with some of the land races, and these increases may be related to plant growth promotion effects . Additionally, from within maize grown in monoculture we have also recovered R . etli isolates recognizable by their 16S rRNA gene types, which lack nif genes and are incapable of nodulating bean . These strains are presumed to correspond to the earlier described non-symbiotic R . etli obtained from bean rhizosphere.

Mikrobiologiia, 2001 Jul-Aug, 70(4), 519 - 24
{Role of the agglutinating proteins of bacilli and rhizobia in bacterial interaction}; Karpunina LV et al.; The surface agglutinating proteins of the soil nitrogen-fixing bacteria Bacillus polymyxa 1460 and Rhizobium leguminosarum 252 were found to be able to interact with the polysaccharide complexes of certain azospirilla and rhizobia . Such interactions are most likely involved in the formation of nitrogen-fixing plant-bacterial associations in the rhizosphere.

Mol Ecol, 2001 Sep, 10(9), 2297 - 305
Genetic diversity of indigenous Rhizobium leguminosarum bv . viciae isolates nodulating two different host plants during soil restoration with alfalfa; Zhang XX et al.; A total of 360 Rhizobium leguminosarum bv . viciae strains was isolated from three brown-coal mining restoration fields of different age and plant cover (without and in the first and second year of alfalfa, Medicago sativa, cultivation) using two host species (Vicia hirsuta and Pisum sativum) as capture plants . The strains were genetically typed by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-generated 16S-23S ribosomal DNA intergenic spacer regions (IGS-RFLP) and characterized by plasmid profiles and RFLP analysis of amplified nodABC genes . The R . leguminosarum bv . viciae population was dominated by the same group of strains (irrespective of the trap plant used) . According to type richness, the genetic diversity of indigenous R . leguminosarum in the second year of restoration was lower than in the first year and it resembled that of the fallow field, except for plasmid types, in which it was higher than that of the fallow field . Some of the less frequent nodABC genotypes were associated with distinct chromosomal IGS genotypes and symbiotic plasmids (pSyms) of different sizes, indicating that horizontal transfer and rearrangements of pSym can occur in natural environments . However, the dominant pSym and chromosomal genotypes were strictly correlated suggesting a genetically stable persistence of the prevailing R . leguminosarum bv . viciae genotypes in the absence of its host plant.

Can J Microbiol, 2001 Jul, 47(7), 595 - 600
Rhizobium population dynamics in the pea rhizosphere of rhizobial inoculant strain applied in different formulations; Hynes RK et al.; The effect of inoculant formulation on the population dynamics of rhizobia in the pea rhizosphere was investigated using a streptomycin-resistant mutant of Rhizobium leguminosarum bv . viceae NITRAGIN128C56G (128C56G strR) . The isolate was formulated into liquid, peat powder, and granular peat carriers, and was tested on pea at field sites near Saskatoon, Saskatchewan, and Beaverlodge, Alberta, in 1996 and 1997 . The liquid and peat powder formulations were applied to seed while the granular inoculant was applied to soil . In three out of four site years, population dynamics were similar among formulations: an initial decline or lag period lasting 2-5 days followed by an increase to approximately 10(5) colony-forming units (CFU)/seedling by 14-28 days after planting (DAP) and, where sampled, a continuing increase from 10(7) to 10(8) CFU/plant at 63 DAP . In these same site years, nodule number (not determined at Beaverlodge in 1997) and nodule occupancy at 60 days were not significantly different among formulations . In contrast, soil populations of 128C56G strR from the liquid formulation declined to near zero by 28 DAP at Beaverlodge in 1996, when soil moisture was excessive in spring because of high rainfall . Populations increased in this treatment after this time, but remained significantly lower than the populations of the other two formulations throughout the sampling period . Pea seed yields were not significantly different among treatments in either year at Beaverlodge, but were significantly higher with granular inoculant than the noninoculated control in Saskatoon . Within inoculated treatments at Saskatoon, there were no significant differences in grain yield.

Adv Space Res, 1999, 24(3), 297 - 302
The use of microcosms as an experimental approach to understanding terrestrial ecosystem functioning; Fraser LH; Since 1986, a series of microcosm experiments has been conducted at the Unit of Comparative Plant Ecology (UCPE) in an attempt to test our understanding of the principles controlling the structure and dynamics of plant communities and ecosystems . In each experiment microcosms have been seeded with a common pool of organisms, and systems have been allowed to assemble under replicated controlled conditions . Experiment variables have included mineral nutrient supply, temperature, moisture supply, soil depth, carbon dioxide concentration, mycorrhizas, rhizobia, herbivores and carnivores . Results from these experiments are presented to illustrate the value of synthesised ecosystems in ecological research.

Acta Hortic, 1996 Dec, 440, 193 - 8
Study of the microbial dynamics in the root environment of closed, hydroponic cultivation systems for tomato using phospholipid fatty acid profiles; Waechter-Kristensen B et al.; A more basic understanding of the microbial dynamics of closed, hydroponic cultivation systems is needed . We therefore initiated a study of the microbial community inhabiting the root environment, using phospholipid fatty acid (PLFA) profiles, and started to examine whether changes in the microbial population structure would result from the introduction of selected isolates of plant growth-promoting rhizobacteria (PGPR) . Tomato were cultured in deep-flow systems with circulating nutrient solution . Bacteria were sampled from tomato roots at three locations, longitudinally, in the gutters of a control system and in two systems inoculated with PGPR . In the beginning of the gutters the PLFA profiles were similar in all systems, whereas the profiles differed in the gutter ends (following the direction of flow) . In the control system, and in a treatment inoculated with two Gram-negative and one Gram-positive PGPR strain, the relative proportion of PLFAs characteristic to Gram-positive bacteria was highest at the end of the gutter . In a treatment inoculated only with a Gram-negative PGPR strain, the relative proportion of PLFAs characteristic of Gram-negative bacteria was highest at the end of the gutter . The results indicate a complex situation with different micro-environments distributed along the root mat . It can also be concluded that PLFA profiles may be useful tools in the study of the microbiology of closed hydroponic plant cultivation systems.

Acta Astronaut, 1995 Jul, 36(2), 129 - 33
Effects of microgravity on the binding of acetylsalicylic acid by Rhizobium leguminosarum bv . trifolii; Urban JE et al.; Bacteroids can be induced in vitro by treating growing Rhizobium leguminosarum bv . trifolii with succinic acid or succinic acid structural analogs like acetysalicylic acid . Quantitating bacteroid induction by measuring acetylsalicylic binding under normal (1 g) conditions showed two forms of binding to occur . In one form of binding cells immediately bound comparatively high levels of acetylsalicylic acid, but the binding was quickly reversed . The second form of binding increased with time by first order kinetics and reached saturation in 40 s . Similar experiments performed in the microgravity environment aboard the NASA 930 aircraft showed only one form of binding and total acetylsalicyclic acid bound was 32% higher than at 1 g.

Plant Physiol, 1990, 92, 262 - 4
Binding of isolated plant lectin by rhizobia during episodes of reduced gravity obtained by parabolic flight; Henry RL et al.; Development of a legume root nodule is a complex process culminating in a plant/bacterial symbiosis possessing the capacity for biological dinitrogen fixation . Formation of root nodules is initiated by the binding and stabilization of rhizobia to plant root hairs, mediated in part by a receptor/ligand recognition system composed of lectins on the plant root surface and lectin-binding sites on the rhizobial cell surface . The dinitrogen fixation activity of these root nodules may be an important feature of enclosed, space-based life support systems, and may provide an ecological method to recycle nitrogen for amino acid production . However, the effects on nodule development of varied gravitational fields, or of root nutrient delivery hardware, remain unknown . We have investigated the effects of microgravity on root nodule formation, with preliminary experiments focused upon the receptor/ligand component . Microgravity, obtained during parabolic flight aboard NASA 930, has no apparent effect on the binding of purified lectin to rhizobia, a result that will facilitate forthcoming experiments using intact root tissues.

Adv Space Res, 1994, 14(8), 173 - 6
Clover development during spaceflight: a model system; Guikema JA et al.; The development of legume root nodules was studied as a model system for the examination of gravitational effects on plant root development . In order to examine whether rhizobial association with clover roots can be achieved in microgravity, experiments were performed aboard the KC-135 parabolic aircraft and aboard the sounding rocket mission Consort 3 . Binding of rhizobia to roots and the initial stages of root nodule development successfully occurred in microgravity . Seedling germination experiments were performed in the sliding block device, the Materials Dispersion Apparatus, aboard STS-37 . When significant hydration of the seeds was achieved, normal rates of germination and seedling development were observed.

Microbiology, 2001 Sep, 147(Pt 9), 2553 - 60
Evidence for redundancy in cysteine biosynthesis in Rhizobium leguminosarum RL3841: analysis of a cysE gene encoding serine acetyltransferase; Parker G et al.; A cysE gene encoding a serine acetyltransferase (SAT) potentially involved in the biosynthesis of cysteine was identified approximately 4 kb upstream of the previously described aapJQMP gene cluster that encodes an amino acid permease in Rhizobium leguminosarum strain 3841 . The gene exhibits >40% identity to the family of SATs containing N-terminal extensions that have been described for other bacteria and plants . The ORF has three possible translation initiation sites which potentially encode polypeptides of 311, 277 and/or 259 amino acid residues, respectively . All three ORFs complemented the cysE mutation in an Escherichia coli cysteine auxotroph, strain JM39 . Insertion of Tn5-lacZ into cysE in the genome of R . leguminosarum (strain RU632) lowered SAT activity in crude extracts by >95% . However, RU632 was not a cysteine auxotroph, which suggests that R . leguminosarum possesses some redundancy in cysteine biosynthesis . Additional copies of cysE could not be detected in the genome when the R . leguminosarum cysE gene was used as a hybridization probe . Therefore it is possible that R . leguminosarum possesses an alternative pathway for cysteine biosynthesis which avoids O-acetylserine . Strain RU632 was unaffected in its ability to nodulate Pisum sativum, and the nodules were effective for N(2) fixation (measured by C(2)H(2) reduction) . Transcriptional activity of cysE was determined by measuring the beta-galactosidase arising from cysE::Tn5-lacZ fusions . Maximal levels of expression were observed during early exponential growth and were not influenced by the level of sulphur (supplied as sulphate) . However, transcription was repressed by approximately twofold in ammonium-grown, as opposed to glutamate-grown, cultures . Repression by ammonium was not seen in a strain defective for ntrC.

Mol Microbiol, 2001 Aug, 41(4), 801 - 16
The Rhizobium leguminosarum tonB gene is required for the uptake of siderophore and haem as sources of iron; Wexler M et al.; In the N2-fixing bacterium Rhizobium leguminosarum, mutations in a homologue of tonB (tonB(Rl)) block the import of vicibactin and haem as iron sources in free-living bacteria . TonB(Rl) mutants were normal for growth with ferric dicitrate and slightly reduced for growth with haemoglobin as sole iron sources . The deduced TonB(Rl) product is larger than that of (for example) Escherichia coli, on account of an extended N-terminal domain . Transcription of tonB(Rl) was enhanced in low-Fe growth conditions; this was not controlled by Fur, nor RpoI, an Fe-regulated extracytoplasmic sigma factor . Upstream of tonB(Rl) and transcribed divergently is an operon, hmuPSTUV, whose products are homologous to ABC transporters involved in haem uptake in pathogenic bacteria . Expression of hmuPSTUV was enhanced in low-Fe conditions, and hmu mutants show slightly diminished growth on haem as sole Fe source, suggesting that there is more than one system for the uptake of this molecule . hmuPSTUV expression appears to be from three closely linked promoters . Downstream of hmuPSTUV, a gene that may encode an extracytoplasmic sigma factor was identified, but this gene, rpoZ, did not affect the transcription of tonB(Rl) or hmuPSTUV . Mutations in tonB(Rl), hmu genes and rpoZ did not affect symbiotic N(2) fixation in peas.

Appl Environ Microbiol, 2001 Sep, 67(9), 3860 - 5
Construction and environmental release of a Sinorhizobium meliloti strain genetically modified to be more competitive for alfalfa nodulation; van Dillewijn P et al.; Highly efficient nitrogen-fixing strains selected in the laboratory often fail to increase legume production in agricultural soils containing indigenous rhizobial populations because they cannot compete against these populations for nodule formation . We have previously demonstrated, with a Sinorhizobium meliloti PutA- mutant strain, that proline dehydrogenase activity is required for colonization and therefore for the nodulation efficiency and competitiveness of S . meliloti on alfalfa roots (J . I . Jimenez-Zurdo, P . van Dillewijn, M . J . Soto, M . R . de Felipe, J . Olivares, and N . Toro, Mol . Plant-Microbe Interact . 8:492-498, 1995) . In this work, we investigated whether the putA gene could be used as a means of increasing the competitiveness of S . meliloti strains . We produced a construct in which a constitutive promoter was placed 190 nucleotides upstream from the start codon of the putA gene . This resulted in an increase in the basal expression of this gene, with this increase being even greater in the presence of the substrate proline . We found that the presence of multicopy plasmids containing this putA gene construct increased the competitiveness of S . meliloti in microcosm experiments in nonsterile soil planted with alfalfa plants subjected to drought stress only during the first month . We investigated whether this construct also increased the competitiveness of S . meliloti strains under agricultural conditions by using it as the inoculum in a contained field experiment at Leon, Spain . We found that the frequency of nodule occupancy was higher with inoculum containing the modified putA gene for samples that were analyzed after 34 days but not for samples that were analyzed later.

Nucleic Acids Res, 2001 Sep 1, 29(17), 3459 - 68
A critical evaluation of differential display as a tool to identify genes involved in legume nodulation: looking back and looking forward; Lievens S et al.; Screening for differentially expressed genes is a straightforward approach to study the molecular basis of a biological system . In the last 10 years, differential screening technology has evolved rapidly and currently high-throughput tools for genome-wide transcript profiling, such as expressed sequence tags and microarray analysis, are becoming widely available . Here, an overview of this (r)evolution is given with emphasis on the differential display method, which for many years has been the preferred technique of scientists in diverse fields of research . Differential display has also been the method of choice for the identification of genes involved in the symbiotic interaction between Azorhizobium caulinodans and Sesbania rostrata . The advantages with respect to tissue specificity of this particular model system for legume nodulation and the results of a screening for early nodulation-related genes have been considered in the context of transcriptome analyses in other rhizobium-legume interactions.

Antonie Van Leeuwenhoek, 2001 Jun, 79(2), 189 - 97
Characterization of two novel Rhizobium leguminosarum bacteriophages from a field release site of genetically-modified rhizobia; Mendum TA et al.; Two Rhizobium leguminosarum biovar viceae bacteriophages with contrasting properties were isolated from a field site in which the survival of genetically modified R . leguminosarum inoculants had been monitored for several years . Inoculant strain RSM2004 was used as the indicator for phage isolation and propagation . One phage, RL1RES, was temperate and could not replicate in any of the 42 indigenous R . leguminosarum field isolates tested although nested PCR indicated that phage sequences were present in six of the isolates . The second phage, RL2RES, was virulent, capable of generalised transduction, contained DNA with modified cytosine residues, and was capable of infecting all field isolates tested although the GM inoculant strain CT0370 was resistant . Sequence with homology to RL2RES was detected by nested PCR in six of the 42 field-isolates . These were not the same isolates that showed homology to RL1RES . The implication of these findings for the survival of rhizobial inoculants, and the ecology of phages and their host bacteria, are discussed.

Antonie Van Leeuwenhoek, 2001 Jun, 79(2), 119 - 25
Characterization of Rhizobium loti strains from the Salado River Basin; Fulchieri MM et al.; Thirty indigenous rhizobia strains, isolated from Lotus tenuis in the area of Chascomus and other regions of the Salado River Basin (Argentina), were characterized based on generation time, acid production, carbon utilization, protein profile, and molecular characterization by restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes amplified by the polymerase chain reaction (PCR) . The results indicated that native rhizobia isolates from the Chascomus area are predominantly fast and intermediate-growers . The unclassified rhizobia examined by PCR-RFLP were found to be closely related to the reference strains of validly described Rhizobium species.

Syst Appl Microbiol, 2001 Jul, 24(2), 192 - 205
Description of two biovars in the Rhizobium galegae species: biovar orientalis and biovar officinalis; Radeva G et al.; Twenty-six Rhizobium galegae strains, representing the center of origin of the host plants Galega orientalis and G . officinalis as well as other geographic regions, were used in a polyphasic analysis of the relationships of R . galegae strains . Phage typing, lipopolysaccharide (LPS) profiling, pulsed field gel electrophoresis (PFGE) profiling and rep-PCR (use of repetitive sequences as PCR primers for genomic fingerprinting) with REP and ERIC primers investigated nonsymbiotic properties, whereas plasmid profiling and hybridisation with a nif gene probe, and with nodB, nodD, nod box and an IS sequence from the symbiotic region as probes, were used to reveal the relationships of symbiotic genes . The results were used in pairwise calculations of distances between the strains, and the distances were visualised as a dendrogram . Indexes of association were compared for all tests pooled, and for chromosomal tests and symbiotic markers separately, to display the input of the different categories of tests on the grouping of the strains . Our study shows that symbiosis related genetic traits in R . galegae divide strains belonging to the species into two groups, which correspond to strains forming an effective symbioses with G . orientalis and G . officinalis respectively . We therefore propose that Rhizobium galegae strains forming an effective symbiosis with Galega orientalis are called R . galegae bv . orientalis and strains forming an effective symbiosis with Galega officinalis are called R . galegae bv . officinalis.

J Bacteriol, 2001 Sep, 183(18), 5334 - 42
Mutational analysis of the Rhizobium lupini H13-3 and Sinorhizobium meliloti flagellin genes: importance of flagellin A for flagellar filament structure and transcriptional regulation; Scharf B et al.; Complex flagellar filaments are unusual in their fine structure composed of flagellin dimers, in their right-handed helicity, and in their rigidity, which prevents a switch of handedness . The complex filaments of Rhizobium lupini H13-3 and those of Sinorhizobium meliloti are composed of three and four flagellin (Fla) subunits, respectively . The Fla-encoding genes, named flaA through flaD, are separately transcribed from sigma(28)-specific promoters . Mutational analysis of the fla genes revealed that, in both species, FlaA is the principal flagellin and that FlaB, FlaC, and FlaD are secondary . FlaA and at least one secondary Fla protein are required for assembling a functional flagellar filament . Western analysis revealed a ratio close to 1 of FlaA to the secondary Fla proteins (= FlaX) present in wild-type extracts, suggesting that the complex filament is assembled from FlaA-FlaX heterodimers . Whenever a given mutant combination of Fla prevented the assemblage of an intact filament, the biosynthesis of flagellin decreased dramatically . As shown in S . meliloti by reporter gene analysis, it is the transcription of flaA, but not of flaB, flaC, or flaD, that was down-regulated by such abortive combinations of Fla proteins . This autoregulation of flaA is unusual . We propose that any combination of Fla subunits incapable of assembling an intact filament jams the flagellar export channel and thus prevents the escape of an (as yet unidentified) anti-sigma(28) factor that antagonizes the sigma(28)-dependent transcription of flaA.

Indian J Exp Biol, 2001 May, 39(5), 401 - 9
Lipochitooligosaccharides and legume Rhizobium symbiosis--a new concept; Chimote V et al.; Legume-Rhizobium symbiosis is a multistep process characterized by the formation of root nodules on the host plant . A number of genes from both symbiotic partners share information during the interaction process . Nodulation genes (nod, nol and noe) have been classified as common nodulation genes and host specific (hsn) nodulation genes . Though common nodulation genes are enough to form root nodules, host specific nodulation genes are needed for specific interaction leading to formation of functional nodules . Core lipochitooligosaccharides (LCOs), the products of common nodulation genes are modified by the action of host specific nodulation genes . LCOs seem to be present in legumes as well as nonlegume and are known to act as a morphogen by acting as auxin-transport inhibitor . The understanding of Nod factor may contribute to reveal complex biological functions such as developmental regulation, signal transduction and plant morphogenesis.

Mol Plant Microbe Interact, 2001 Aug, 14(8), 1016 - 25
Investigation of myo-inositol catabolism in Rhizobium leguminosarum bv . viciae and its effect on nodulation competitiveness; Fry J et al.; Three discrete loci required for growth on myo-inositol in Rhizobium leguminosarum bv . viciae have been characterized . Two of these are catabolic loci that code for malonate semialdehyde dehydrogenase (iolA) and malonate semialdehyde dehydrogenase (iolD) . IolD is part of a possible operon, iolDEB, although the functions of IolE and IolB are unknown . The third locus, int, codes for an ABC transport system that is highly specific for myo-inositol . LacZ analysis showed that mutation of iolD, which codes for one of the last steps in the catabolic pathway, prevents increased transcription of the entire pathway . It is likely that the pathway is induced by an end product of catabolism rather than myo-inositol itself . Mutants in any of the loci nodulated peas (Pisum sativum) and vetch (Vicia sativa) at the same rate as the wild type . Acetylene reduction rates and plant dry weights also were the same in the mutants and wild type, indicating no defects in nitrogen fixation . When wild-type 3841 was coinoculated onto vetch plants with either catabolic mutant iolD (RU360) or iolA (RU361), however, >95% of the nodules were solely infected with the wild type . The competitive advantage of the wild type was unaffected, even when the mutants were at 100-fold excess . The myo-inositol transport mutant (RU1487), which grows slowly on myo-inositol, was only slightly less competitive than the wild type . The nodulation advantage of the wild type was not the result of superior growth in the rhizosphere . Instead, it appears that the wild type may displace the mutants early on in the infection and nodulation process, suggesting an important role for myo-inositol catabolism.

Plant Cell, 2001 Aug, 13(8), 1835 - 49
Ethylene inhibits the Nod factor signal transduction pathway of Medicago truncatula; Oldroyd GE et al.; Legumes form a mutualistic symbiosis with bacteria collectively referred to as rhizobia . The bacteria induce the formation of nodules on the roots of the appropriate host plant, and this process requires the bacterial signaling molecule Nod factor . Although the interaction is beneficial to the plant, the number of nodules is tightly regulated . The gaseous plant hormone ethylene has been shown to be involved in the regulation of nodule number . The mechanism of the ethylene inhibition on nodulation is unclear, and the position at which ethylene acts in this complex developmental process is unknown . Here, we used direct and indirect ethylene application and inhibition of ethylene biosynthesis, together with comparison of wild-type plants and an ethylene-insensitive supernodulating mutant, to assess the effect of ethylene at multiple stages of this interaction in the model legume Medicago truncatula . We show that ethylene inhibited all of the early plant responses tested, including the initiation of calcium spiking . This finding suggests that ethylene acts upstream or at the point of calcium spiking in the Nod factor signal transduction pathway, either directly or through feedback from ethylene effects on downstream events . Furthermore, ethylene appears to regulate the frequency of calcium spiking, suggesting that it can modulate both the degree and the nature of Nod factor pathway activation.

Biotechnol Prog, 2001 Jul-Aug, 17(4), 612 - 7
Enhancing the atom economy of polyketide biosynthetic processes through metabolic engineering; Lombo F et al.; Polyketides, a large family of bioactive natural products, are synthesized from building blocks derived from alpha-carboxylated Coenzyme A thioesters such as malonyl-CoA and (2S)-methylmalonyl-CoA . The productivity of polyketide fermentation processes in natural and heterologous hosts is frequently limited by the availability of these precursors in vivo . We describe a metabolic engineering strategy to enhance both the yield and volumetric productivity of polyketide biosynthesis . The genes matB and matC from Rhizobium trifolii encode a malonyl-CoA synthetase and a putative dicarboxylate transport protein, respectively . These proteins can directly convert exogenous malonate and methylmalonate into their corresponding CoA thioesters with an ATP requirement of 2 mol per mol of acyl-CoA produced . Heterologous expression of matBC in a recombinant strain of Streptomyces coelicolor that produces the macrolactone 6-deoxyerythronolide B results in a 300% enhancement of macrolactone titers . The unusual efficiency of the bioconversion is illustrated by the fact that approximately one-third of the methylmalonate units added to the fermentation medium are converted into macrolactones . The direct conversion of inexpensive feedstocks such as malonate and methylmalonate into polyketides represents the most carbon- and energy-efficient route to these high value natural products and has implications for cost-effective fermentation of numerous commercial and development-stage small molecules.

Arch Microbiol, 2001 Jul, 176(1-2), 44 - 51
ROSE elements occur in disparate rhizobia and are functionally interchangeable between species; Nocker A et al.; Expression of at least ten genes in Bradyrhizobium japonicum, seven of which code for small heat shock proteins (sHsps), is under the control of ROSE (repression of heat shock gene expression) . This negatively cis-acting DNA element confers temperature control to a sigma(70)-type promoter . Here, we show that ROSE elements are not restricted to B . japonicum but are also present in Bradyrhizobium sp . (Parasponia), Rhizobium sp . strain NGR234 and Mesorhizobium loti . An overall alignment of all ROSE sequences reveals a highly conserved and probably functionally important region towards the 3'-end of the element . Moreover, we provide genetic evidence for the previously proposed presence of multiple sHsps in these organisms . Primer-extension data of five newly identified ROSE-associated operons show that transcription is repressed at low temperatures and induced after a temperature upshift . Translational ROSE-hsp'-'lacZ fusions of Bradyrhizobium sp . (Parasponia) and Rhizobium sp . strain NGR234 integrated into the chromosome of B . japonicum were heat-responsive . The functionality of these heterologous ROSE elements hints at a common regulatory principle conserved in various rhizobia.

Science, 2001 Jul 27, 293(5530), 668 - 72
The composite genome of the legume symbiont Sinorhizobium meliloti; Galibert F et al.; The scarcity of usable nitrogen frequently limits plant growth . A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N2) to ammonium (NH4+) . We present here the annotated DNA sequence of the alpha-proteobacterium Sinorhizobium meliloti, the symbiont of alfalfa . The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids . Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution . The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.

Environ Microbiol, 2001 Jun, 3(6), 363 - 70
Direct amplification of nodD from community DNA reveals the genetic diversity of Rhizobium leguminosarum in soil; Zeze A et al.; Sequences of nodD, a gene found only in rhizobia, were amplified from total community DNA isolated from a pasture soil . The polymerase chain reaction (PCR) primers used, Y5 and Y6, match nodD from Rhizobium leguminosarum biovar trifolii, R . leguminosarum biovar viciae and Sinorhizobium meliloti . The PCR product was cloned and yielded 68 clones that were identified by restriction pattern as derived from biovar trifolii {11 restriction fragment length polymorphism (RFLP) types} and 15 clones identified as viciae (seven RFLP types) . These identifications were confirmed by sequencing . There were no clones related to S . meliloti nodD . For comparison, 122 strains were isolated from nodules of white clover (Trifolium repens) growing at the field site, and 134 from nodules on trap plants of T . repens inoculated with the soil . The nodule isolates were of four nodD RFLP types, with 77% being of a single type . All four of these patterns were also found among the clones from soil DNA, and the same type was the most abundant, although it made up only 34% of the trifolii-like clones . We conclude that clover selects specific genotypes from the available soil population, and that R . leguminosarum biovar trifolii was approximately five times more abundant than biovar viciae in this pasture soil, whereas S . meliloti was rare.

Microb Comp Genomics, 2000, 5(4), 181 - 8
Characterization of IS666, a newly described insertion element of Mycobacterium avium; Sangari FJ et al.; The insertion sequence IS666 was isolated from Mycobacterium avium strain 101 . IS666 is a 1474 bp insertion sequence belonging to the IS256 family, that includes IS6120 from Mycobacterium smegmatis, IS1166 and IS1295 from Rhodococcus sp . IGTS8, IST2 from Thiobacillus ferrooxidans, IS256 from Staphylococcus aureus, and ISRm3 from Rhizobium meliloti . IS666 has 24 bp imperfect inverted repeats that fit the consensus described for the family, and generates 9 bp duplications upon insertion into the host DNA with no apparent specificity in the target sequence . In contrast with its two closest homologues, IS1166 and IS6120, IS666 contains a single ORF that would codify a transposase of 434 aa . IS666 is restricted to M . avium, where it is present in 21% of the isolates in a number ranging between 1 to 7 copies.

Proc Natl Acad Sci U S A, 2001 Aug 14, 98(17), 9736 - 41 Epub 2001 Jul 24.
A conserved RNA structure (thi box) is involved in regulation of thiamin biosynthetic gene expression in bacteria; Miranda-Rios J et al.; The thiCOGE genes of Rhizobium etli code for enzymes involved in thiamin biosynthesis . These genes are transcribed with a 211-base untranslated leader that contains the thi box, a 38-base sequence highly conserved in the 5' regions of thiamin biosynthetic and transport genes of Gram-positive and Gram-negative organisms . A deletion analysis of thiC-lacZ fusions revealed an unexpected relationship between the degree of repression shown by the deleted derivatives and the length of the thiC sequences present in the transcript . Three regions were found to be important for regulation: (i) the thi box sequence, which is absolutely necessary for high-level expression of thiC; (ii) the region immediately upstream to the translation start codon of thiC, which can be folded into a stem-loop structure that would mask the Shine-Dalgarno sequence; and (iii) the proximal part of the coding region of thiC, which was shown to contain a putative Rho-independent terminator . A comparative phylogenetic analysis revealed a possible folding of the thi box sequence into a hairpin structure composed of a hairpin loop, two helices, and an interior loop . Our results show that thiamin regulation of gene expression involves a complex posttranscriptional mechanism and that the thi box RNA structure is indispensable for thiCOGE expression.

Can J Microbiol, 2001 Jun, 47(6), 590 - 3
{Effects of inoculation with Rhizobium leguminosarum biovar trifolii on wheat cultivated in clover crop rotation agricultural soil in Morocco.}; Hilali A et al.; One hundred strains of Rhizobium leguminosarum bv . trifolii were isolated from roots of wheat cultivated in rotation with clover in two different regions of Morocco . The isolates were first screened for their effect on the growth of the cultivar Rihane of wheat cultivated in an agricultural soil under greenhouse conditions . After 5 weeks of growth, 14 strains stimulating the fresh or dry matter yield of shoots were selected and used in a second pot inoculation trial performed with two different agricultural soils . The results show that the strains behaved differently according to the soil used . In the loamy sand Rabat, strain IAT 168 behaved potentially like a plant growth promoting rhizobacteria (PGPR), as indicated by the 24% increases (P < 0.1) observed in wheat shoot dry matter and grain yields . In the silty clay Merchouch, no PGPR activity was observed, and 6 strains showed a significant deleterious effect on yields . These observations suggest that it is very important in a crop rotation system to choose a R . leguminosarum bv . trifolii strain that is effective with clover and shows PGPR activity with wheat to avoid deleterious effects on wheat yields.

Can J Microbiol, 2001 Jun, 47(6), 548 - 58
{IRepeat sequences of genomes of Rhizobium and Sinorhizobium meliloti: a comparative analysis.}; Perret X et al.; Amongst prokaryotic genomes, those of nitrogen-fixing members of the Rhizobiaceae family are relatively large (6-9 Mb), often include mega-plasmids of 1.5-2 Mb, and contain numerous families of repeated DNA sequences . Although most essential nodulation and nitrogen fixation genes are well characterized, these represent only a small fraction of the DNA content . Little is known about the detailed structure of rhizobial genomes . With the development of sequencing techniques and new bio-informatic tools such studies become possible, however . Using the 2275 shotgun sequences of ANU265 (a derivative of NGR234 cured of pNGR234a), we have identified numerous families of repeats . Amongst these, the 58-bp-long NGRREP-4 represents the third most abundant DNA sequence after the RIME1 and RIME2 repeats, all of which are also found in Sinorhizobium meliloti . Surprisingly, studies on the distribution of these elements showed that in proportion to its size, the chromosome of NGR234 carries many more RIME modules than pNGR234a or pNGR234b . Together with the presence in NGR234 and S . meliloti 1021 of an insertion sequence (IS) element more conserved than essential nodulation and nitrogen fixation genes, these results give new insights into the origin and evolution of rhizobial genomes.

Can J Microbiol, 2001 Jun, 47(6), 503 - 8
{ Nodulation of certain legumes of the genus Crotalaria by the new species Methylobacterium.}; Sy A et al.; We studied a collection of 126 rhizobial isolates from eight species of Crotalaria (C . comosa, C . glaucoides, C . goreensis, C . hyssopifolia, C . lathyroides, C . perrottetii, C . podocarpa, and C . retusa) growing in Senegal . Nodulation and nitrogen-fixation tests on nine Crotalaria species revealed two specificity groups within the genus Crotalaria . Group I consists of plants solely nodulated by very specific fast-growing strains . Group II plants are nodulated by slow-growing strains similar to promiscuous Bradyrhizobium spp . strains already reported to nodulate many tropical legumes . SDS-PAGE studies showed that slow-growing strains grouped with Bradyrhizobium while fast-growing strains constituted a homogeneous group distinct from all known rhizobia . Amplified ribosomal DNA restriction analysis (ARDRA) of 10 representative strains of this group using four restriction enzymes showed a single pattern for each enzyme confirming the high homogeneity of group I . The 16S rDNA sequence analysis revealed that this specific group belonged to the genus Methylobacterium, thus constituting a new branch of nodulating bacteria.

Can J Microbiol, 2001 Jun, 47(6), 585 - 9
Rhizobial survival and nodulation of chickpea as influenced by fungicide seed treatment; Kyei-Boahen S et al.; The survival of Rhizobium ciceri on chickpea (Cicer arietinum cv . Myles) seed, treated separately with 1 of 4 commercial fungicides, i.e., Apron, Arrest 75W, Crown, or Captan, was examined under laboratory conditions using standard serial dilution and plate count techniques . The resulting effects of fungicide-Rhizobium interactions on nodulation, N2 fixation, and plant growth were assessed in a controlled environment . Fungicide treatment decreased the number of viable rhizobia on the seed . In general, the toxicity of the fungicides in terms of rhizobial viability increased in the following order: Control = Crown < Arrest = Apron < Captan . Although Crown had no effect on rhizobial viability assessed under laboratory conditions, it significantly reduced nodulation, percent N derived from the atmosphere (%Ndfa), and shoot dry matter . Seed treated with Arrest and Captan decreased nodule dry weight and %Ndfa, but only Arrest reduced dry matter yield . Apron had no effect on any of the parameters measured at the early pod-filling stage and was compatible with the chickpea inoculum used in this study.

Can J Microbiol, 2001 Jun, 47(6), 580 - 4
Comparative strain typing of Rhizobium leguminosarum bv . viciae natural populations; Corich V et al.; 372 natural isolates of Rhizobium leguminosarum bv . viciae, rescued from nodules of pea plants grown in an agricultural field in northern Italy, were analyzed by different methods . Three DNA-based fingerprinting techniques were lined up to compare their relative degree of resolution and possible advantages of each approach . The methods included (i) Eckhardt gel plasmid profiles, (ii) pulsed-field gel electrophoresis (PFGE) of genomic large fragment digests, and (iii) random amplified polymorphic DNA (RAPD) profiles, generated with arbitrary primers . The scheme also involved the isolation of a number of different isolates per nodule to estimate the level of intra-nodular variability . It was therefore possible to evaluate the frequency of double and multiple occupancies, and the proportion of the alternative profiles sharing the same nodule, generally resulting in a numerically dominant, main representative accompanied by a secondary one with a slightly different fingerprint . This finding revealed that the different profiles within a nodule are normally due to bacteria derived from the same single invader following genetic alterations possibly occurred during infection, e.g., by plasmid loss . The analysis of 31 nodules revealed 16 different patterns, representing the most frequently occurring nodulation-proficient isolates of the natural soil examined, five of which were found with frequencies around 15% . The sensitivity of the methods in differentiating isolates was compared . The relatedness of the different natural rhizobial isolates was investigated by densitometrical gel analysis of the fingerprints, allowing a comparison of the results . One of the most interesting conclusions was that the degree of information yielded by the plasmid gel profiling alone, carried out by simple visual inspection without software-aided analyses, was surprisingly high, as it enabled a placement of the isolates, whose accuracy, in terms of relatedness, was subsequently confirmed by each of the two genomic methods.

Can J Microbiol, 2001 Jun, 47(6), 574 - 9
Regulation of nod factor sulphation genes in Rhizobium tropici CIAT899; Manyani H et al.; Rhizobium tropici CIAT899 is a tropical symbiont able to nodulate various legumes such as Leucaena, Phaseolus, and Macroptilium . Broad host range of this species is related to its Nod factors wide spectrum . R . tropici contains Nod factors sulphation nod genes, nodHPQ genes, which control nodulation efficiency in Leucaena . To study nodHPQ regulation, we carried out different interposon insertions in its upstream region . One of these generated interruptions, nodI mutant produced nonsulphated Nod factors suggesting a possible dependence of these genes on nodI upstream region . Moreover, analysis results of lacZ transcriptional fusions with these genes in symbiotic plasmid showed dependence of these genes on NodD protein . In order to determine nodHPQ organization, we studied the effect of interposon insertion upstream of each lacZ transcriptional fusion, and the data obtained was used to indicate that nodHPQ belong to the nodABCSUIJ operon . However, comparison between nodP::lacZ beta-galactosidase activity in the symbiotic plasmid and in the pHM500 plasmid (containing nodHPQ genes) suggested constitutive expression in free living, and flavonoid inducible expression in symbiotic conditions . Constitutive nodHPQ expression may play a role in bacterial house-keeping metabolism . On the other hand, the transference of R . tropici nodHPQ genes to other rhizobia that do not present sulphated substitutions demonstrated that NodH protein sulphotransference is specific to C6 at the reducing end.

Can J Microbiol, 2001 Jun, 47(6), 559 - 66
Biotransformation and partial mineralization of the explosive 2,4,6-trinitrotoluene (TNT) by rhizobia; Labidi M et al.; Three strains, T10, B5, and M8, each belonging to a different species of the family Rhizobiaceae and isolated from atrazine-contaminated soils, were tested for their ability to transform 2,4,6-trinitrotoluene (TNT) (50 microg x mL(-1)) in liquid cultures using glucose as the C-source . All three strains were able to transform TNT to hydroxylaminodinitrotoluenes (2-HADNT, 4-HADNT), aminodinitrotoluenes (2-ADNT, 4-ADNT), and diaminonitrotoluene (2,4-DANT) . The transformation was significantly faster in the presence of glutamate . Furthermore, the major metabolites that accumulated in cultures were 2-ADNT with glucose, and 4-ADNT with glutamate plus glucose . Rhizobium trifolii T10 was also tested for its ability to transform high levels of TNT (approximately 350 microg x mL(-1)) in a soil slurry . Almost 60% of the TNT was transformed within 2 days in bioaugmented soil slurries, and up to 90% when cultures were supplemented with glucose and glutamate . However, mineralization was minimal in all cases, less than 2% in 78 days . This is the first report on the degradation of TNT by rhizobial strains, and our findings suggest that rhizobia have the potential to play an important role in the safe decontamination of soils and sites contaminated with TNT if bioaugmentation with rhizobia is shown to have no ecotoxicological consequence.

Can J Microbiol, 2001 Jun, 47(6), 535 - 40
Hopanoid lipids in Frankia: identification of squalene-hopene cyclase gene sequences; Dobritsa SV et al.; In Frankia, the microsymbiont in actinorhizal root nodules, nitrogen fixation takes place in specialized structures called vesicles . The lipidic vesicle envelope forms a barrier to oxygen diffusion, an essential part of the nitrogenase oxygen protection system . We have shown previously that the vesicle envelope is composed primarily of two species of hopanoid lipids, sterol-like molecules that are synthesized in a wide range of bacteria, including Frankia, several cyanobacteria, and rhizobia . The levels of hopanoid found in Frankia are among the highest of any organism known to date . Here we report that short (328-bp) DNA sequences from several strains of Frankia spp . have been identified that are homologous to a portion of the coding region of squalene-hopene cyclase (shc) genes . The fragments and corresponding polymerase chain reaction (PCR) primers can be used in phylogenetic comparisons of Frankia, both within Frankiaceae and among bacteria that synthesize hopanoids.

Can J Microbiol, 2001 Jun, 47(6), 526 - 34
Diversity in the rhizobia associated with Phaseolus vulgaris L . in Ecuador, and comparisons with Mexican bean rhizobia; Bernal G et al.; Common beans (Phaseolus vulgaris L.) have centers of origin in both Mesoamerica and Andean South America, and have been domesticated in each region for perhaps 5000 years . A third major gene pool may exist in Ecuador and Northern Peru . The diversity of the rhizobia associated with beans has also been studied, but to date with an emphasis on the Mesoamerican center of origin . In this study we compared bean rhizobia from Mexico and Andean South America using both phenotypic and phylogenetic approaches . When differences between the rhizobia of these two regions were shown, we then examined the influence of bean cultivar on the most probable number (MPN) count and biodiversity of rhizobia recovered from different soils . Three clusters of bean rhizobia were distinguished using phenotypic analysis and principal-component analysis of Box AIR-PCR banding patterns . They corresponded principally to isolates from Mexico, and the northern and southern Andean regions, with isolates from southern Ecuador exhibiting significant genetic diversity . Rhizobia from Dalea spp., which are infective and effective on beans, may have contributed to the apparent diversity of rhizobia recovered from the Mesoamerican region, while the rhizobia of wild Phaseolus aborigineus from Argentina showed only limited similarity to the other bean rhizobia tested . Use of P . vulgaris cultivars from the Mesoamerican and Andean Phaseolus gene pools as trap hosts did not significantly affect MPN counts of bean rhizobia from the soils of each region, but did influence the diversity of the rhizobia recovered . Such differences in compatibility of host and Rhizobium could be a factor in the poor reputation for nodulation and N2 fixation in this crop.

Can J Microbiol, 2001 Jun, 47(6), 519 - 25
Characterization of soybean bradyrhizobia for which serogroup affinities have not been identified; van Berkum P et al.; The USDA, ARS National Rhizobium Germplasm Collection contains 143 accessions of slow-growing soybean strains among which there are 17 distinct serological groups . However, 11 strains appear to have no serological affinity with the 17 serogroups . Therefore, we determined whether these strains were diverse and examined their phylogenetic placement . Nine strains formed nitrogen-fixing symbioses with soybean indicating that these accessions were not contaminants . We concluded from results of amplified fragment length polymorphism (AFLP) analysis, using 3 selective primers with 8 strains, that they were genetically dissimilar . Nine strains were examined for their fatty acid composition using fatty acid methyl ester (FAME) derivatives . The FAME results with 5 strains and serotype strains of Bradyrhizobium elkanii were similar, while results with each of the remaining 2 pairs were either similar to the type strain of Bradyrhizobium japonicum (USDA 6) or to USDA 110 . Evolutionary history of 9 strains was reconstructed from sequence divergence of a combination of the complete 16S rRNA gene, the internally transcribed spacer region, and about 400 bases of the 5' end of the 23S rRNA gene . Placement of 5 strains was nested within B . elkanii, 2 with USDA 110, and the other 2 with USDA 6 . We concluded that soybean isolates that cannot be placed within one of the 17 established serogroups are phenotypically and genetically as diverse as the serotype strains.

Can J Microbiol, 2001 Jun, 47(6), 509 - 18
Identification of a system that allows a Rhizobium tropici dctA mutant to grow on succinate, but not on other C4-dicarboxylates; Batista S et al.; A defined insertion mutant of a gene encoding a homolog of the rhizobial C4-dicarboxylate permease (dctA) was constructed in Rhizobium tropici strain CIAT899 . This mutant (GA1) was unable to grow on fumarate or malate; however, in contrast with other rhizobial dctA mutants, it retained a limited ability to grow on succinate with ammonia as a nitrogen source . Our results suggest the presence of a novel succinate-specific transport system in R . tropici . Biochemical characterization indicated that this alternative transport system in GAI is active and dependent on an energized membrane . It was also induced by succinate and aspartate, and was repressed by glucose and glycerol . Bean plants inoculated with GA1 showed a reduced nitrogen-fixing ability, achieving only 29% of the acetylene reduction activity determined in CIAT899 strain nodules, 33 days after inoculation . Also, bean plants inoculated with GA1 had reduced shoot dry weight compared with plants inoculated with the wild-type strain.

Can J Microbiol, 2001 Jun, 47(6), 488 - 94
Selection of several classes of mimosine-degradation-defective Tn3Hogus-insertion mutants of Rhizobium sp . strain TAL1145 on the basis of mimosine-inducible GUS activity; Fox PM et al.; Rhizobium sp . strain TAL1145 that nodulates Leucaena leucocephala degrades mimosine, a toxin produced by this tree legume . A cosmid clone, pUHR263, containing approximately 25 kb cloned DNA was isolated by plating Escherichia coli cells containing the cosmid clone library of TAL1145 on a minimal medium in which 3-hydroxy-4-pyridone (HP), a degradation product of mimosine, was used as the source of nitrogen . Cosmid pUHR263 was mutagenized by random insertions of Tn3Hogus, a transposon that makes transcriptional gus fusions when it is inserted in a gene in the correct orientation . Various pUHR263::Tn3Hogus derivatives that showed mimosine-inducible or mimosine-repressible GUS activities when transferred to the Rhizobium sp . strain TAL1145 were selected . Mutants of TAL1145 were constructed by transferring these Tn3Hogus insertions into the TAL1145 chromosome through double-homologous recombination . These mutants were classified into five classes on the basis of defects in mimosine degradation . The growth of these mutants was inhibited to different extents by mimosine applied to the growth medium . Mimosine forms a red-colored Fe-mimosine complex when FeCI3 is added to the medium . The inhibitory effect of Fe-mimosine on growth of the mutants was much less than that of mimosine.

Can J Microbiol, 2001 Jun, 47(6), 475 - 87
Erosion of root epidermal cell walls by Rhizobium polysaccharide-degrading enzymes as related to primary host infection in the Rhizobium-legume symbiosis; Mateos PF et al.; A central event of the infection process in the Rhizobium-legume symbiosis is the modification of the host cell wall barrier to form a portal of entry large enough for bacterial penetration . Transmission electron microscopy (TEM) indicates that rhizobia enter the legume root hair through a completely eroded hole that is slightly larger than the bacterial cell and is presumably created by localized enzymatic hydrolysis of the host cell wall . In this study, we have used microscopy and enzymology to further clarify how rhizobia modify root epidermal cell walls to shed new light on the mechanism of primary host infection in the Rhizobium-legume symbiosis . Quantitative scanning electron microscopy indicated that the incidence of highly localized, partially eroded pits on legume root epidermal walls that follow the contour of the rhizobial cell was higher in host than in nonhost legume combinations, was inhibited by high nitrate supply, and was not induced by immobilized wild-type chitolipooligosaccharide Nod factors reversibly adsorbed to latex beads . TEM examination of these partially eroded, epidermal pits indicated that the amorphous, noncrystalline portions of the wall were disrupted, whereas the crystalline portions remained ultrastructurally intact . Further studies using phase-contrast and polarized light microscopy indicated that (i) the structural integrity of clover root hair walls is dependent on wall polymers that are valid substrates for cell-bound polysaccharide-degrading enzymes from rhizobia, (ii) the major site where these rhizobial enzymes can completely erode the root hair wall is highly localized at the isotropic, noncrystalline apex of the root hair tip, and (iii) the degradability of clover root hair walls by rhizobial polysaccharide-degrading enzymes is enhanced by modifications induced during growth in the presence of chitolipooligosaccharide Nod factors from wild-type clover rhizobia . The results suggest a complementary role of rhizobial cell-bound glycanases and chitolipooligosaccharides in creating the localized portals of entry for successful primary host infection.

Can J Microbiol, 2001 Jun, 47(6), 467 - 74
Acid and alkaline treatments for enhancing the growth of rhizobia in sludge; Rebah FB et al.; Wastewater sludges have been proposed as an effective media for the production of rhizobia . The effect of total suspended solid (TSS) concentrations and pretreatments of sludge on the growth of Sinorhizobium meliloti were investigated . Acid (pH 2.0-6.0 obtained with H2SO4) and alkaline (50-200 mequiv.wt./L of NaOH) treatments were applied to enhance the biodegradability of primary (0.325%-3.2% TSS obtained by dilution of original sample) and secondary (0.2%-0.4% TSS obtained by concentration of original sample) sludges . In primary sludge without pretreatment, the highest cell count (11.10 x 10(9) cfu/mL) was obtained with 1.3% TSS . However, a maximum cell count of 13.00 x 10(9) cfu/mL was reached using an acid treatment of pH 2.0 and a 0.325% TSS concentration . Moreover, the alkaline treatment with 100 mequiv.wt./L of NaOH and 0.65% TSS increased the cell yield to 21.00 x 10(9) cfu/mL . For secondary sludge without pretreatment, no enhancement of growth was observed while increasing TSS concentration . This may be due to the increase of inhibitory substances, such as heavy metals, and of the Ca and Mg concentrations . As in primary sludge, some acid and alkaline treatments of secondary sludge tend to improve the cell count of S . meliloti . However, the highest value of 9.80 x 10(9) cfu/mL obtained with 0.4% TSS at pH 2.0 was lower than that obtained with primary sludge . It was also observed that S . meliloti grown in treated sludges maintained its capacity to nodulate alfalfa.

J Exp Bot, 2001 Jul, 52(360), 1537 - 43
Specific flavonoids induced nod gene expression and pre-activated nod genes of Rhizobium leguminosarum increased pea (Pisum sativum L.) and lentil (Lens culinaris L.) nodulation in controlled growth chamber environments; Begum AA et al.; The gram-negative soil bacteria Rhizobium spp . infect and establish a nitrogen-fixing symbiosis with legume crops which involves the mutual exchange of diffusable signal molecules . In this study, Rhizobium leguminosarum containing a nod-lacZ gene fusion was used to screen the most effective plant-to-bacteria signal molecules for pea and lentil and the induction conditions . Out of a number of signal compounds including apigenin, daidzein, genistein, hesperetin, kaempferol, luteolin, naringenin, and rutin, hesperetin and naringenin were found to be the most effective plant-to-bacteria signal molecules . The induction of nod genes was temperature-dependent, where nod gene induction was decreased with dropping incubation temperature . The combination of hesperetin at 7 microM and naringenin at 3 microM resulted in better induction of nod gene activities compared to either hesperetin or naringenin alone . Nodulation and plant dry matter accumulation of pea and lentil plants receiving preinduced R . leguminosarum were higher than those of plants receiving uninduced R . leguminosarum cells in controlled environment growth chamber conditions . Preinduced Rhizobium with hesperetin at a concentration of 10 microM increased nodule number on average by 60.5% and dry matter accumulation by 14% in field pea at 17 degrees C, while it was 32% and 9% at 24 degrees C, respectively . Similarly, averaged over two rhizobial strains, a 59% and 6% increase in nodule number and biomass production at 17 degrees C, and a 39% and 27% at 24 degrees C, were obtained from lentil inoculated with hesperetin-induced R . leguminosarum, respectively.

Mikrobiologiia, 2001 May-Jun, 70(3), 348 - 51
{Effect of Rhizobium leguminosarum 252 agglutinins on the activity of some plant cell enzymes}; Karpunina LV et al.; The incubation of pea seedling roots with the surface agglutinins R1 and R2 of Rhizobium leguminosarum 252 brought about an increase in the activity of proteases, beta-glucosidase, and, especially, succinate dehydrogenase in the roots . The data presented show that rhizobial agglutinins play an important part in the formation and functioning of legume-rhizobial associations.

Adv Microb Physiol, 2001, 45, 113 - 56
Metals and the rhizobial-legume symbiosis--uptake, utilization and signalling; Johnston AW et al.; In this review, we consider how the nitrogen-fixing root nodule bacteria, the 'rhizobia', acquire various metals, paying particular attention to the uptake of iron . We also review the literature pertaining to the roles of molybdenum and nickel in the symbiosis with legumes . We highlight some gaps in our knowledge, for example the lack of information on how rhizobia acquire molybdenum . We examine the means whereby different metals affect rhizobial physiology and the role of metals as signals for gene regulation . We describe the ways in which genetics has shown (or not) if, and how, particular metal uptake and/or metal-mediated signalling pathways are required for the symbiotic interaction with legumes.

Chemistry, 2001 Jun 1, 7(11), 2390 - 7
Heterologous over-expression of alpha-1,6-fucosyltransferase from Rhizobium sp.: application to the synthesis of the trisaccharide beta-D-GlcNAc(1-->4)-{alpha-L-Fuc-(1-->6)}-D-GLcNAc, study of the acceptor specificity and evaluation of polyhydroxylated indolizidines as inhibitors; Bastida A et al.; An efficient heterologous expression system for overproduction of the enzyme alpha-1,6-Fucosyltransferase (alpha-1,6-FucT) from Rhizobium sp . has been developed . The gene codifying for the alpha-1,6-FucT was amplified by PCR using specific primers . After purification, the gene was cloned in the plasmid pKK223-3 . The resulting plasmid, pKK1,6FucT, was transformed into the E . coli strain XL1-Blue MRF' . The protein was expressed both as inclusion bodies and in soluble form . Changing the induction time a five-fold increase of enzyme expressed in soluble form was obtained . In this way five units of enzyme alpha-1,6-FucT can be obtained per liter of culture . A crude preparation of the recombinant enzyme was used for the synthesis of the branched trisaccharide alpha-D-GlcNAc-(1-->4)-{alpha-L-Fuc-(1-->6)}-D-GlcNAc (3), from chitobiose (2) and GDP-Fucose (1) . After purification, the trisaccharide 3 was obtained in a 84% overall yield . In order to elucidate the structural requirements for the acceptors, the specificity of the enzyme was studied towards mono-, di- and trisaccharides, which are structurally related to chitobiose . The enzyme uses, among others, the disaccharide N-acetyl lactosamine as a good substrate; the monosaccharide GlcNAc is a weak acceptor . Finally, several racemic polyhydroxylated indolizidines have been tested as potential inhibitors of the enzyme . Indolizidine 21 was the best inhibitor with an IC50 of 4.5 x 10(-5) M . Interestingly, this compound turned out to be the best mimic for the structural features of the fucose moiety in the presumed transition state.

Mol Microbiol, 2001 Jun, 40(6), 1449 - 59
Solute-binding protein-dependent ABC transporters are responsible for solute efflux in addition to solute uptake; Hosie AH et al.; The ATP-binding cassette (ABC) transporter superfamily is one of the most widespread of all gene families and currently has in excess of 1100 members in organisms ranging from the Archaea to manQ1 . The movement of the diverse solutes of ABC transporters has been accepted as being strictly unidirectional, with recent models indicating that they are irreversible . However, contrary to this paradigm, we show that three solute-binding protein-dependent (SBP) ABC transporters of amino acids, i.e . the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra) of Rhizobium leguminosarum and the histidine permease (His) of Salmonella typhimurium, are bidirectional, being responsible for efflux in addition to the uptake of solutes . The net solute movement measured for an ABC transporter depends on the rates of uptake and efflux, which are independent; a plateau is reached when both are saturated . SBP ABC transporters promote active uptake because, although the Vmax values for uptake and efflux are not significantly different, there is a 103-104 higher affinity for uptake of solute compared with efflux . Therefore, the SBP ABC transporters are able to support a substantial concentration gradient and provide a net uptake of solutes into bacterial cells.

Biosci Biotechnol Biochem, 2001 May, 65(5), 1265 - 9
Evidence for the presence of DNA-binding proteins involved in regulation of the gene expression of indole-3-pyruvic acid decarboxylase, a key enzyme in indole-3-acetic acid biosynthesis in Azospirillum lipoferum FS; Yagi K et al.; We isolated the ipdc gene coding for indole-3-pyruvic acid decarboxylase (IPDC), a key enzyme in the indole-3-pyruvic acid pathway for indole-3-acetic acid biosynthesis, in the plant growth-promoting rhizobacterium Azospirillum lipoferum FS . Gel mobility-shift assay showed the presence of two DNA-binding proteins that might be involved in regulation of the ipdc gene expression.

Mol Plant Microbe Interact, 2001 Jul, 14(7), 918 - 24
Analysis of a symbiosis-specific cytochrome P450 homolog in Rhizobium sp . BR816; Luyten E et al.; Sequence analysis of the DNA region upstream of nodO in Rhizobium sp . BR816 revealed an open reading frame in which the deduced amino acid sequence shows homology with cytochrome P450 . Because the BR816 P450 homolog shows 73% amino acid similarity with CYP127A1(Y4vG), which is identified on the symbiotic plasmid of Rhizobium sp . NGR234, it is named CYP127A2 . Transcriptional analysis of CYP127A2 revealed high expression in bacteroids, whereas no or hardly any expression was observed under free-living conditions . Low-level, free-living expression, however was noticed when cells were grown microoxically at acid pH levels . A number of possible substrates that may induce P450 gene expression were analyzed, but only the addition of short-chain alcohols to cultures slightly increased CYP127A2 expression . High levels of CYP127A2 expression observed in bacteroids of a nifH mutant strain, which formed non-fixing nodules on bean, indicated that the genuine substrate for CYP127A2 is not a metabolite resulting from N2-fixation . Nevertheless, expression analysis pointed to a NifA- and sigma54-dependent transcription.

Mol Plant Microbe Interact, 2001 Jul, 14(7), 839 - 47
Cell biological changes of outer cortical root cells in early determinate nodulation; van Spronsen PC et al.; In the symbiosis of leguminous plants and Rhizobium bacteria, nodule primordia develop in the root cortex . This can be either in the inner cortex (indeterminate-type of nodulation) or outer cortex (determinate-type of nodulation), depending upon the host plant . We studied and compared early nodulation stages in common bean (Phaseolus vulgaris) and Lotus japonicus, both known as determinate-type nodulation plants . Special attention was paid to the occurrence of cytoplasmic bridges, the influence of rhizobial Nod factors (lipochitin oligosaccharides {LCOs}) on this phenomenon, and sensitivity of the nodulation process to ethylene . Our results show that i) both plant species form initially broad, matrix-rich infection threads; ii) cytoplasmic bridges occur in L . japonicus but not in bean; iii) formation of these bridges is induced by rhizobial LCOs; iv) formation of primordia starts in L . japonicus in the middle root cortex and in bean in the outer root cortex; and v) in the presence of the ethylene-biosynthesis inhibitor aminoethoxyvinylglycine (AVG), nodulation of L . japonicus is stimulated when the roots are grown in the light, which is consistent with the role of cytoplasmic bridges during nodulation of L . japonicus.

Mol Plant Microbe Interact, 2001 Jul, 14(7), 823 - 31
The Rhizobium GstI protein reduces the NH4+ assimilation capacity of Rhizobium leguminosarum; Tate R et al.; We show that the protein encoded by the glutamine synthetase translational inhibitor (gstI) gene reduces the NH4+ assimilation capacity of Rhizobium leguminosarum . In this organism, gstI expression is regulated by the ntr system, including the PII protein, as a function of the nitrogen (N) status of the cells . The GstI protein, when expressed from an inducible promoter, inhibits glutamine synthetase II (glnII) expression under all N conditions tested . The induction of gstI affects the growth of a glutamine synthetase I (glnA-) strain and a single amino acid substitution (W48D) results in the complete loss of GstI function . During symbiosis, gstI is expressed in young differentiating symbiosomes (SBs) but not in differentiated N2-fixing SBs . In young SBs, the PII protein modulates the transcription of NtrC-regulated genes such as gstI and glnII . The evidence presented herein strengthens the idea that the endocytosis of bacteria inside the cytoplasm of the host cells is a key step in the regulation of NH4+ metabolism.

Carbohydr Res, 2001 May 18, 332(2), 167 - 73
Structure of a polysaccharide from a Rhizobium species containing 2-deoxy-beta-D-arabino-hexuronic acid; Guentas L et al.; The structure of the extracellular polysaccharide (EPS) produced by the Rhizobium sp . B strain isolated from atypical nodules on alfalfa has been determined using a combination of chemical and physical techniques (methylation analysis, high pH-anion exchange chromatography (HPAEC), mass spectrometry and 1-D and 2-D NMR spectroscopy) . As opposed to the EPS from other strains of Rhizobium, the EPS from the sp . B strain contains D-Glc together with L-Rha and 2-deoxy-D-arabino-hexuronic acid . It is a polymer of a repeating unit having the following structure: --> 4)-beta-D-Glcp-(1 --> 4)-alpha-L-Rhap -(1 --> 3)-beta-D-Glcp-(1 --> 4)-2-deoxy-beta-D-GlcpA-(1 --> . The polysaccharide also contains 0.6 O-acetyl groups per sugar which have not been located.

Appl Environ Microbiol, 2001 Jul, 67(7), 3264 - 8
Nitrogen-fixing nodules with Ensifer adhaerens harboring Rhizobium tropici symbiotic plasmids; Rogel MA et al.; Ensifer adhaerens is a soil bacterium that attaches to other bacteria and may cause lysis of these other bacteria . Based on the sequence of its small-subunit rRNA gene, E . adhaerens is related to Sinorhizobium spp . E . adhaerens ATCC 33499 did not nodulate Phaseolus vulgaris (bean) or Leucaena leucocephala, but with symbiotic plasmids from Rhizobium tropici CFN299 it formed nitrogen-fixing nodules on both hosts . The nodule isolates were identified as E . adhaerens isolates by growth on selective media.

FEMS Microbiol Lett, 2001 Jun 25, 200(2), 207 - 13
Rhodobacter capsulatus nifA mutants mediating nif gene expression in the presence of ammonium; Paschen A et al.; Expression of nitrogen fixation genes in Rhodobacter capsulatus is repressed by ammonium at different regulatory levels including an NtrC-independent mechanism controlling NifA activity . In contrast to R . capsulatus NifA, heterologous NifA proteins of Klebsiella pneumoniae and Rhizobium meliloti, respectively, were not subjected to this posttranslational ammonium control in R . capsulatus . The characterization of ammonium-tolerant R . capsulatus NifA1 mutants indicated that the N-terminal domain of NifA was involved in posttranslational regulation . Analysis of a double mutant carrying amino acid substitutions in both the N-terminal domain and the C-terminal DNA-binding domain gave rise to the hypothesis that an interaction between these two domains might be involved in ammonium regulation of NifA activity . Western analysis demonstrated that both constitutively expressed wild-type and ammonium-tolerant NifA1 proteins exhibited high stability and accumulated to comparable levels in cells grown in the presence of ammonium excluding the possibility that proteolytic degradation was responsible for ammonium-dependent inactivation of NifA.

Carbohydr Res, 2001 Jun 22, 333(1), 73 - 8
Biotin labeling of the symbiotically important succinoglycan oligosaccharides of Rhizobium meliloti for identification of putative plant receptors; Wang L et al.; The symbiotically important trimer of the succinoglycan octasaccharide subunit was labeled with a biotin tag through coupling with a 6-biotinamidohexan hydrazide and subsequent reduction with borane . The acetyl and succinyl groups in the molecule were stable to the two-step sequence, while a small percentage of the ketal in the pyruvate groups was reduced to an ether-linked lactic acid moiety attached to either the O-4 or O-6 position of the sugar residue under the reaction conditions.

Nature, 2001 Jun 21, 411(6840), 948 - 50
Nodulation of legumes by members of the beta-subclass of Proteobacteria; Moulin L et al.; Members of the Leguminosae form the largest plant family on Earth, with around 18,000 species . The success of legumes can largely be attributed to their ability to form a nitrogen-fixing symbiosis with specific bacteria known as rhizobia, manifested by the development of nodules on the plant roots in which the bacteria fix atmospheric nitrogen, a major contributor to the global nitrogen cycle . Rhizobia described so far belong exclusively to the alpha-subclass of Proteobacteria, where they are distributed in four distinct phylogenetic branches . Although nitrogen-fixing bacteria exist in other proteobacterial subclasses, for example Herbaspirillum and Azoarcus from the phylogenetically distant beta-subclass, none has been found to harbour the nod genes essential for establishing rhizobial symbiosis . Here we report the identification of proteobacteria from the beta-subclass that nodulate legumes . This finding shows that the ability to establish a symbiosis with legumes is more widespread in bacteria than anticipated to date.

Curr Opin Plant Biol, 2001 Aug, 4(4), 343 - 50
Molecular basis of plant growth promotion and biocontrol by rhizobacteria; Bloemberg GV et al.; Plant-growth-promoting rhizobacteria (PGPRs) are used as inoculants for biofertilization, phytostimulation and biocontrol . The interactions of PGPRs with their biotic environment, for example with plants and microorganisms, are often complex . Substantial advances in elucidating the genetic basis of the beneficial effects of PGPRs on plants have been made, some from whole-genome sequencing projects . This progress will lead to a more efficient use of these strains and possibly to their improvement by genetic modification.

Curr Opin Plant Biol, 2001 Aug, 4(4), 336 - 42
Rhizobium type III secretion systems: legume charmers or alarmers?
Marie C, Broughton WJ, Deakin WJ.
Mutagenesis and sequence analyses of rhizobial genomes have revealed the presence of genes encoding type III secretion systems . Considered as a machine used by plant and animal pathogens to deliver virulence factors into their hosts, this secretion apparatus has recently been proven to play a role in symbiotic bacteria-leguminous plant interactions.

Curr Opin Plant Biol, 2001 Aug, 4(4), 328 - 35
Genetics and genomics of root symbiosis; Stougaard J; Model genetics and genomics have been developed as tools for studying the third largest family of flowering plants, the Leguminosae, which includes important crop plants . Functional genomics strategies for the global analysis of gene expression, the elucidation of pathways and reverse genetics are established . These approaches provide new possibilities for investigating rhizobial as well as mycorrhizal endosymbiosis . Plant genes with central functions in these mutualistic interactions have been identified by positional cloning and gene tagging . With progress in Lotus japonicus genome sequencing, which was recently initiated by Japanese researchers, comparative genomics will contribute to our understanding of symbiosis, pathogenesis and the evolution of plant genomes.

Plant Mol Biol, 2001 Mar, 45(5), 609 - 18
Substrate specificity and antifungal activity of recombinant tobacco class I chitinases; Suarez V et al.; Endochitinases contribute to the defence response of plants against chitin-containing pathogens . The vacuolar class I chitinases consist of an N-terminal cysteine-rich domain (CRD) linked by a glycine-threonine-rich spacer with 4-hydroxylated prolyl residues to the catalytic domain . We examined the functional role of the CRD and spacer region in class I chitinases by comparing wild-type chitinase A (CHN A) of Nicotiana tabacum with informative recombinant forms . The chitinases were expressed in transgenic N . sylvestris plants, purified to near homogeneity, and their structures confirmed by mass spectrometry and partial sequencing . The enzymes were tested for their substrate preference towards chitin, lipo-chitooligosaccharide Nod factors of Rhizobium, and bacterial peptidoglycans (lysozyme activity) as well as for their capacity to inhibit hyphal growth of Trichoderma viride . Deletion of the CRD and spacer alone or in combination resulted in a modest <50% reduction of hydrolytic activity relative to CHN A using colloidal chitin or M . lysodeikticus walls as substrates; whereas, antifungal activity was reduced by up to 80% . Relative to CHN A, a variant with two spacers in tandem, which binds chitin, showed very low hydrolytic activity towards chitin and Nod factors, but comparable lysozyme activity and enhanced antifungal activity . Neither hydrolytic activity, substrate specificity nor antifungal activity were strictly correlated with the CRD-mediated capacity to bind chitin . This suggests that the presence of the chitin-binding domain does not have a major influence on the functions of CHN A examined . Moreover, the results with the tandem-spacer variant raise the possibility that substantial chitinolytic activity is not essential for inhibition of T . viride growth by CHN A.

Lett Appl Microbiol, 2001 Jun, 32(6), 379 - 83
Tolerance of Bradyrhizobium sp . (Lupini) strains to salinity, pH, CaCO3 and antibiotics; Raza S et al.; AIMS: Ten rhizobial isolates obtained from different locations in Egypt were examined for their ability to survive under stress conditions and their growth response to increasing levels of NaCl (1-8% w/v), pH (4-10), CaCO3 (1-10% w/v) and 12 antibiotics . METHODS AND RESULTS: All the rhizobial isolates tolerated a NaCl concentration up to 5% and were divided into two groups with respect to NaCl tolerance . The rhizobial isolates from group two showed significantly (P < 0.05) better survival under high NaCl concentration . All the tested isolates survived acidic (pH 4-5) and alkaline conditions (pH 9-10) and CaCO3 (up to 10% w/v) in liqued YEM medium . CONCLUSION: Antibiotic resistance patterns did not correlate to NaCl, pH or CaCO3 tolerance . Variations among different strains showed that there is potential to improve strain performance under stress conditions . Significance and Impact of the Study: The results suggest that selection of adapted strains under stress conditions is possible and can be used as inoculants for successful lupin growth.

Eur J Histochem, 2001, 45(1), 39 - 49
Extracellular polysaccharides are involved in the attachment of Azospirillum brasilense and Rhizobium leguminosarum to arbuscular mycorrhizal structures; Bianciotto V et al.; Arbuscular mycorrhizal (AM) fungi, one of the most important component of the soil microbial community, establish physical interactions with naturally occurring and genetically modified bacterial biofertilizers and biopesticides, commonly referred to as plant growth-promoting rhizobacteria (PGPR) . We have used a genetic approach to investigate the bacterial components possibly involved in the attachment of two PGPR (Azospirillum and Rhizobium) to AM roots and AM fungal structures . Mutants affected in extracellular polysaccharides (EPS) have been tested in in vitro adhesion assays and shown to be strongly impaired in the attachment to both types of surfaces as well as to quartz fibers . Anchoring of rhizobacteria to AM fungal structures may have special ecological and biotechnological significance because it may facilitate colonisation of new rhizospheres by the bacteria, and may be an essential trait for the development of mixed inocula.

Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 909 - 14
Rhizobium yanglingense sp . nov., isolated from arid and semi-arid regions in China; Tan ZY et al.; A novel rhizobial group, cluster 9, defined in previous research {Tan, Z . Y., Wang, E . T., Peng, G . X., Zhu, M . E., Martinez-Romero, E . & Chen, W . X . (1999) . Int J Syst Bacteriol 49, 1457-1469}, was further characterized by determination of DNA base composition, whole-cell protein SDS-PAGE analysis, DNA-DNA hybridization, 16S rRNA gene sequencing and host specificity . These isolates were collected from the wild legumes Amphicarpaea trisperma, Coronilla varia and Gueldenstaedtia multiflora growing in arid and semi-arid regions in northwestern China . Isolates within cluster 9 grouped into a single cluster above a similarity level of 90.6% in a cluster analysis based on protein SDS-PAGE, and they were differentiated from defined rhizobial species . Comparative analysis of 16S rRNA gene sequences showed that isolate CCBAU 71623T, representing cluster 9, was most related to Rhizobium gallicum and Rhizobium mongolense . The DNA-DNA homologies were lower than 42.4% among cluster 9 and defined species, including R . gallicum and R . mongolense . These data indicated that cluster 9 was a unique genomic species . Isolates within this cluster could share their host plants . They could not nodulate Galega orientalis and Leucaena leucocephala and formed ineffective nodules on Phaseolus vulgaris . This group could also be differentiated from defined species by phenotypic characteristics . It is therefore proposed as a new species, Rhizobium yanglingense, with isolate CCBAU 71623 as the type strain.

Indian J Exp Biol, 2000 Dec, 38(12), 1245 - 50
Influence of metham sodium on suppression of collar rot disease of peanut, in vitro antibiosis, siderophore production and root colonization by a fluorescent pseudomonad strain FPO4; Dileep C et al.; A hydroxamate type siderophore producing fluorescent Pseudomonas strain, isolated from the rhizoplane of paddy root showing plant growth promoting activity, exhibited a decreased in vitro antibiosis, production of siderophore and suppression of collar rot in presence of metham sodium . Use of herbicide had a detrimental effect on the plant growth promoting activity of this organism . The multiple drug resistant mutant strain derived from this rhizobacteria colonized the roots, but the herbicide application had a negative effect on their population . HPLC analysis of the siderophore showed five peaks of which the peak number three confirmed the antifungal activity.

Gene, 2001 May 30, 270(1-2), 237 - 43
Functional characterization of an ammonium transporter gene from Lotus japonicus; Salvemini F et al.; NH(4)(+) is the main product of symbiotic nitrogen fixation and the external concentration of combined nitrogen plays a key regulatory role in all the different step of plant-rhizobia interaction . We report the cloning and characterization of the first member of the ammonium transporter family, LjAMT1;1 from a leguminous plant, Lotus japonicus . Sequence analysis reveals a close relationship to plant transporters of the AMT1 family . The wild type and two mutated versions of LjAMT1;1 were expressed and functionally characterized in yeast . LjAMT1;1 is transcribed in roots, leaves and nodules of L . japonicus plants grown under low nitrogen conditions, consistent with a role in uptake of NH(4)(+) by the plant cells.

Curr Microbiol, 2001 Sep, 43(3), 182 - 6
Cyanide production by rhizobacteria and potential for suppression of weed seedling growth; Kremer RJ et al.; Rhizobacteria strains were characterized for ability to synthesize hydrogen cyanide and for effects on seedling root growth of various plants . Approximately 32% of bacteria from a collection of over 2000 isolates were cyanogenic, evolving HCN from trace concentrations to > 30 nmoles/mg cellular protein . Cyanogenesis was predominantly associated with pseudomonads and was enhanced when glycine was provided in the culture medium . Concentrations of HCN produced by rhizobacteria were similar to exogenous concentrations inhibiting seedling growth in bioassays, suggesting that cyanogenesis by rhizobacteria in the rhizosphere can adversely affect plant growth . Growth inhibition of lettuce and barnyardgrass by volatile metabolites of the cyanogenic rhizobacteria confirmed that HCN was the major inhibitory compound produced . Our results suggest that HCN produced in the rhizospheres of seedlings by selected rhizobacteria is a potential and environmentally compatible mechanism for biological control of weeds.

Environ Int, 2001 May, 26(5-6), 417 - 23
Relationships between chromium biomagnification ratio, accumulation factor, and mycorrhizae in plants growing on tannery effluent-polluted soil; Khan AG; Heavy metal-contaminated land is increasingly becoming an important environmental, health, economic, and planning issue in Pakistan . The unplanned disposal of industrial effluent from tannery, for example, has resulted in a many fold increase in chromium (Cr) in the land near a tannery . This study was undertaken to compare the total and the DTPA-available Cr contents in the soil and the roots and leaves of tree species growing on it with those on the nearby noncontaminated reference site at Kala Shah Kakoo, Panjab, Pakistan . A very reduced plant cover on the tannery effluent-contaminated site was noted and there was a sharp boundary between the polluted and nonpolluted reference sites, suggesting a strong selection pressure . Polluted soil contained considerable higher amounts of Cr as compared to the reference soil but no correlation was found between Cr contents in the dried plant tissue and the total DTPA-extractable Cr . Roots of all the three tree species, i.e . Dalbergia sissoo, Acacia arabica, and Populus euroamericana, growing on both the contaminated as well reference site possessed arbuscular mycorrhizal fungal (AMF) infection in their roots and AMF propagules in the associated rhizospheres . D . sissoo and A . arabica roots were also studded with nitrogen-fixing rhizobial root nodules, while those of P . euroamericana possessed AMF as well as ectomycorrhizal infections . The dual infection would encourage mineral nutrition, including Cr . AMF community varied, i.e . trees growing on the reference site were exposed to a wide variety of AMF such as Glomus, Scutellospora, and Acaulospora, whereas those on the contaminated site contained only Gigaspora spp . in their mycorrhizospheres, suggesting a selection pressure . Typical Glomus infection patterns in the roots of D . sissoo growing on the contaminated soil but absence of spores of Glomus spp . in the associated rhizospheres indicate the potential error of using AMF spores to extrapolate the root infection . High Cr contents adversely affected the size, diversity, and species richness of AMF as measured by Shannon-Weiner index . The potential of mycorrhizae in protecting the host plant against the harmful effect of heavy metals and in phytoremediation of the Cr-polluted soil is discussed.

Microb Ecol, 2001 Apr, 41(3), 252 - 263
The Diversity of Archaea and Bacteria in Association with the Roots of Zea mays L; Chelius MK et al.; The diversity of bacteria and archaea associating on the surface and interior of maize roots (Zea mays L.) was investigated . A bacterial 16S rDNA primer was designed to amplify bacterial sequences directly from maize roots by PCR to the exclusion of eukaryotic and chloroplast DNA . The mitochondrial sequence from maize was easily separated from the PCR-amplified bacterial sequences by size fractionation . The culturable component of the bacterial community was also assessed, reflecting a community composition different from that of the clone library . The phylogenetic overlap between organisms obtained by cultivation and those identified by direct PCR amplification of 16S rDNA was 48% . Only 4 bacterial divisions were found in the culture collection, which represented 27 phylotypes, whereas 6 divisions were identified in the clonal analysis, comprising 74 phylotypes, including a member of the OP10 candidate division originally described as a novel division level lineage in a Yellowstone hot spring . The predominant group in the culture collection was the actinobacteria and within the clone library, the a-proteobacteria predominated . The population of maize-associated proteobacteria resembled the proteobacterial population of a typical soil community within which resided a subset of specific plant-associated bacteria, such as Rhizobium- and Herbaspirillum-related phylotypes . The representation of phylotypes within other divisions (OP10 and Acidobacterium) suggests that maize roots support a distinct bacterial community . The diversity within the archaeal domain was low . Of the 50 clones screened, 6 unique sequence types were identified, and 5 of these were highly related to each other (sharing 98% sequence identity) . The archaeal sequences clustered with good bootstrap support near Marine group I (crenarchaea) and with Marine group II (euryarchaea) uncultured archaea . The results suggest that maize supports a diverse root-associated microbial community composed of species that for the first time have been described as inhabitants of a plant-root environment.

Mol Plant Microbe Interact, 2001 Jun, 14(6), 737 - 48
Medicago truncatula ENOD11: a novel RPRP-encoding early nodulin gene expressed during mycorrhization in arbuscule-containing cells; Journet EP et al.; Leguminous plants establish endosymbiotic associations with both rhizobia (nitrogen fixation) and arbuscular mycorrhizal fungi (phosphate uptake) . These associations involve controlled entry of the soil microsymbiont into the root and the coordinated differentiation of the respective partners to generate the appropriate exchange interfaces . As part of a study to evaluate analogies at the molecular level between these two plant-microbe interactions, we focused on genes from Medicago truncatula encoding putative cell wall repetitive proline-rich proteins (RPRPs) expressed during the early stages of root nodulation . Here we report that a novel RPRP-encoding gene, MtENOD11, is transcribed during preinfection and infection stages of nodulation in root and nodule tissues . By means of reverse transcription-polymerase chain reaction and a promoter-reporter gene strategy, we demonstrate that this gene is also expressed during root colonization by endomycorrhizal fungi in inner cortical cells containing recently formed arbuscules . In contrast, no activation of MtENOD11 is observed during root colonization by a nonsymbiotic, biotrophic Rhizoctonia fungal species . Analysis of transgenic Medicago spp . plants expressing pMtENOD11-gusA also revealed that this gene is transcribed in a variety of nonsymbiotic specialized cell types in the root, shoot, and developing seed, either sharing high secretion/metabolite exchange activity or subject to regulated modifications in cell shape . The potential role of early nodulins with atypical RPRP structures such as ENOD11 and ENOD12 in symbiotic and nonsymbiotic cellular contexts is discussed.

Environ Exp Bot, 2001 Aug, 46(1), 37 - 46
Nitrate reductase activity, distribution, and response to nitrate in two contrasting Phaseolus species inoculated with Rhizobium spp; Silveira JA et al.; The nitrate reductase activity distribution and response of two nodulated species of Phaseolus (Phaseolus vulgaris-common bean, and Phaseolus lunatus-lima bean) to different exogenous nitrate levels were studied during the vegetative period . These Phaseolus species showed to be very contrasting in respect to the pattern of nitrate reductase (NR) activity distribution thought the plant . The highest level of NR activity in P . vulgaris was clearly shown to occur in leaves in contrast with the lowest one detected in roots and nodules as widely seen for other tropical species of the Phaseoleae tribe . Conversely, P . lunatus had higher NR activity in the nodules, whereas its leaves exhibited a steadily decrease during the plant development . Indeed, at 32 days after emergence (pre-flowering stage), the nodulated P . vulgaris had approximately 95% of the total NR activity localized in its leaves, whereas in P . lunatus it was equally distributed in the nodules and in the leaves . Under long-term exposure to increasing exogenous level of nitrate, the leaf-NR activity of nodulated P . vulgaris presented a positive response, whereas the enzyme activity was very low and unresponsive in P . lunatus . In contrast, the nodule-NR activity showed a reverse response to the increasing NO(3)(-) level . The nodule-NR activity of P . lunatus significantly increased whereas in the P . vulgaris nodules it was very low and unresponsive . This present study suggests that P . lunatus inoculated with Rhizobium tropici presents a singular pattern of nitrate reduction distribution among leaves and nodules during the vegetative development . It is speculated that the nodulated Phaseolus lunatus may have different NR isoforms in their leaves (at least a constitutive type) and an inducible form in their nodules, responsive to long-term exposure to nitrate.

Appl Environ Microbiol, 2001 Jun, 67(6), 2718 - 22
Bacteria mediate methylation of iodine in marine and terrestrial environments; Amachi S et al.; Methyl iodide (CH(3)I) plays an important role in the natural iodine cycle and participates in atmospheric ozone destruction . However, the main source of this compound in nature is still unclear . Here we report that a wide variety of bacteria including terrestrial and marine bacteria are capable of methylating the environmental level of iodide (0.1 microM) . Of the strains tested, Rhizobium sp . strain MRCD 19 was chosen for further analysis, and it was found that the cell extract catalyzed the methylation of iodide with S-adenosyl-L-methionine as the methyl donor . These results strongly indicate that bacteria contribute to iodine transfer from the terrestrial and marine ecosystems into the atmosphere.

Appl Environ Microbiol, 2001 Jun, 67(6), 2545 - 54
Exploiting genotypic diversity of 2,4-diacetylphloroglucinol-producing Pseudomonas spp.: characterization of superior root-colonizing P . fluorescens strain Q8r1-96; Raaijmakers JM et al.; The genotypic diversity that occurs in natural populations of antagonistic microorganisms provides an enormous resource for improving biological control of plant diseases . In this study, we determined the diversity of indigenous 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas spp . occurring on roots of wheat grown in a soil naturally suppressive to take-all disease of wheat . Among 101 isolates, 16 different groups were identified by random amplified polymorphic DNA (RAPD) analysis . One RAPD group made up 50% of the total population of DAPG-producing Pseudomonas spp . Both short- and long-term studies indicated that this dominant genotype, exemplified by P . fluorescens Q8r1-96, is highly adapted to the wheat rhizosphere . Q8r1-96 requires a much lower dose (only 10 to 100 CFU seed(-1) or soil(-1)) to establish high rhizosphere population densities (10(7) CFU g of root(-1)) than Q2-87 and 1M1-96, two genotypically different, DAPG-producing P . fluorescens strains . Q8r1-96 maintained a rhizosphere population density of approximately 10(5) CFU g of root(-1) after eight successive growth cycles of wheat in three different, raw virgin soils, whereas populations of Q2-87 and 1M1-96 dropped relatively quickly after five cycles and were not detectable after seven cycles . In short-term studies, strains Q8r1-96, Q2-87, and 1M1-96 did not differ in their ability to suppress take-all . After eight successive growth cycles, however, Q8r1-96 still provided control of take-all to the same level as obtained in the take-all suppressive soil, whereas Q2-87 and 1M1-96 gave no control anymore . Biochemical analyses indicated that the superior rhizosphere competence of Q8r1-96 is not related to in situ DAPG production levels . We postulate that certain rhizobacterial genotypes have evolved a preference for colonization of specific crops . By exploiting diversity of antagonistic rhizobacteria that share a common trait, biological control can be improved significantly.

Microbiol Res, 2001, 156(1), 71 - 4
Interaction of pea (Pisum sativum L.) lectins with rhizobial strains; Bajaj M et al.; Lectins from two varieties (PG-3 and LFP-48) of pea have been purified by affinity chromatography on Sephadex G-50 . The specific activity increased by 23 and 25 folds, respectively . These lectins from both the varieties were found to be specific for mannose . The purified fluorescein isothiocyanate (FITC)-labelled lectins showed binding reaction with homologous as well as heterologous strains of Rhizobium spp . The results revealed that pea lectins are not highly specific to their respective rhizobia . Moreover, these lectins showed a greater stimulatory effect on homologous Rhizobium leguminosarum strains.

J Bacteriol, 2001 Jun, 183(12), 3721 - 8
Unusual methyl-branched alpha,beta-unsaturated acyl chain substitutions in the Nod Factors of an arctic rhizobium, Mesorhizobium sp . strain N33 (Oxytropis arctobia); Poinsot V et al.; Mesorhizobium sp . strain N33 (Oxytropis arctobia), a rhizobial strain isolated in arctic Canada, is able to fix nitrogen at very low temperatures in association with a few arctic legume species belonging to the genera Astragalus, Onobrychis, and Oxytropis . Using mass spectrometry and nuclear magnetic resonance spectroscopy, we have determined the structure of N33 Nod factors, which are major determinants of nodulation . They are pentameric lipochito-oligosaccharides 6-O sulfated at the reducing end and exhibit other original substitutions: 6-O acetylation of the glucosamine residue next to the nonreducing terminal glucosamine and N acylation of the nonreducing terminal glucosamine by methyl-branched acyl chains of the iso series, some of which are alpha,beta unsaturated . These unusual substitutions may contribute to the peculiar host range of N33 . Analysis of N33 whole-cell fatty acids indicated that synthesis of the methyl-branched fatty acids depended on the induction of bacteria by plant flavonoids, suggesting a specific role for these fatty acids in the signaling process between the plant and the bacteria . Synthesis of the methyl-branched alpha,beta-unsaturated fatty acids required a functional nodE gene.

Biometals, 2001 Mar, 14(1), 59 - 66
Alleviation of aluminum toxicity to Rhizobium leguminosarum bv . viciae by the hydroxamate siderophore vicibactin; Rogers NJ et al.; Acid rain solubilises aluminum which can exert toxic effects on soil bacteria . The root nodule bacterium Rhizobium leguminosarum biovar viciae synthesises the hydroxamate siderophore vicibactin in response to iron limitation . We report the effect of vicibactin on the toxicity of aluminum(III) to R . leguminosarum and kinetic studies on the reaction of vicibactin with Al(III) and Fe(III) . Aluminum (added as the nitrate) completely inhibited bacterial growth at 25 microM final concentration, whereas the preformed Al-vicibactin complex had no effect . When aluminum and vicibactin solutions were added separately to growing cultures, growth was partly inhibited at 25 microM final concentration of each, but fully inhibited at 50 microM final concentration of each . Growth was not inhibited at 50 microM Al and 100 microM vicibactin, probably reflecting the slow reaction between Al and vicibactin; this results in some aluminum remaining uncomplexed long enough to exert toxic effects on growth, partly at 25 microM Al and vicibactin and fully at 50 microM Al and vicibactin . At 100 microM vicibactin and 50 microM Al, Al was complexed more effectively and there was no toxic effect . It was anticipated that vicibactin might enhance the toxicity of Al by transporting it into the cell, but the Al-vicibactin complex was not toxic . Several explanations are possible: the Al-vicibactin complex is not taken up by the cell; the complex is taken up but Al is not released from vicibactin; Al is released in the cell but is precipitated immediately . However, vicibactin reduces the toxicity of Al by complexing it outside the cell.

Environ Microbiol, 2001 Apr, 3(4), 273 - 80
Ribotyping of rhizobia nodulating Acacia mangium and Paraserianthes falcataria from different geographical areas in Indonesia using PCR-RFLP-SSCP (PRS) and sequencing; Clapp JP et al.; Acacia mangium and Paraserianthes falcataria are leguminous tree species widely grown for timber in Indonesia and other tropical countries, yet little is known about the identity of their rhizobial symbionts . Polymerase chain reaction-restriction fragment length polymorphism-single-strand conformational polymorphism (PRS) analysis of the 16S rRNA gene was used along with sequencing to assess the diversity of 57 rhizobia isolated from nodules of A . mangium and P . falctaria in Indonesia . In total, 26 rhizobia isolated from A . mangium were analysed by PRS and sequencing . The PRS patterns indicated that 12 (46%) clustered with Bradyrhizobium elkanii, 13 (50%) with B . lianoningense/japonicum and one (4%) with Mesorhizobium loti . Thirty-one isolates were analysed from P . falcataria: five (16%) clustered with B . elkanii and 26 (84%) with B . lianoningense/japonicum . These results were confirmed by phylogenetic analysis of sequences . Intraspecific diversity of the 16S rRNA genes from rhizobia nodulating A . mangium and P . falcataria revealed by PRS was low, only one genotype was found within the isolates that clustered with B . elkanii and two within the B . liaoningense/japonicum group . These Bradyrhizobium species are apparently ubiquitous throughout the Indonesian archipelago and it is clear why the two tree species are able to successfully establish outside their native range without the need for inoculation with indigenous rhizobia.

Plant Physiol, 2001 May, 126(1), 133 - 44
Sugar-binding activity of pea lectin enhances heterologous infection of transgenic alfalfa plants by Rhizobium leguminosarum biovar viciae; van Rhijn P et al.; Transgenic alfalfa (Medicago sativa L . cv Regen) roots carrying genes encoding soybean lectin or pea (Pisum sativum) seed lectin (PSL) were inoculated with Bradyrhizobium japonicum or Rhizobium leguminosarum bv viciae, respectively, and their responses were compared with those of comparably inoculated control plants . We found that nodule-like structures formed on alfalfa roots only when the rhizobial strains produced Nod factor from the alfalfa-nodulating strain, Sinorhizobium meliloti . Uninfected nodule-like structures developed on the soybean lectin-transgenic plant roots at very low inoculum concentrations, but bona fide infection threads were not detected even when B . japonicum produced the appropriate S . meliloti Nod factor . In contrast, the PSL-transgenic plants were not only well nodulated but also exhibited infection thread formation in response to R . leguminosarum bv viciae, but only when the bacteria expressed the complete set of S . meliloti nod genes . A few nodules from the PSL-transgenic plant roots were even found to be colonized by R . leguminosarum bv viciae expressing S . meliloti nod genes, but the plants were yellow and senescent, indicating that nitrogen fixation did not take place . Exopolysaccharide appears to be absolutely required for both nodule development and infection thread formation because neither occurred in PSL-transgenic plant roots following inoculation with an Exo(-) R . leguminosarum bv viciae strain that produced S . meliloti Nod factor.

Indian J Exp Biol, 2001 Jan, 39(1), 90 - 4
Influence of hexaconazole, carbofuran and ethion on soil microflora and dehydrogenase activities in soil and intact cell; Kalam A et al.; Total microbial count was highly affected (up to 61% at 1000 micrograms level) in presence of hexaconazole and persisted up to 21 days . Bacteria were more susceptible than actinomycetes . Carbofuran and ethion were moderately toxic to soil microflora . Inhibitory effects of all the three pesticides gradually decreased after 21 days as was evident by increase in total microbial count except in carbofuran . GDH activity in soil was also affected initially (up to 14 days) by all the three pesticides (60.3% in hexaconazole at 1000 micrograms level) and inhibition gradually decreased to zero except in carbofuran (15-20% toxicity persisted up to 35 days) . GDH and LDH activity in presence of hexaconazole was strongly affected in intact cells of some standard culture of bacteria like Rhizobium sp . (host Dolichos sp., 32.1 and 72.5%), Bacillus subtilis Cohn (86.75 and 76.5%), Azotobacter sp . (36.9 and 55.4%) and B . sphaericus (67.6% GDH) respectively . Carbofuran inhibited the enzyme activity in B . subtilis (55.55 and 35.3%) and to some extent in B . sphaericus . Ethion moderately inhibited LDH activity in Rhodococcus sp . AK1 (17.1 and 33.3%), Rhizobium (27.6% LDH), E . coli HB 101 (34.2% LDH) as evidenced by formazan formation . From the result, it might be concluded that among the above three pesticides tested hexaconazole strongly inhibited the dehydrogenase system in bacteria including nitrogen fixing bacteria of soil and thus may affect soil fertility . It was concluded that hexaconazole was more toxic than ethion to dehydrogenase enzymes.

J Bacteriol, 2001 Jun, 183(11), 3408 - 16
Rhizobial NodL O-acetyl transferase and NodS N-methyl transferase functionally interfere in production of modified Nod factors; Lopez-Lara IM et al.; The products of the rhizobial nodulation genes are involved in the biosynthesis of lipochitin oligosaccharides (LCOs), which are host-specific signal molecules required for nodule formation . The presence of an O-acetyl group on C-6 of the nonreducing N-acetylglucosamine residue of LCOs is due to the enzymatic activity of NodL . Here we show that transfer of the nodL gene into four rhizobial species that all normally produce LCOs that are not modified on C-6 of the nonreducing terminal residue results in production of LCOs, the majority of which have an acetyl residue substituted on C-6 . Surprisingly, in transconjugant strains of Mesorhizobium loti, Rhizobium etli, and Rhizobium tropici carrying nodL, such acetylation of LCOs prevents the endogenous nodS-dependent transfer of the N-methyl group that is found as a substituent of the acylated nitrogen atom . To study this interference between nodL and nodS, we have cloned the nodS gene of M . loti and used its product in in vitro experiments in combination with purified NodL protein . It has previously been shown that a chitooligosaccharide N deacetylated on the nonreducing terminus (the so-called NodBC metabolite) is the preferred substrate for NodS as well as for NodL . Here we show that the NodBC metabolite, acetylated by NodL, is not used by the NodS protein as a substrate while the NodL protein can acetylate the NodBC metabolite that has been methylated by NodS.

Enzyme Microb Technol, 2001 May 7, 28(7-8), 682 - 688
Purification and some characteristics of a recombinant dimeric rhizobium meliloti beta-galactosidase expressed in escherichia coli; Leahy M et al.; A recombinant Rhizobium meliloti beta-galactosidase was purified to homogeneity from an Escherichia coli expression system . The gene for the enzyme was cloned into a pKK223-3 plasmid which was then used to transform E . coli JM109 cells . The enzyme was purified 35-fold with a yield of 34% by a combination of DEAE-cellulose (pH 8.0) and two sequential Mono Q steps (at pH 8.0 and 6.0, respectively) . The purified enzyme had an apparent molecular mass of 174 kDa and a subunit molecular weight of 88 kDa, indicating that it is a dimer . It was active with both synthetic substrates p-nitrophenyl beta-D-galactopyranoside (PNPG) and o-nitrophenyl beta-D-galactopyranoside (ONPG) with K(m)(PNPG) and K(m)(ONPG) of 1 mM at 25 degrees C . The k(cat)/K(m) ratios for both substrates were approximately 70 mM(-1) sec(-1), indicating no clear preference for either PNPG or ONPG, unlike E . coli beta-galactosidase . After non-denaturing electrophoresis, active beta-galactosidase bands were identified using 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) or 6-bromo-2-naphthyl beta-D-galactopyranoside (BNG) and diazo blue B.

Mol Plant Microbe Interact, 2001 May, 14(5), 678 - 84
Rhizobium sp . BR816 produces a complex mixture of known and novel lipochitooligosaccharide molecules; Snoeck C et al.; Rhizobial lipochitooligosaccharide (LCO) signal molecules induce various plant responses, leading to nodule development . We report here the LCO structures of the broadhost range strain Rhizobium sp . BR816 . The LCOs produced are all pentamers, carrying common C18:1 or C18:0 fatty acyl chains, N-methylated and C-6 carbamoylated on the nonreducing terminal N-acetylglucosamine and sulfated on the reducing/terminal residue . A second acetyl group can be present on the penultimate N-acetylglucosamine from the nonreducing terminus . Two novel characteristics were observed: the reducing/terminal residue can be a glucosaminitol (open structure) and the degree of acetylation of this glucosaminitol or of the reducing residue can vary.

Mol Plant Microbe Interact, 2001 May, 14(5), 663 - 70
Lectin-like glycoprotein PsNLEC-1 is not correctly glycosylated and targeted in boron-deficient pea nodules; Bolanos L et al.; Symbiosome development was studied in pea root nodules from plants growing in the absence of boron (B) . Rhizobia released into the host cells of nodules from B-deficient plants developed to abnormal endophytic forms with an altered electrophoretic lipopolysaccharide pattern . Immunostaining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting of nodule homogenates with antibodies that recognize glycoprotein components showed that two previously described lectin-like glycoproteins (PsNLEC-1A and PsNLEC-1B) did not harbor the carbohydrate epitope normally recognized by specific monoclonal antibodies . Material derived from B-deficient nodules, however, still contained three antigenic isoforms with similar electrophoretic mobilities to PsNLEC-1 isoforms A, B, and C . These could be detected following immunoblotting and immunostaining with a specific antiserum originating from the purified PsNLEC protein that had been heterologously expressed in Escherichia coli . Immunogold localization of PsNLEC-1 sugar epitopes in B-deficient nodules showed that they were associated mostly with cytoplasmic vesicles rather than normal localization in the symbiosome compartment of mature infected cells . These results suggest that a modification of the glycosyl-moieties of PsNLEC-1 and an alteration of vesicle targeting occur during the development of pea nodules in the absence of B, and that these changes are associated with the development of aberrant nonfunctional symbiosomes.

J Exp Bot, 2001 Mar, 52(Spec Issue), 435 - 43
Nitrogen nutrition and the role of root-shoot nitrogen signalling particularly in symbiotic systems; Parsons R et al.; To obtain and concentrate reduced N from the environment, plants have evolved a diverse array of adaptations to utilize soil, biotic and atmospheric N . In symbiotic N(2)-fixing systems the potential for oversupply exists and regulation of activity to match demand is crucial . N status in plants is likely to be most strongly sensed in the shoot and signals translocated to the roots may involve phloem transported amino compounds or very low concentrations of specific signal molecules . The mechanism for sensing N status in plant cells is not understood at the molecular level although it may be expected to be similar in all plants . Mechanisms for the regulation of symbiotic N(2) fixation may be very different in the different symbiotic types . Rhizobia, Frankia and cyanobacteria are all symbiotic with different species of plants and the provision of O(2), carbohydrate or other nutrients may control symbiotic activity to varying extents in the different symbioses.

Indian J Exp Biol, 2000 Oct, 38(10), 1041 - 9
Isolation and symbiotic characterization of aromatic amino acid auxotrophs of Sinorhizobium meliloti; Prasad CK et al.; Ten aromatic amino acid auxotrophs of Sinorhizobium meliloti (previously called Rhizobium meliloti) Rmd201 were generated by random mutagenesis with transposon Tn5 and their symbiotic properties were studied . Normal symbiotic activity, as indicated by morphological features, was observed in the tryptophan synthase mutants and the lone tyrosine mutant . The trpE and aro mutants fixed trace amounts of nitrogen whereas the phe mutant was completely ineffective in nitrogen fixation . Histology of the nodules induced by trpE and aro mutants exhibited striking similarities . Each of these nodules contained an extended infection zone and a poorly developed nitrogen fixation zone . Transmission electron microscopic studies revealed that the bacteroids in the extended infection zone of these nodules did not show maturation tendency . A leaky mutant, which has a mutation in trpC, trpD, or trpF gene, was partially effective in nitrogen fixation . The histology of the nodules induced by this strain was like that of the nodules induced by the parental strain but the inoculated plants were stunted . These studies demonstrated the involvement of anthranilic acid and at least one more intermediate of tryptophan biosynthetic pathway in bacteroidal maturation and nitrogen fixation in S . meliloti . The alfalfa plant host seems to provide tryptophan and tyrosine but not phenylalanine to bacteroids in nodules.

Mol Plant Microbe Interact, 2001 Apr, 14(4), 572 - 6
A purine-related metabolite negatively regulates fixNOQP expression in Sinorhizobium meliloti by modulation of fixK expression; Soberon M et al.; 5-aminoimidazole-4-carboxamide nucleotide (AICAR) is a negative effector of cytochrome terminal oxidase cbb3 production in Rhizobium etli . In this work, the effect of AICAriboside (AICAr), the precursor of AICAR on the expression of the Sinorhizobium meliloti fixNOQP operon encoding the symbiotic terminal oxidase cbb3, was analyzed . AICAr reduced the microaerobic induction levels of fixN-lacZ and fixT-lacZ gene fusions 18- and seven-fold respectively, and both genes were activated by the transcriptional activator FixK . A fixK-lacZ fusion presented 14-fold-reduced induction levels in microaerobic cell cultures in the presence of AICAr . AICAr also reduced three-fold the microaerobic expression levels of the nifA-lacZ fusion, whose expression as well as that of fixK is controlled by the two-component system FixL-FixJ . In contrast, AICAr had no effect on the expression levels of a hemA-lacZ fusion . These data suggest that AICAr prevents fixNOQP induction by the inhibition of fixK transcription.

Mol Plant Microbe Interact, 2001 Apr, 14(4), 555 - 61
Purification of the major outer membrane protein of Azospirillum brasilense, its affinity to plant roots, and its involvement in cell aggregation; Burdman S et al.; The major outer membrane protein (MOMP) of the nitrogen-fixing rhizobacterium Azospirillum brasilense strain Cd was purified and isolated by gel filtration, and antiserum against this protein was obtained . A screening of the binding of outer membrane proteins (OMPs) of A . brasilense to membrane-immobilized root extracts of various plant species revealed different affinities for the MOMP, with a stronger adhesion to extracts of cereals in comparison with legumes and tomatoes . Moreover, this protein was shown to bind to roots of different cereal seedlings in an in vitro adhesion assay . Incubation of A . brasilense cells with MOMP-antiserum led to fast agglutination, indicating that the MOMP is a surface-exposed protein . Cells incubated with Fab fragments obtained from purified MOMP-antiserum immunoglobulin G exhibited significant inhibition of bacterial aggregation as compared with controls . Bacteria preincubated with Fab fragments showed weaker adhesion to corn roots in comparison to controls without Fab fragments . These findings suggest that the A . brasilense MOMP acts as an adhesin involved in root adsorption and cell aggregation of this bacterium.

Mol Plant Microbe Interact, 2001 Apr, 14(4), 471 - 6
Effect of mutations in Pisum sativum L . genes blocking different stages of nodule development on the expression of late symbiotic genes in Rhizobium leguminosarum bv . viciae; Voroshilova VA et al.; In this report, the expression of late symbiotic genes (fnrN, fixN, and nifA) of Rhizobium leguminosarum bv . viciae was studied in nodules of mutant pea lines blocked at four successive stages of nodule development . Bacterial gene expression was analyzed in situ with transcriptional gusA reporter gene fusions . As a control, a constitutively expressed gusA gene was included . In the nodules of Nop(nodule persistence) mutants (mutant in gene sym13), which had not yet exhibited signs of premature senescence, the expression patterns observed were identical to those in wild-type nodules . Normal expression of fusions also occurred in nodules defective at the infection droplet differentiation stage (mutant in gene sym40) in which bacteria are endocytosed, but infection threads and infection droplets are hypertrophied . In contrast, in Itn- (infection thread formation inside the nodule tissue) mutants (mutant gene sym33), in which there is no endocytosis of bacteria, expression of the constitutive fusion was only in infection threads and no activity was shown for the other fusions . From this it can be concluded that functionality of the plant gene Sym33, i.e., bacterial endocytosis, is a prerequisite for the expression of late symbiotic genes in the microsymbiont . No morphologically distinct interzone II-III could be detected in nodules blocked at the bacteroid differentiation stage (mutants in gene sym31) . The constitutive fusion was expressed equally throughout the nodule tissue (except for the meristem), and the activity of fusions to late symbiotic genes increased gradually with a maximal expression level at the base of the nodule . This is consistent with an altered oxygen barrier previously reported for these nodules . By including double mutants, earlier results on sequential functioning of gene pairs sym33-sym40 and sym31-sym13 could be confirmed and it could be demonstrated that the developmental epistasis found at the morphological level also is reflected in the expression pattern of late symbiotic genes in the microsymbiont.

J Appl Microbiol, 2001 Apr, 90(4), 662 - 7
Rhizobia of chickpea from southern Portugal: symbiotic efficiency and genetic diversity; Laranjo M et al.; AIMS: In order to evaluate differences between chickpea rhizobial populations from three geographical areas in southern Portugal (Beja, Elvas and Evora), isolates from the three regions were obtained and analysed . METHODS AND RESULTS: The genetic characterization of the isolates was done by plasmid profiles and restriction analysis of the nifH gene . Symbiotic efficiency of the isolates was also determined . Relationships between geographical origin, symbiotic efficiency and molecular characteristics were established . Beja soil revealed a larger rhizobia population as well as the presence of some of the isolates with higher symbiotic efficiency values . Isolates with a single plasmid showed a significantly higher symbiotic efficiency . CONCLUSION: Genetic and phenotypic differences were detected between the natural rhizobial populations from the three locations . SIGNIFICANCE AND IMPACT OF THE STUDY: The different yield potential with cultivars of chickpea usually obtained in the three regions of southern Portugal could be due to their different natural rhizobial populations.

Plant Physiol, 2001 Apr, 125(4), 2104 - 19
Differential regulation of a family of apyrase genes from Medicago truncatula; Cohn JR et al.; Four putative apyrase genes were identified from the model legume Medicago truncatula . Two of the genes identified from M . truncatula (Mtapy1 and Mtapy4) are expressed in roots and are inducible within 3 h after inoculation with Sinorhizobium meliloti . The level of mRNA expression of the other two putative apyrases, Mtapy2 and Mtapy3, was unaffected by rhizobial inoculation . Screening of a bacterial artificial chromosome library of M . truncatula genomic DNA showed that Mtapy1, Mtapy3, and Mtapy4 are present on a single bacterial artificial chromosome clone . This apyrase cluster was mapped to linkage group seven . A syntenic region on soybean linkage group J was found to contain at least two apyrase genes . Screening of nodulation deficient mutants of M . truncatula revealed that two such mutants do not express apyrases to any detectable level . The data suggest a role for apyrases early in the nodulation response before the involvement of root cortical cell division leading to the nodule structure.

Microbiol Res, 2001 Mar, 155(4), 325 - 9
Symbiotic effectiveness of spontaneous antibiotic-resistant mutants of Rhizobium sp . Cicer nodulating chickpea (Cicer arietinum); Sindhu SS et al.; Spontaneous streptomycin-resistant mutants were isolated from two fast growing gum-producing strains Ca85 and Ca401 and from two moderately growing strains Ca181 and Ca534 of Rhizobium sp . Cicer . The nodulation ability and symbiotic effectiveness of the mutants relative to parent strains were evaluated on chickpea (Cicer arietinum) grown in sterilized chillum jars . Some mutants of strains Ca85 and Ca401 showed Nod phenotype whereas some mutants of strains Ca181 and Ca534 showed Nod(+) fix(-) phenotype . Other mutants also showed decreased nodule number and reduction in nitrogenase activity as well as in shoot dry weight as compared to inoculation with parental strains . The results showed that acquisition of streptomycin resistance in Rhizobium sp . Cicer strains is associated with decreased symbiotic effectiveness in chickpea, suggesting that antibiotic-resistant mutants first should be analyzed for symbiotic effectiveness before using these mutants for ecological studies or nodulation competitiveness.

Acta Microbiol Pol, 2000, 49(3-4), 201 - 6
Microbiological and electron microscopic studies of urea treated Rhizobium sp . cells; Bhattacharya R et al.; Microbiological investigation of urea treated Rhizobium sp . cells showed a gradual decrease of colony forming unit from initial 100% to 2.13% value . Maximum effect was reached at 90 min onwards . The liquid holding recovery in phosphate buffer saline at pH 7.0 also was studied . Electron microscopic studies revealed important structural changes in treated cells.

Gene, 2001 Mar 21, 266(1-2), 77 - 84
Cloning and expression of a down-regulated gene (TrEnodDR1) of white clover responded by the nod genes derived from Rhizobium leguminosarum bv . trifolii strain 4S; Suzuki A et al.; The nodulation genes of Rhizobium leguminosarum bv . trifolii 4S (strain 4S) were cloned into cosmid vector pLAFR1 named pC4S8 which was contained nodNMLFEDABCIJ and a part of nodT as an insert . The pC4S8 was transferred to strain H1, Sym plasmid (pRt4Sa) cured strain of strain 4S, and isolated as Tc resistant and nodulation restored mutant, strain H1(pC4S8) . During infection process of this strain, visible symbiotic features, such as root hair curling (Hac), root hair deformation (Had) and infection thread formation (Inf) were also restored . The nodule forming ability of strain H1(pC4S8) was increased 3-4 times in nodule number than that of strain 4S . Then, to investigate the effect of Rhizobium nod genes on the host plant (Trifolium repens L.) gene expression, cDNAs which were responded to the inoculation of rhizobia were differentially screened based on the presence or absence of nod genes treated with strains H1(pC4S8) or H1, respectively . The cDNA, TrEnodDR1 (Trifolium repens early nodulin down regulation 1) gene was isolated from cDNA library prepared from white clover seedlings treated with nod- strain H1, but didn't exhibit in nod+ treated cDNA library, as a down-regulated gene . Expression analysis of TrEnodDR1 was performed in various tissues of white clover, it is suppressed in root nodule and also strongly suppressed by the inoculation of rhizobia in the seedlings . It is discussed that TrEnodDR1 gene is suppressed when the white clover comes into symbiosis with rhizobia.

Development, 2001 May, 128(9), 1507 - 18
The HCL gene of Medicago truncatula controls Rhizobium-induced root hair curling; Catoira R et al.; The symbiotic infection of the model legume Medicago truncatula by Sinorhizobium meliloti involves marked root hair curling, a stage where entrapment of the microsymbiont occurs in a chamber from which infection thread formation is initiated within the root hair . We have genetically dissected these early symbiotic interactions using both plant and rhizobial mutants and have identified a M . truncatula gene, HCL, which controls root hair curling . S . meliloti Nod factors, which are required for the infection process, induced wild-type epidermal nodulin gene expression and root hair deformation in hcl mutants, while Nod factor induction of cortical cell division foci was reduced compared to wild-type plants . Studies of the position of nuclei and of the microtubule cytoskeleton network of hcl mutants revealed that root hair, as well as cortical cells, were activated in response to S . meliloti . However, the asymmetric microtubule network that is typical of curled root hairs, did not form in the mutants, and activated cortical cells did not become polarised and did not exhibit the microtubular cytoplasmic bridges characteristic of the pre-infection threads induced by rhizobia in M . truncatula . These data suggest that hcl mutations alter the formation of signalling centres that normally provide positional information for the reorganisation of the microtubular cytoskeleton in epidermal and cortical cells.

Arch Microbiol, 2001 Feb, 175(2), 152 - 60
Mutants in the nodFEL promoter of Rhizobium leguminosarum bv . viciae reveal a role of individual nucleotides in transcriptional activation and protein binding; Okker RJ et al.; The highly conserved nod box sequence in the promoters of the inducible nodulation genes of rhizobia is required for transcription activation together with NodD, a LysR-type transcriptional regulator, and a flavonoid as a coinducer . DNA fragments containing nod box sequences form two binding complexes when crude preparations of Rhizobium leguminosarum bv . viciae are used: a NodD-dependent and an additional, NodD-independent complex . The role of individual nucleotides in the conserved nod box sequence in complex formation and in nodulation gene expression was investigated by introducing 13 individual base-pair substitutions in the nodF nod box of R . leguminosarum bv . viciae and studying their effect on promoter activity and protein-DNA complex formation . Two mutants showed decreased NodD binding and decreased promoter activity . Five mutants showed a NodD-dependent complex as with the wild-type nodF nod box, whereas their promoter activity was severely reduced after induction . This result is in agreement with earlier observations that NodD DNA binding also occurs in the absence of inducer . Four mutants were impaired in the formation of the NodD-independent retardation complex . Three of them showed no alterations in promoter activity, meaning that no specific role for the protein forming the NodD-independent complex could be established . The two mutants in the highly conserved LysR motif of the nod box were unable to direct coinducer-dependent promoter activity but, unexpectedly, their retardation patterns were not altered . The remaining two mutants showed constitutive promoter activity . The results are discussed in terms of the relevance of conserved nucleotides and motifs identified in the nod box.

Arch Microbiol, 2001 Feb, 175(2), 143 - 51
The Rhizobium leguminosarum bv . trifolii pssB gene product is an inositol monophosphatase that influences exopolysaccharide synthesis; Janczarek M et al.; The pssB gene of Rhizobium leguminosarum bv . trifolii encodes a protein of 284 amino acids with sequence similarity to eukaryotic inositol monophosphatases . The gene was cloned and overexpressed in Escherichia coli . The purified gene product of pssB showed inositol monophosphatase activity with a Km of 0.23 mM, and a Vmax of 3.27 mumol Pi min-1 (mg protein)-1 . Its substrate specificity, Mg+2 requirement, Li+ inhibition, and subunit association (dimerization) were studied and compared to those of other inositol monophosphatases . Western immunoblotting with anti-PssB antibodies showed the presence of PssB in R . leguminosarum bv . trifolii strain TA1 and lack of this protein in the pssB mutant strain Rt12A . The presence of PssB protein in R . leguminosarum bv . trifolii TA1 was correlated with phosphatase activity with myo-inositol 1-phosphate as a substrate . Evidence for a regulatory function of PssB protein in exopolysaccharide (EPS) synthesis is presented . The mutation in pssB caused EPS overproduction, and introduction of pssB into the wild-type TA1 strain reduced EPS synthesis . The changes in the level of EPS production were correlated with a non-nitrogen-fixing phenotype of rhizobia.

Microbiology, 2001 Apr, 147(Pt 4), 981 - 93
Classification of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts; Laguerre G et al.; The nodC and nifH genes were characterized in a collection of 83 rhizobial strains which represented 23 recognized species distributed in the genera Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium, as well as unclassified rhizobia from various host legumes . Conserved primers were designed from available nucleotide sequences and were able to amplify nodC and nifH fragments of about 930 bp and 780 bp, respectively, from most of the strains investigated . RFLP analysis of the PCR products resulted in a classification of these rhizobia which was in general well-correlated with their known host range and independent of their taxonomic status . The nodC and nifH fragments were sequenced for representative strains belonging to different genera and species, most of which originated from Phaselous vulgaris nodules . Phylogenetic trees were constructed and revealed close relationships among symbiotic genes of the Phaseolus symbionts, irrespective of their 16S-rDNA-based classification . The nodC and nifH phylogenies were generally similar, but cases of incongruence were detected, suggesting that genetic rearrangements have occurred in the course of evolution . The results support the view that lateral genetic transfer across rhizobial species and, in some instances, across Rhizobium and Sinorhizobium genera plays a role in diversification and in structuring the natural populations of rhizobia.

Protein Expr Purif, 2001 Apr, 21(3), 432 - 7
Overexpression and purification of Rhizobium etli glutaminase A by recombinant and conventional procedures; Huerta-Saquero A et al.; Rhizobium etli glutaminase A was purified to homogeneity by conventional procedures that included ammonium sulfate differential precipitation, ion-exchange chromatography, hydrophobic interaction chromatography, gel filtration, and dye-ligand chromatography . Alternatively, the structural glsA gene that codifies for glutaminase A was amplified by PCR and cloned in the expression vector pTrcHis . The recombinant protein was purified to homogeneity by affinity chromatography . This protein showed the same kinetic properties as native glutaminase A (K(m) for glutamine of 1.5 mM and V(max) of 80 micromol ammonium min(-1) mg protein(-1)) . Physicochemical and biochemical properties of native and recombinant glutaminase were identical . The molecular mass of recombinant glutaminase A (M(r) 106.8 kDa) and the molecular mass of the subunits (M(r) 26.9 kDa) were estimated by mass spectrometry . These results suggest that R . etli glutaminase A is composed of four identical subunits . The high-level production of recombinant glutaminase A elevates the possibilities for determination of its three-dimensional structure through X-ray crystallography .

J Biol Chem, 2001 May 18, 276(20), 17190 - 8 Epub 2001 Feb 09.
Identification of an ATP-binding cassette transporter for export of the O-antigen across the inner membrane in Rhizobium etli based on the genetic, functional, and structural analysis of an lps mutant deficient in O-antigen; Lerouge I et al.; For O-antigen lipopolysaccharide (LPS) synthesis in bacteria, transmembrane migration of undecaprenyl pyrophosphate-bound O-antigen oligosaccharide subunits or polysaccharide occurs before ligation to the core region of the LPS molecule . In this study, we identified by mutagenesis an ATP-binding cassette transporter in Rhizobium etli CE3 that is likely responsible for the translocation of the O-antigen across the inner plasma membrane . Mutant FAJ1200 LPS lacks largely the O-antigen, as shown by SDS-polyacrylamide gel electrophoresis and confirmed by immunoblot analysis . Furthermore, LPS isolated from FAJ1200 is totally devoid of any O-chain glycosyl residues and contains only those glycosyl residues that can be expected for the inner core region . The membrane component and the cytoplasmic ATP-binding component of the ATP-binding cassette transporter are encoded by wzm and wzt, respectively . The Tn5 transposon in mutant FAJ1200 is inserted in the wzm gene . This mutation resulted in an Inf- phenotype in bean plants.

Mol Plant Microbe Interact, 2001 Mar, 14(3), 426 - 30
Stable RK2-derived cloning vectors for the analysis of gene expression and gene function in gram-negative bacteria; Dombrecht B et al.; The construction of several stable RK2-derived cloning vectors for the analysis of gene expression and function in gram-negative bacteria is reported . Plasmid stability is conferred by the RK2 par locus or by insertion of the spsAB or spsCD symbiotic plasmid stability loci from pNGR234a of Rhizobium sp . NGR234 . The vectors carry multiple cloning sites with protection against read-through transcriptional activity of vector sequences . Vector derivatives with the constitutive nptII promoter or a promoterless gusA gene are suitable for the study of gene function or regulation in bacteria.

Mol Plant Microbe Interact, 2001 Mar, 14(3), 349 - 57
The nodulation protein NodG shows the enzymatic activity of an 3-oxoacyl-acyl carrier protein reductase; Lopez-Lara IM et al.; The acyl carrier protein NodF is required for the synthesis of unusual polyunsaturated fatty acids that confer specificity to lipochitin oligosaccharide nodulation (Nod) factors of Rhizobium leguminosarum . In this study, homogeneous NodF protein was used as a ligand to identify proteins of R . leguminosarum that specifically interact with NodF and presumably are involved in the biosynthesis or transfer of the unusual fatty acids . The N-terminal amino acid sequence of a 29-kDa protein that interacts strongly with NodF revealed high similarity to NodG of Rhizobium sp . N33 and to NodG of Sinorhizobium meliloti We cloned and sequenced the gene coding for the NodG-like protein of R . leguminosarum and found it to be the product of the constitutively expressed gene fabG . FabG is the 3-oxoacyl-acyl carrier protein reductase that catalyzes the first reduction step in each cycle of fatty acid elongation . FabG of R . leguminosarum and NodG of Rhizobium sp . N33 were expressed in Escherichia coli . In both cases, the purified protein showed 3-oxoacyl-acyl carrier protein reductase activity in vitro . Therefore, NodG has the same biochemical function as FabG, and the high degree of similarity at the protein and DNA level suggest that nodG is a duplication of the housekeeping genefabG.

J Bacteriol, 2001 Apr, 183(8), 2595 - 604
A functional myo-inositol dehydrogenase gene is required for efficient nitrogen fixation and competitiveness of Sinorhizobium fredii USDA191 to nodulate soybean (Glycine max {L.} Merr.); Jiang G et al.; Inositol derivative compounds provide a nutrient source for soil bacteria that possess the ability to degrade such compounds . Rhizobium strains that are capable of utilizing certain inositol derivatives are better colonizers of their host plants . We have cloned and determined the nucleotide sequence of the myo-inositol dehydrogenase gene (idhA) of Sinorhizobium fredii USDA191, the first enzyme responsible for inositol catabolism . The deduced IdhA protein has a molecular mass of 34,648 Da and shows significant sequence similarity with protein sequences of Sinorhizobium meliloti IdhA and MocA; Bacillus subtilis IolG, YrbE, and YucG; and Streptomyces griseus StrI . S . fredii USDA191 idhA mutants revealed no detectable myo-inositol dehydrogenase activity and failed to grow on myo-inositol as a sole carbon source . Northern blot analysis and idhA-lacZ fusion expression studies indicate that idhA is inducible by myo-inositol . S . fredii USDA191 idhA mutant was drastically affected in its ability to reduce nitrogen and revealed deteriorating bacteroids inside the nodules . The number of bacteria recovered from such nodules was about threefold lower than the number of bacteria isolated from nodules initiated by S . fredii USDA191 . In addition, the idhA mutant was also severely affected in its ability to compete with the wild-type strain in nodulating soybean . Under competitive conditions, nodules induced on soybean roots were predominantly occupied by the parent strain, even when the idhA mutant was applied at a 10-fold numerical advantage . Thus, we conclude that a functional idhA gene is required for efficient nitrogen fixation and for competitive nodulation of soybeans by S . fredii USDA191.

J Bacteriol, 2001 Apr, 183(8), 2576 - 85
Genetic organization of the region encoding regulation, biosynthesis, and transport of rhizobactin 1021, a siderophore produced by Sinorhizobium meliloti; Lynch D et al.; Eight genes have been identified that function in the regulation, biosynthesis, and transport of rhizobactin 1021, a hydroxamate siderophore produced under iron stress by Sinorhizobium meliloti . The genes were sequenced, and transposon insertion mutants were constructed for phenotypic analysis . Six of the genes, named rhbABCDEF, function in the biosynthesis of the siderophore and were shown to constitute an operon that is repressed under iron-replete conditions . Another gene in the cluster, named rhtA, encodes the outer membrane receptor protein for rhizobactin 1021 . It was shown to be regulated by iron and to encode a product having 61% similarity to IutA, the outer membrane receptor for aerobactin . Transcription of both the rhbABCDEF operon and the rhtA gene was found to be positively regulated by the product of the eighth gene in the cluster, named rhrA, which has characteristics of an AraC-type transcriptional activator . The six genes in the rhbABCDEF operon have interesting gene junctions with short base overlaps existing between the genes . Similarities between the protein products of the biosynthesis genes and other proteins suggest that rhizobactin 1021 is synthesized by the formation of a novel siderophore precursor, 1,3-diaminopropane, which is then modified and attached to citrate in steps resembling those of the aerobactin biosynthetic pathway . The cluster of genes is located on the pSyma megaplasmid of S . meliloti 2011 . Reverse transcription-PCR with RNA isolated from mature alfalfa nodules yielded no products for rhbF or rhtA at a time when the nifH gene was strongly expressed, indicating that siderophore biosynthesis and transport genes are not strongly expressed when nitrogenase is being formed in root nodules . Mutants having transposon insertions in the biosynthesis or transport genes induced effective nitrogen-fixing nodules on alfalfa plants.

Folia Microbiol (Praha), 2000, 45(2), 177 - 82
Cold stress induces switchover of respiratory pathway to lactate glycolysis in psychrotrophic Rhizobium strains; Sardesai N et al.; Two psychrotrophic strains of Rhizobium, DDSS69, a non-cold acclimated strain, and ATR1, a cold acclimated strain, were subjected to cold stress . A 4-fold increase in the specific activity of lactate dehydrogenase (LDH) was characteristic for cold stressed cells of DDSS69, whereas ATR1 showed a higher LDH activity in general, which increased 1.5-fold under cold stress . Cold sensitive mutants of DDSS69 which could not grow below 15 degrees C, in contrast to the wild type which could grow at 5 degrees C, were isolated using Tn5-tagged mutagenesis . These mutants showed a 40% lower LDH activity than the wild type grown at 5 degrees C that was comparable to the wild type grown at 15 degrees C . High specific activity of succinic dehydrogenase (SDH) at 28 degrees C in both strains and mutants indicated that aerobic respiration via the citrate cycle is the normal mode of saccharide utilization . Shifts to lower temperatures decreased the specific activity of SDH . However, alcohol dehydrogenase (ADH) activity remained very low in both the strains and the mutants at low temperatures indicating that a shift from aerobic saccharide metabolism to anaerobic one under cold stress involves lactate glycolysis rather than alcohol fermentation . There was an increase in membrane-bound ATPase activity under cold stress which is correlated to higher LDH activity . These data show that, in psychrotrophic Rhizobium strains, cold stress induces a switchover of respiratory metabolism from aerobic to anaerobic pathway, especially lactate glycolysis.

Can J Microbiol, 2001 Feb, 47(2), 165 - 71
Early production of rhizopine in nodules induced by Sinorhizobium meliloti strain L5-30; Heinrich K et al.; The rhizopine L-3-O-methyl-scyllo-inosamine (3-O-MSI) is metabolized by approximately 10% of the strains of Rhizobium leguminosarum by . viciae and Sinorhizobium meliloti . Rhizopine strains enjoy a substantial competitive advantage in nodulation, which is manifest before 14 days post-inoculation, implying that rhizopine is produced before this time . We were able to detect this compound in the roots of alfalfa (Medicago sativum L . cv . Hunter River) four days after germination (six days post-infection) with S . meliloti strain L5-30 by gas chromatography-mass spectrometry (GC-MS) . At four days, nodules were not visible, and the concentration of rhizopine was extremely low, estimated at 67 pg/gfw (picograms/gram fresh weight) . The amount increased gradually but remained low until 16 days, when there was a 50-fold increase from day four, by which time nodules were well established . This pattern of synthesis is consistent with previous studies indicating that rhizopine synthesis is regulated by nifA/ntrA regulatory genes, which are maximally expressed in bacteroids at the onset of nitrogen fixation . However, the low level of rhizopine synthesis must be responsible for the early effects on competition for nodulation . Production of rhizopine at this time most likely results from micro-aerobic induction of mos genes in free-living bacteria, either in the infection threads or in the rhizosphere.

Can J Microbiol, 2001 Feb, 47(2), 139 - 47
Genetic diversity of Sinorhizobium populations recovered from different medicago varieties cultivated in Tunisian soils; Jebara M et al.; A collection of 468 rhizobial isolates was obtained from different ecological areas of Tunisia by trapping them on Medicago sativa cv . Gabes, Medicago scutelleta cv . Kelson, Medicago truncatula, and Medicago ciliaris . A subsample of 134 rhizobia was chosen to determine their plasmid profile, and 89 isolates were subjected to multilocus enzyme electrophoresis (MLEE) and PCR/RFLP analysis using 16S, IGS (inter genic spacer), and nifKD probes . Twenty-five representatives from these isolates were evaluated for their nodulation and nitrogen fixation capacities . MLEE studies revealed two groups with highly heterogeneous host specificity and geographical origin . The discriminatory power was found to be slightly better with the amplified ribosomal intergenic region, than the nifKD genes . Divisions detected by nifKD amplified DNA analysis matched those established by ribosomal PCR- RFLPs . The comparison between different analyses revealed that MLEE illustrated better phenotypic properties of isolates than PCR-RFLP or plasmid content analysis . Clear distinction between Sinorhizobium meliloti and Sinorhizobium medicae were observed by analysis of the IGS symbiotic regions between nifD and nifK genes . Were able to distinguish three inoculation groups; isolates trapped from M . sativa cv . Gabes and M . scutelleta cv . Kelson formed one inoculation group which was more closely related to isolates trapped from M . truncatula than those trapped from M . ciliaris.

Can J Microbiol, 2001 Feb, 47(2), 118 - 22
Genetic diversity of Bradyrhizobium strains isolated from Arachis hypogaea; Saleena LM et al.; Rhizobia are used exclusively in agricultural systems for enhancing the ability of legumes to fix atmospheric nitrogen . Knowledge about the indigenous population is necessary for the selection and application of inoculant strains . In this study, we have assessed the genetic diversity of Bradyrhizobium strains isolated from the host plant, Arachis hypogaea along the coastline of Tamil Nadu . Different populations collected from varying environmental conditions were analysed for salt and pH tolerance . Genetic diversity among the strains was studied using RAPD markers and PCR-RFLP of 16S rDNA and nifD genes . The approaches used in this study yielded consistent results, which revealed a high degree of heterogeneity among strains and detection of two distinct genetic groups.

Electrophoresis, 2001 Feb, 22(3), 586 - 98
Proteome analysis of cultivar-specific interactions between Rhizobium leguminosarum biovar trifolii and subterranean clover cultivar Woogenellup; Morris AC et al.; Proteome analysis was used to identify proteins that are involved in the early stages of nodulation between the subterranean clover cultivar Woogenellup and the Rhizobium leguminosarum bv . trifolii strains ANU843 and ANU794 . Strain ANU843 induces nitrogen-fixing nodules whereas strain ANU794 forms aberrant nodules on the roots of cv . Woogenellup that fail to develop beyond an early stage . Our aim was to identify proteins that might be involved in the early stages of nodulation over a 48 h period and to identify proteins that are differentially displayed during the interactions between the host and the two microbes . Proteome maps from control Woogenellup roots and inoculated roots were generated and compared at 24 and 48 h post inoculation . Over 1500 spots were resolved on all gels . Of the 16 protein spots that were differentally displayed or developmentally regulated, 10 were assigned putative identities . These included an alpha-fucosidase, several ethylene-induced proteins, a Cu/Zn superoxide dismutase, a hypothetical 16.5 kDa protein, tubulin alpha-chain, chaperonin 21 precursor and triosephosphate isomerase . Of the 22 constitutively expressed proteins spots examined, eight spots were assigned putative protein homologies through N-terminal sequencing and included several pathogenesis and stress-related proteins . The result may suggest that ethylene levels are upregulated during the early stages of infection but that this does not result in the induction of common pathogenesis-related proteins . The specific induction of alpha-fucosidase by ANU794 may be important in the nodulation failure phenotype of strain ANU794.

Genetika, 2001 Feb, 37(2), 215 - 22
{Hydrocarbon-binding peptides from bean plant lectins in connection with various host specific macrosymbionts during development of symbiosis with rhizobium bacteria}; Baimiev AKh et al.; The nucleotide sequences of 280-360-bp domains of lectin genes from 20 legume species belonging to 17 genera have been determined . A computer analysis of the sequences has been performed with the LASERGENE package . Based on this analysis, we constructed the phylogenetic tree of the lectins, which reflects their phylogenetic and evolutionary relationships, and predicted the amino-acid sequences of the corresponding protein domains . Features of the structure of the hydrocarbon-binding lectin domains were elucidated in some species of legume genera from the temperate climatic zone . The domains were highly variable and contained the consensus sequence AspTrePheXxxAsxXxxXxxTrpAspProXxxXxxIns/DelArgHis bearing the bulk of amino acid replacements, insertions, and deletions . An association between legume groups (including species from different genera and tribes) symbiotic with the same rhizobium species and the similarity between the hydrocarbon-binding domains of lectins from these plants was found.

Mol Microbiol, 2001 Mar, 39(5), 1186 - 98
Phosphatidylcholine levels in Bradyrhizobium japonicum membranes are critical for an efficient symbiosis with the soybean host plant; Minder AC et al.; Phosphatidylcholine (PC), the major membrane phospholipid in eukaryotes, is found in only some bacteria including members of the family Rhizobiaceae . For this reason, it has long been speculated that rhizobial PC might be required for a successful interaction of rhizobia with their legume host plants in order to allow the formation of nitrogen-fixing root nodules . A major pathway for PC formation in prokaryotes involves a threefold methylation of the precursor phosphatidylethanolamine (PE) . Here, we report on the isolation of a Bradyrhizobium japonicum gene (pmtA) encoding the phospholipid N-methyltransferase PmtA . Upon expression of the bradyrhizobial pmtA gene in Escherichia coli, predominantly monomethylphosphatidylethanolamine was formed from PE . PmtA-deficient B . japonicum mutants still produced low levels of PC by a second methylation pathway . The amount of PC formed in such mutants (6% of total phospholipids) was greatly decreased compared with the wild type (52% of total phospholipids) . Root nodules of soybean plants infected with B . japonicum pmtA mutants showed a nitrogen fixation activity of only 18% of the wild-type level . The interior colour of the nodules was beige instead of red, suggesting decreased amounts of leghaemoglobin . Moreover, ultrastructure analysis of these nodules demonstrated a greatly reduced number of bacteroids within infected plant cells . These data suggest that the biosynthesis of wild-type amounts of PC are required to allow for an efficient symbiotic interaction of B . japonicum with its soybean host plant.

Mikrobiol Z, 2000 Nov-Dec, 62(6), 44 - 50
{Effect of heavy metals and reclaimers on formation and functioning of legume-Rhizobium symbiosis}; Antipchuk AF et al.; The influence of a mixture of salts of heavy metals Cd2+, Cu2+, Pb2+, Zn2+ on the efficiency of legume-rhizobial symbiosis and a part of reclaimers in the decrease of toxic effect of pollutants have been studied under conditions of the vegetative and field experiment on small plots . Concentrations of salts of heavy metals were tested in the interval of 1 to 20 MPC (maximum permissible concentrations) . Negative effect of the tested substances on the growth, photo-assimilation properties of soybean plants, nodulation intensity, nitrogenase activity and yield (correlation coefficients made, respectively, -0.58; -0.86; -0.88; -0.84 and -0.86 under reliability 95-99.9%) has been established . The increase of concentration of heavy metals was accompanied by the decrease of the indices of symbiosis efficiency . The complete suppression of symbiotic system of soy-beans was registered under the maximum of test doses which was equal to 20 MPC . The reclaimer white zeolite has displayed protective properties with respect to legume-rhizobial symbiosis against a background of soil contamination with high doses of heavy metals . The use of combined reclaimer gypsum-streptomycete may be also considered promising for decreasing negative effect of heavy metals on legume-rhizobial symbiosis.

Microbiology, 2001 Mar, 147(Pt 3), 549 - 59
A unipolarly located, cell-surface-associated agglutinin, RapA, belongs to a family of Rhizobium-adhering proteins (Rap) in Rhizobium leguminosarum bv . trifolii; Ausmees N et al.; The phage-display cloning technique was used to find rhizobial proteins that bind to receptors located on the bacterial cell surface . The aim was to clone the gene(s) encoding rhicadhesin, a universal rhizobial adhesion protein, and/or other cell-surface-binding proteins . Four such Rhizobium-adhering proteins (Rap) were revealed in Rhizobium leguminosarum bv . trifolii strain R200 . The binding is mediated by homologous Ra domains in these proteins . One member of the Rap protein family, named RapA1, is a secreted calcium-binding protein, which are also properties expected for rhicadhesin . However, the size of the protein (24 kDa instead of 14 kDa) and its distribution among different rhizobia (present in only Rhizobium leguminosarum biovars and R . etli instead of all members of Rhizobiaceae argue against RapA1 being rhicadhesin . Protein RapA1 consists of two homologous Ra domains and agglutinates R200 cells by binding to specific receptors located at one cell pole during exponential growth . Expression of these cell-surface receptors was detected only in rhizobia that produce the RapA proteins . The authors propose that the homologous Ra domains, found to be present also in other proteins with different structure, represent lectin domains, which confer upon these proteins the ability to recognize their cognate carbohydrate structures.

Cell Mol Life Sci, 2001 Jan, 58(1), 61 - 71
Ammonia and amino acid transport across symbiotic membranes in nitrogen-fixing legume nodules; Day DA et al.; Biological nitrogen fixation involves the reduction of atmospheric N2 to ammonia by the bacterial enzyme nitrogenase . In legume-rhizobium symbioses, the nitrogenase-producing bacteria (bacteroids) are contained in the infected cells of root nodules within which they are enclosed by a plant membrane to form a structure known as the symbiosome . The plant provides reduced carbon to the bacteroids in exchange for fixed nitrogen, which is exported to the rest of the plant . This exchange is controlled by plant-synthesised transport proteins on the symbiosome membranes . This review summarises our current understanding of these transport processes, focusing on ammonia and amino acid transport.

J Bacteriol, 2001 Mar, 183(6), 2141 - 4
Symbiotic plasmid rearrangement in Rhizobium leguminosarum bv . viciae VF39SM; Zhang XX et al.; A rearrangement between the symbiotic plasmid (pRleVF39d) and a nonsymbiotic plasmid (pRleVF39b) in Rhizobium leguminosarum bv . viciae VF39 was observed . The rearranged derivative showed the same plasmid profile as its parent strain, but hybridization to nod, fix, and nif genes indicated that most of the symbiotic genes were now present on a plasmid corresponding in size to pRleVF39b instead of pRleVF39d . On the other hand, some DNA fragments originating from pRleVF39b now hybridized to the plasmid band at the position of pRleVF39d . These results suggest that a reciprocal but unequal DNA exchange between the two plasmids had occurred.

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