J Bacteriol, 1999 Aug, 181(15), 4576 - 83 Cloning, sequencing, and characterization of the cgmB gene of Sinorhizobium meliloti involved in cyclic beta-glucan biosynthesis; Wang P et al.; Periplasmic cyclic beta-glucans of Rhizobium species provide important functions during plant infection and hypo-osmotic adaptation . In Sinorhizobium meliloti (also known as Rhizobium meliloti), these molecules are highly modified with phosphoglycerol and succinyl substituents . We have previously identified an S . meliloti Tn5 insertion mutant, S9, which is specifically impaired in its ability to transfer phosphoglycerol substituents to the cyclic beta-glucan backbone (M . W . Breedveld, J . A . Hadley, and K . J . Miller, J . Bacteriol . 177:6346-6351, 1995) . In the present study, we have cloned, sequenced, and characterized this mutation at the molecular level . By using the Tn5 flanking sequences (amplified by inverse PCR) as a probe, an S . meliloti genomic library was screened, and two overlapping cosmid clones which functionally complement S9 were isolated . A 3.1-kb HindIII-EcoRI fragment found in both cosmids was shown to fully complement mutant S9 . Furthermore, when a plasmid containing this 3.1-kb fragment was used to transform Rhizobium leguminosarum bv . trifolii TA-1JH, a strain which normally synthesizes only neutral cyclic beta-glucans, anionic glucans containing phosphoglycerol substituents were produced, consistent with the functional expression of an S . meliloti phosphoglycerol transferase gene . Sequence analysis revealed the presence of two major, overlapping open reading frames within the 3.1-kb fragment . Primer extension analysis revealed that one of these open reading frames, ORF1, was transcribed and its transcription was osmotically regulated . This novel locus of S . meliloti is designated the cgm (cyclic glucan modification) locus, and the product encoded by ORF1 is referred to as CgmB. Development, 1999 Aug, 126(16), 3617 - 28 Refined analysis of early symbiotic steps of the Rhizobium-Medicago interaction in relationship with microtubular cytoskeleton rearrangements; Timmers AC et al.; In situ immunolocalization of tubulin revealed that important rearrangements occur during all the early symbiotic steps in the Medicago/R . meliloti symbiotic interaction . Microtubular cytoskeleton (MtC) reorganizations were observed in inner tissues, first in the pericycle and then in the inner cortex where the nodule primordium forms . Subsequently, major MtC changes occurred in outer tissues, associated with root hair activation and curling, the formation of preinfection threads (PITs) and the initiation and the growth of an infection network . From the observed sequence of MtC changes, we propose a model which aims to better define, at the histological level, the timing of the early symbiotic stages . This model suggests the existence of two opposite gradients of cell differentiation controlling respectively the formation of division centers in the inner cortex and plant preparation for infection . It implies that (i) MtC rearrangements occur in pericycle and inner cortex earlier than in the root hair, (ii) the infection process proceeds prior to the formation of the nodule meristem, (iii) the initial primordium prefigures the future zone II of the mature nodule and (iv) the nodule meristem derives from the nodule primordium . Finally, our data also strongly suggest that in alfalfa PIT differentiation, a stage essential for successful infection, requires complementary signaling additional to Nod factors. Biochim Biophys Acta, 1999 Jul 13, 1432(2), 275 - 85 Characterization of two members of a novel malic enzyme class; Voegele RT et al.; The Gram-negative bacterium Rhizobium meliloti contains two distinct malic enzymes . We report the purification of the two isozymes to homogeneity, and their in vitro characterization . Both enzymes exhibit unusually high subunit molecular weights of about 82 kDa . The NAD(P)(+) specific malic enzyme {EC 1.1.1.39} exhibits positive co-operativity with respect to malate, but Michaelis-Menten type behavior with respect to the co-factors NAD(+) or NADP(+) . The enzyme is subject to substrate inhibition, and shows allosteric regulation by acetyl-CoA, an effect that has so far only been described for some NADP(+) dependent malic enzymes . Its activity is positively regulated by succinate and fumarate . In contrast to the NAD(P)(+) specific malic enzyme, the NADP(+) dependent malic enzyme {EC 1.1.1.40} shows Michaelis-Menten type behavior with respect to malate and NADP(+) . Apart from product inhibition, the enzyme is not subjected to any regulatory mechanism . Neither reductive carboxylation of pyruvate, nor decarboxylation of oxaloacetate, could be detected for either malic enzyme . Our characterization of the two R . meliloti malic enzymes therefore suggests a number of features uncharacteristic for malic enzymes described so far. J Biotechnol, 1999 Jun 11, 72(1-2), 33 - 47 Production of O-acetylated and sulfated chitooligosaccharides by recombinant Escherichia coli strains harboring different combinations of nod genes; Samain E et al.; High cell density cultivation of recombinant Escherichia coli strains harboring the nodBC genes (encoding chitooligosaccharide synthase and chitooligosaccharide N-deacetylase, respectively) from Azorhizobium caulinodans has been previously described as a practical method for the preparation of gram-scale quantities of penta-N-acetyl-chitopentaose and tetra-N-acetylchitopentaose (Samain, E., Drouillard, S., Heyraud, A., Driguez, H., Geremia, R.A., 1997 . Carbohydr . Res . 30, 235-242) . We have now extended this method to the production of sulfated and O-acetylated derivatives of these two compounds by coexpressing nodC or nodBC with nodH and/or nodL that encode chitooligosaccharide sulfotransferase and chitooligosaccharide O-acetyltransferase, respectively . In addition, these substituted chitooligosaccharides were also obtained as tetramers by using nodC from Rhizobium meliloti instead of nodC from A . caulinodans . These compounds should be useful precursors for the preparation of Nod factor analogues by chemical modification. J Bacteriol, 1999 Jul, 181(14), 4185 - 92 Identification of site-specific recombination genes int and xis of the Rhizobium temperate phage 16-3; Semsey S et al.; Phage 16-3 is a temperate phage of Rhizobium meliloti 41 which integrates its genome with high efficiency into the host chromosome by site-specific recombination through DNA sequences of attB and attP . Here we report the identification of two phage-encoded genes required for recombinations at these sites: int (phage integration) and xis (prophage excision) . We concluded that Int protein of phage 16-3 belongs to the integrase family of tyrosine recombinases . Despite similarities to the cognate systems of the lambdoid phages, the 16-3 int xis att system is not active in Escherichia coli, probably due to requirements for host factors that differ in Rhizobium meliloti and E . coli . The application of the 16-3 site-specific recombination system in biotechnology is discussed. J Bacteriol, 1999 Jul, 181(14), 4176 - 84 A novel Sinorhizobium meliloti operon encodes an alpha-glucosidase and a periplasmic-binding-protein-dependent transport system for alpha-glucosides; Willis LB et al.; The most abundant carbon source transported into legume root nodules is photosynthetically produced sucrose, yet the importance of its metabolism by rhizobia in planta is not yet known . To identify genes involved in sucrose uptake and hydrolysis, we screened a Sinorhizobium meliloti genomic library and discovered a segment of S . meliloti DNA which allows Ralstonia eutropha to grow on the alpha-glucosides sucrose, maltose, and trehalose . Tn5 mutagenesis localized the required genes to a 6.8-kb region containing five open reading frames which were named agl, for alpha-glucoside utilization . Four of these (aglE, aglF, aglG, and aglK) appear to encode a periplasmic-binding-protein-dependent sugar transport system, and one (aglA) appears to encode an alpha-glucosidase with homology to family 13 of glycosyl hydrolases . Cosmid-borne agl genes permit uptake of radiolabeled sucrose into R . eutropha cells . Analysis of the properties of agl mutants suggests that S . meliloti possesses at least one additional alpha-glucosidase as well as a lower-affinity transport system for alpha-glucosides . It is possible that the Fix+ phenotype of agl mutants on alfalfa is due to these additional functions . Loci found by DNA sequencing to be adjacent to aglEFGAK include a probable regulatory gene (aglR), zwf and edd, which encode the first two enzymes of the Entner-Doudoroff pathway, pgl, which shows homology to a gene encoding a putative phosphogluconolactonase, and a novel Rhizobium-specific repeat element. Acta Biochim Pol, 1998, 45(4), 1067 - 73 Molecular characterization and symbiotic importance of prsD gene of Rhizobium leguminosarum bv . trifolii TA1; Mazur A et al.; The prsD, prsE and orf3 genes of Rhizobium leguminosarum bv . trifolii strain TA1 encode the proteins which are significantly related to the family of bacterial ABC transporters type I secretion systems . The prsD:Km(r) mutant of strain TA1 induced non-nitrogen-fixing nodules on Trifolium pratense . Microscopic analysis of the nodules induced by prsD mutant did not reveal major abberations in the bacteroid appearance . The exopolysaccharide of prsD mutant was produced in increased amount and its level of polymerization was changed . SDS/PAGE of the proteins from the culture supernatants showed a lack of the 47-kDa protein in the culture of prsD mutant . Thus, PrsD may play a role in the export of this protein. Acta Biochim Pol, 1998, 45(4), 1001 - 9 Occurrence of lipopolysaccharide alterations among Tn5 mutants of Rhizobium leguminosarum bv . trifolii strain 24.1 with altered colony morphology; Szyprowska S et al.; Transposon mutants of Rhizobium leguminosarum bv . trifolii 24.1 showing less glossy or smaller colonies were screened for properties usually associated with lipopolysaccharide (LPS) defects in R . leguminosarum, i.e . motility, growth rate, tendency to agglutination in liquid media and symbiotic efficiency . Neither any of the above mutants nor the earlier isolated 24.12 strain, defective in LPS, showed all these properties changed simultaneously . According to PAGE/sodium deoxycholate analysis the mutant 24.12 was the only one producing defective lipopolysaccharide . GC-MS analysis revealed in this mutant qualitative changes in composition of its LPS in comparison with LPS isolated from the parent strain . Other Tn5 mutants produced LPSs similar in composition, however the proportion between LPS I and LPS II differed from that in the parent strain. Plant Mol Biol, 1999 May, 40(1), 65 - 77 A new class of plant homeobox genes is expressed in specific regions of determinate symbiotic root nodules; Jorgensen JE et al.; A cDNA containing a homeobox sequence was isolated from a soybean nodule-specific expression library . This homeobox cDNA, Ndx (nodulin homeobox), represents a small gene family with at least two members in soybean (Glycine max) and three in Lotus japonicus . One complete 3304 bp Ndx cDNA from L . japonicus encodes a protein, NDX, of 958 amino acids . An unusual type of homeodomain that differs in two of the most conserved amino acid positions in the consensus sequence is located close to the C-terminal and appears to be the only DNA-binding domain . Weak Ndx gene expression in the root increases very shortly after infection with Rhizobium and remains throughout nodule development . In situ hybridizations show cell-specific expression patterns that suggest developmentally separate regions in maturing determinate nodules . Thus in the maturing nodule Ndx and leghemoglobin genes are expressed in a mutually exclusive fashion . The Ndx transcript is also detectable in the young nodule primordium . Ndx expression is not confined to the root nodule since Ndx is also expressed in shoot and root meristems, indicating that the Ndx gene products might also be involved in developmental processes in other plant tissues. J Biol Chem, 1999 Jul 9, 274(28), 20011 - 6 Plant-exuded choline is used for rhizobial membrane lipid biosynthesis by phosphatidylcholine synthase; de Rudder KE et al.; Phosphatidylcholine is a major lipid of eukaryotic membranes, but found in only few prokaryotes . Enzymatic methylation of phosphatidylethanolamine by phospholipid N-methyltransferase was thought to be the only biosynthetic pathway to yield phosphatidylcholine in bacteria . However, mutants of the microsymbiotic soil bacterium Sinorhizobium (Rhizobium) meliloti, defective in phospholipid N-methyltransferase, form phosphatidylcholine in wild type amounts when choline is provided in the growth medium . Here we describe a second bacterial pathway for phosphatidylcholine biosynthesis involving the novel enzymatic activity, phosphatidylcholine synthase, that forms phosphatidylcholine directly from choline and CDP-diacylglycerol in cell-free extracts of S . meliloti . We further demonstrate that roots of host plants of S . meliloti exude choline and that the amounts of exuded choline are sufficient to allow for maximal phosphatidylcholine biosynthesis in S . meliloti via the novel pathway. Br J Cancer, 1999 Apr, 80(1-2), 110 - 6 Anti-tumour promoter activity in Malaysian ginger rhizobia used in traditional medicine; Vimala S et al.; Zingiberaceae rhizomes commonly used in the Malaysian traditional medicine were screened for anti-tumour promoter activity using the short-term assay of inhibition of 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced Epstein-Barr virus early antigen (EBV-EA) in Raji cells . The inhibition of TPA-induced EBV-EA was detected using the indirect immunofluorescence assay (IFA) and Western blot technique . The indirect IFA detected the expression/inhibition of EBV-EA-D (diffused EA antigen), whereas the Western blot technique detected the expression/inhibition of both EBV-EA-D and EA-R (restricted EA antigen) . Seven rhizomes were found to possess inhibitory activity towards EBV activation, induced by TPA; they are: Curcuma domestica, C . xanthorrhiza, Kaempferia galanga, Zingiber cassumunar, Z . officinale, Z . officinale (red variety), and Z . zerumbet . A cytotoxicity assay was carried out to determine the toxicity of the Zingiberaceae rhizome extracts . The rhizome extracts that exhibited EBV activation inhibitory activity had no cytotoxicity effect in Raji cells . Therefore, the present study shows that several Zingiberaceae species used in Malaysian traditional medicine contain naturally occurring non-toxic compounds that inhibit the EBV activation, which, if further investigated, could contribute in the development of cancer prevention methods at the tumour-promoting stage. Appl Environ Microbiol, 1999 Jul, 65(7), 2907 - 11 Purification and characterization of an alpha-glucosidase from Rhizobium sp . (Robinia pseudoacacia L.) strain USDA 4280; Berthelot K et al.; A novel alpha-glucosidase with an apparent subunit mass of 59 +/- 0 . 5 kDa was purified from protein extracts of Rhizobium sp . strain USDA 4280, a nodulating strain of black locust (Robinia pseudoacacia L), and characterized . After purification to homogeneity (475-fold; yield, 18%) by ammonium sulfate precipitation, cation-exchange chromatography, hydrophobic chromatography, dye chromatography, and gel filtration, this enzyme had a pI of 4.75 +/- 0.05 . The enzyme activity was optimal at pH 6.0 to 6.5 and 35 degrees C . The activity increased in the presence of NH4+ and K+ ions but was inhibited by Cu2+, Ag+, Hg+, and Fe2+ ions and by various phenyl, phenol, and flavonoid derivatives . Native enzyme activity was revealed by native gel electrophoresis and isoelectrofocusing-polyacrylamide gel electrophoresis with fluorescence detection in which 4-methylumbelliferyl alpha-glucoside was the fluorogenic substrate . The enzyme was more active with alpha-glucosides substituted with aromatic aglycones than with oligosaccharides . This alpha-glucosidase exhibited Michaelis-Menten kinetics with 4-methylumbelliferyl alpha-D-glucopyranoside (Km, 0.141 microM; Vmax, 6.79 micromol min-1 mg-1) and with p-nitrophenyl alpha-D-glucopyranoside (Km, 0.037 microM; Vmax, 2.92 micromol min-1 mg-1) . Maltose, trehalose, and sucrose were also hydrolyzed by this enzyme. Appl Environ Microbiol, 1999 Jul, 65(7), 2833 - 40 Cloning and characterization of a Rhizobium leguminosarum gene encoding a bacteriocin with similarities to RTX toxins; Oresnik IJ et al.; A 3-kb region containing the determinant for bacteriocin activity from Rhizobium leguminosarum 248 was isolated and characterized by Tn5 insertional mutagenesis and DNA sequencing . Southern hybridizations showed that this bacteriocin was encoded on the plasmid pRL1JI and that homologous loci were not found in other unrelated R . leguminosarum strains . Tn5 insertional mutagenesis showed that mutations in the C-terminal half of the bacteriocin open reading frame apparently did not abolish bacteriocin activity . Analysis of the deduced amino acid sequence revealed that, similarly to RTX proteins (such as hemolysin and leukotoxin), this protein contains a characteristic nonapeptide repeated up to 18 times within the protein . In addition, a novel 19- to 25-amino-acid motif that occurred every 130 amino acids was detected . Bacteriocin bioactivity was correlated with the presence of a protein of approximately 100 kDa in the culture supernatants, and the bacteriocin bioactivity demonstrated a calcium dependence in both R . leguminosarum and Sinorhizobium meliloti . A mutant of strain 248 unable to produce this bacteriocin was found to have a statistically significant reduction in competitiveness for nodule occupancy compared to two test strains in coinoculation assays . However, this strain was unable to compete any more successfully with a third test strain, 3841, than was wild-type 248. Microb Ecol, 1999 Jul, 38(1), 39 - 49 Influence of an Elevated Atmospheric CO2 Content on Soil and Rhizosphere Bacterial Communities Beneath Lolium perenne and Trifolium repens under Field Conditions; Marilley L et al.; > Abstract The increase in atmospheric CO2 content alters C3 plant photosynthetic rate, leading to changes in rhizodeposition and other root activities . This may influence the activity, the biomass, and the structure of soil and rhizosphere microbial communities and therefore the nutrient cycling rates and the plant growth . The present paper focuses on bacterial numbers and on community structure . The rhizospheres of two grassland plants, Lolium perenne (ryegrass) and Trifolium repens (white clover), were divided into three fractions: the bulk soil, the rhizospheric soil, and the rhizoplane-endorhizosphere . The elevated atmospheric CO2 content increased the most probable numbers of heterotrophic bacteria in the rhizosphere of L . perenne . However, this effect lasted only at the beginning of the vegetation period for T . repens . Community structure was assessed after isolation of DNA, PCR amplification, and construction of cloned 16S rDNA libraries . Amplified ribosomal DNA restriction analysis (ARDRA) and colony hybridization with an oligonucleotide probe designed to detect Pseudomonas spp . showed under elevated atmospheric CO2 content an increased dominance of pseudomonads in the rhizosphere of L . perenne and a decreased dominance in the rhizosphere of T . repens . This work provides evidence for a CO2-induced alteration in the structure of the rhizosphere bacterial populations, suggesting a possible alteration of the plant-growth-promoting-rhizobacterial (PGPR) effect.http://link.springer-ny.com/link/service/journals/00248/bibs/38n1p39.html Microbiology, 1999 May, 145 ( Pt 5), 1275 - 85 Molecular analysis of the recA gene and SOS box of the purple non-sulfur bacterium Rhodopseudomonas palustris no . 7; Dumay V et al.; The recA gene of the purple non-sulfur bacterium Rhodopseudomonas palustris no . 7 was isolated by a PCR-based method and sequenced . The complete nucleotide sequence consists of 1089 bp encoding a polypeptide of 363 amino acids which is most closely related to the RecA proteins from species of Rhizobiaceae and Rhodospirillaceae . A recA-deficient strain of R . palustris no . 7 was obtained by gene replacement . As expected, this strain exhibited increased sensitivity to DNA-damaging agents . Transcriptional fusions of the recA promoter region to lacZ confirmed that the R . palustris no . 7 recA gene is inducible by DNA damage . Primer extension analysis of recA mRNA located the recA gene transcriptional start . A sequential deletion of the fusion plasmid was used to delimit the promoter region of the recA gene . A gel mobility shift assay demonstrated that a DNA-protein complex is formed at this promoter region . This DNA-protein complex was not formed when protein extracts from cells treated with DNA-damaging agents were used, indicating that the binding protein is a repressor . Comparison of the minimal R . palustris no . 7 recA promoter region with the recA promoter sequences from other alpha-Proteobacteria revealed the presence of the conserved sequence GAACA-N6-G(A/T)AC . Site-directed mutations that changed this consensus sequence abolished the DNA-damage-mediated expression of the R . palustris recA gene, confirming that this sequence is the SOS box of R . palustris and probably plays the same role in other alpha-Proteobacteria. Theor Popul Biol, 1999 Jun, 55(3), 309 - 23 Evolution of mutualistic symbiosis without vertical transmission; Genkai-Kato M et al.; Mutualistic symbioses are considered to evolve from parasitic relationships . Vertical transmission, defined as the direct transfer of infection from a parent organism to its progeny, has been suggested as a key factor causing reduction of symbiont virulence and evolution of mutualism . On the other hand, there are several mutualistic associations without vertical transmission, such as those between plants and mycorrhizal fungi, legumes and rhizobia, and some corals and dinoflagellates . It is expected that all mutualisms evolve perfect vertical transmission if the relationship is really mutualistic, because hosts may fail to acquire symbionts if they do not transmit the symbionts by vertical transmission . We have developed a mathematical model to clarify the conditions under which mutualistic symbiosis without vertical transmission should evolve . The evolution may occur when and only when (i) vertical transmission involves some costs in the host, (ii) the symbiont suffers direct negative effects if it exploits the host too intensively, (iii) the host establishes the ability to make use of waste products from the symbiont, and (iv) the mechanism of vertical transmission is controlled by the host . We also clarify the conditions under which mutualistic symbiosis with vertical transmission evolves . Mol Microbiol, 1999 May, 32(4), 837 - 49 Bacterial genes induced within the nodule during the Rhizobium-legume symbiosis; Oke V et al.; During the symbiosis between the bacterium Rhizobium meliloti and plants such as alfalfa, the bacteria elicit the formation of nodules on the roots of host plants . The bacteria infect the nodule, enter the cytoplasm of plant cells and differentiate into a distinct cell type called a bacteroid, which is capable of fixing atmospheric nitrogen . To discover bacterial genes involved in the infection and differentiation stages of symbiosis, we obtained genes expressed at the appropriate time and place in the nodule by identifying promoters that are able to direct expression of the bacA gene, which is required for bacteroid differentiation . We identified 230 fusions that are expressed predominantly in the nodule . Analysis of 23 sequences indicated that only three encode proteins known to be involved in the Rhizobium-legume symbiosis, six encode proteins with homology to proteins not previously associated with symbiosis, and 14 have no significant similarity to proteins of known function . Disruption of a locus that encodes a protein with homology to a cell adhesion molecule led to a defect in the formation of nitrogen-fixing nodules, resulting in an increased number of nitrogen-starved plants . Our isolation of a large number of nodule-expressed genes will help to open the intermediate stages of nodulation to molecular analysis. Microbiol Res, 1999 May, 154(1), 49 - 55 Inducers of nod genes of Rhizobium ciceri; Srivastava P et al.; Induction of nodABC genes of R . ciceri was studied by constructing nodABC-lacZ fusion . The root exudates of the homologous hosts induced the expression of nodABC genes but those of heterologous hosts failed to do so . The HPLC analysis of the root exudates of C . arietinum showed the presence of 6-7 compounds with retention times matching to flavonoids like naringenin, hesperetin, daidzein, naringin, 7 OH coumarin and luteolin . Induction studies using the standard flavonoids showed naringenin, followed by daidzein, as most potent inducer of the nodABC genes of R . ciceri . Naringenin in combination with daidzein showed a synergistic effect on the expression of nodABC genes. J Bacteriol, 1999 Jun, 181(11), 3582 - 6 Cloning of the glutamyl-tRNA synthetase (gltX) gene from Pseudomonas aeruginosa; Franklund CV et al.; The glutamyl-tRNA synthetase (gltX) gene from Pseudomonas aeruginosa was identified . A plasmid containing a 2.3-kb insert complemented the temperature-sensitive gltX mutation of Escherichia coli JP1449, and GltX activity was demonstrated . The inferred amino acid sequence of this gene showed 50.6% identity with GltX from Rhizobium meliloti. Appl Environ Microbiol, 1999 Jun, 65(6), 2802 - 4 High-efficiency transformation of Rhizobium leguminosarum by electroporation; Garg B et al.; Electrotransformation of Rhizobium leguminosarum was successfully carried out with a 15.1-kb plasmid, pMP154 (Cmr), containing a nodABC-lacZ fusion by electroporation . The maximum transformation efficiency, 10(8) transformants/microg of DNA, was achieved at a field strength of 14 kV/cm with a pulse of 7.3 ms (186 Omega) . The number of transformants was found to increase with increasing cell density, with no sign of saturation . In relation to DNA dosage, the maximum transformation efficiency (5.8 x 10(8) transformants/microg of DNA) was obtained with 0.5 microg of DNA/ml of cell suspension, and a further increase in the DNA concentration resulted in a decline in transformation efficiency. Electrophoresis, 1999 Apr-May, 20(4-5), 818 - 25 Proteome analysis of the model microsymbiont Sinorhizobium meliloti: isolation and characterisation of novel proteins; Guerreiro N et al.; Sinorhizobium meliloti is an agriculturally and ecologically important microbe due to its capacity to establish nitrogen-fixing symbiosis with plant legumes . Two-dimensional gel electrophoresis of total cellular protein was used to establish a proteome reference map for the model microsymbiont Sinorhizobium meliloti strain 1021 . The extent of changes in the gene expression of cells grown in a defined medium at different growth phases was established . After examination of over 2000 resolved protein spots, a minimum of 52 reproducible changes in protein expression levels were detected when early exponential phase cells were compared to late exponential phase cells . In contrast, induction of nodulation gene expression by the addition of the flavonoid luteolin to cells did not result in detectable changes in protein expression at either early or late exponential phase . N-terminal microsequencing of eighteen unknown constitutive proteins plus four proteins, induced or up-regulated in late exponential phase cells, allowed the identification of proteins not previously described in rhizobia . These included an amide-binding protein, a putative hydrolase of the glyoxalase II protein family, a nucleoside diphosphate kinase, and a 5'-nucleotidase . N-terminal microsequencing was also valuable in revealing N-terminal post-translational processing and assigning a subcellular location to the analysed protein . Proteome analysis will provide a powerful analytical tool to complement the sequencing of the genome of strain 1021. Curr Microbiol, 1999 Jun, 38(6), 355 - 9 Primary structure of the DNA polymerase I gene of an alpha-proteobacterium, Rhizobium leguminosarum, and comparison with other family A DNA polymerases; Huang YP et al.; The structural gene for DNA polymerase I of Rhizobium leguminosarum was determined . The rhizobium DNA polymerase I consists of 1016 amino acid residues with a calculated molecular weight of 111,491 Dalton . The amino acid sequence comparison with E . coli DNA polymerase I, Thermus aquaticus DNA polymerase I, and Rickettsia prowazekii DNA polymerase I showed that, although 5'-nuclease and DNA polymerase domains are highly conserved, 3' to 5' exonuclease domains are much less conserved . While both R . leguminosarum and R . prowazekii belong to the alpha subdivision of the Proteobacteria on the basis of 16S ribosomal RNA phylogeny, the primary structure of the DNA polymerase I is quite different; the rhizobium DNA polymerase I has 3' to 5' proofreading exonuclease, but the rickettsia DNA polymerase I does not. Mol Biol Evol, 1999 Jan, 16(1), 98 - 113 Comparison of the evolutionary dynamics of symbiotic and housekeeping loci: a case for the genetic coherence of rhizobial lineages; Wernegreen JJ et al.; In prokaryotes, lateral gene transfer across chromosomal lineages may be mediated by plasmids, phages, transposable elements, and other accessory DNA elements . However, the importance of such transfer and the evolutionary forces that may restrict gene exchange remain largely unexplored in native settings . In this study, tests of phylogenetic congruence are employed to explore the range of horizontal transfer of symbiotic (sym) loci among distinct chromosomal lineages of native rhizobia, the nitrogen-fixing symbiont of legumes . Rhizobial strains isolated from nodules of several host plant genera were sequenced at three loci: symbiotic nodulation genes (nodB and nodC), the chromosomal housekeeping locus glutamine synthetase II (GSII), and a portion of the 16S rRNA gene . Molecular phylogenetic analysis shows that each locus generally subdivides strains into the same major groups, which correspond to the genera Rhizobium, Sinorhizobium, and Mesorhizobium . This broad phylogenetic congruence indicates a lack of lateral transfer across major chromosomal subdivisions, and it contrasts with previous studies of agricultural populations showing broad transfer of sym loci across divergent chromosomal lineages . A general correspondence of the three rhizobial genera with major legume groups suggests that host plant associations may be important in the differentiation of rhizobial nod and chromosomal loci and may restrict lateral transfer among strains . The second major result is a significant incongruence of nod and GSII phylogenies within rhizobial subdivisions, which strongly suggests horizontal transfer of nod genes among congenerics . This combined evidence for lateral gene transfer within, but not between, genetic subdivisions supports the view that rhizobial genera are "reproductively isolated" and diverge independently . Differences across rhizobial genera in the specificity of host associations imply that the evolutionary dynamics of the symbiosis vary considerably across lineages in native settings. Acta Crystallogr D Biol Crystallogr, 1999 Jun, 55 ( Pt 6), 1215 - 8 Dynamic light-scattering and preliminary crystallographic studies of the sensor domain of the haem-based oxygen sensor FixL from Rhizobium meliloti; Miyatake H et al.; FixL is a transmitter protein in a two-component system which acts as an oxygen sensor when the symbiotic Rhizobia resides in root nodules of host plants . The oxygen-sensor domain of Rhizobium meliloti FixL (RmFixLH) was purified by His-tag affinity and isoelectronic focusing chromatographies, without the use of gel-filtration chromatography . Dynamic light-scattering measurements of RmFixLH thus obtained revealed it to be monodispersive and present as a homodimer in solution . A single crystal of RmFixLH in the met (Fe3+) form was grown in 100 mM acetic acid/NaOH buffer at pH 4.6 in the presence of 200 mM ammonium acetate, using 40%(w/v) PEG 4000 as a precipitant . A crystal of the ferrous CO form of RmFixLH was also prepared by reduction of the met crystal with Na2S2O4 in an atmosphere of CO . The crystals (0.2 x 0.05 x 0.01 mm) belong to the monoclinic system (C2) with unit-cell parameters a = 60.94, b = 37.44, c = 54.14 A, beta = 115.29 degrees and diffract X-rays to 1.7 A resolution at station BL44B2 of SPring-8, Japan . Bijvoet difference Patterson maps show a clear peak corresponding to the haem iron in RmFixLH. Proc Natl Acad Sci U S A, 1999 May 11, 96(10), 5856 - 61 A nod factor binding lectin with apyrase activity from legume roots; Etzler ME et al.; A lectin isolated from the roots of the legume, Dolichos biflorus, binds to Nod factors produced by rhizobial strains that nodulate this plant and has a deduced amino acid sequence with no significant homology to any lectin reported to date . This lectin also is an enzyme that catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside di- and triphosphates; the enzyme activity is increased in the presence of carbohydrate ligands . This lectin-nucleotide phosphohydrolase (LNP) has a substrate specificity characteristic of the apyrase category of phosphohydrolases, and its sequence contains four motifs characteristic of this category of enzymes . LNP is present on the surface of the root hairs, and treatment of roots with antiserum to LNP inhibits their ability to undergo root hair deformation and to form nodules on exposure to rhizobia . These properties suggest that this protein may play a role in the rhizobium-legume symbiosis and/or in a related carbohydrate recognition event endogenous to the plant. Mol Microbiol, 1999 Apr, 32(2), 415 - 25 High-resolution transcriptional analysis of the symbiotic plasmid of Rhizobium sp . NGR234; Perret X et al.; Most of the bacterial genes involved in nodulation of legumes (nod, nol and noe ) as well as nitrogen fixation (nif and fix ) are carried on pNGR234a, the 536 kb symbiotic plasmid (pSym) of the broad-host-range Rhizobium sp . NGR234 . Putative transcription regulators comprise 24 of the predicted 416 open reading frames (ORFs) contained on this replicon . Computational analyses identified 19 nod boxes and 16 conserved NifA-sigma54 regulatory sequences, which are thought to co-ordinate the expression of nodulation and nitrogen fixation genes respectively . To analyse transcription of all putative ORFs, the nucleotide sequence of pNGR234a was divided into 441 segments designed to represent all coding and intergenic regions . Each of these segments was amplified by polymerase chain reactions, transferred to filters and probed with radioactively labelled RNA . RNA was extracted from bacterial cultures grown under various experimental conditions, as well as from bacteroids of determinate and indeterminate nodules . Generally, genes involved in the synthesis of Nod factors (e.g . the three hsn loci) were induced rapidly after the addition of flavonoids, whereas others thought to act within the plant (e.g . those encoding the type III secretion system) responded more slowly . Many insertion (IS) and transposon (Tn)-like sequences were expressed strongly under all conditions tested, while a number of loci other than those known to encode nod, noe, nol, nif and fix genes were also transcribed in nodules . Many more diverse transcripts were found in bacteroids of determinate as opposed to indeterminate nodules. FEMS Microbiol Lett, 1999 Apr 15, 173(2), 319 - 25 The pssB gene product of Rhizobium leguminosarum bv . trifolii is homologous to a family of inositol monophosphatases; Janczarek M et al.; The Rhizobium leguminosarum bv . trifolii region encoding pssA and pssB genes was cloned . The pssB gene located upstream of the pssA encoded a 28.36-kDa protein which displayed 97.5% identity with the PssB of R . leguminosarum bv . viciae . Inactivation of the pssB gene by insertion of the lacZ-Gmr cassette resulted in the significant increased production of exopolysaccharide in comparison to the wild-type level . A mutant strain was also defective in nitrogen fixation suggesting a regulatory role of pssB in symbiosis with clover. Mol Plant Microbe Interact, 1999 May, 12(5), 450 - 8 Nanogram amounts of salicylic acid produced by the rhizobacterium Pseudomonas aeruginosa 7NSK2 activate the systemic acquired resistance pathway in bean; De Meyer G et al.; Root colonization by specific nonpathogenic bacteria can induce a systemic resistance in plants to pathogen infections . In bean, this kind of systemic resistance can be induced by the rhizobacterium Pseudomonas aeruginosa 7NSK2 and depends on the production of salicylic acid by this strain . In a model with plants grown in perlite we demonstrated that Pseudomonas aeruginosa 7NSK2-induced resistance is equivalent to the inclusion of 1 nM salicylic acid in the nutrient solution and used the latter treatment to analyze the molecular basis of this phenomenon . Hydroponic feeding of 1 nM salicylic acid solutions induced phenylalanine ammonia-lyase activity in roots and increased free salicylic acid levels in leaves . Because pathogen-induced systemic acquired resistance involves similar changes it was concluded that 7NSK2-induced resistance is mediated by the systemic acquired resistance pathway . This conclusion was validated by analysis of phenylalanine ammonia-lyase activity in roots and of salicylic acid levels in leaves of soil-grown plants treated with Pseudomonas aeruginosa . The induction of systemic acquired resistance by nanogram amounts of salicylic acid is discussed with respect to long-distance signaling in systemic acquired resistance. FEBS Lett, 1999 Apr 16, 449(1), 23 - 7 Co-transcription of Rhizobium meliloti lysyl-tRNA synthetase and glutamyl-tRNA synthetase genes; Pelchat M et al.; An open reading frame encoding a putative polypeptide very similar to several lysyl-tRNA synthetases was found 10 nucleotides downstream of Rhizobium meliloti gltX encoding glutamyl-tRNA synthetase . Expression of this gene complemented a mutation in lysS of Escherichia coli and led to the overexpression of a polypeptide of the expected mass (62 kDa), thus confirming that it encodes R . meliloti lysyl-tRNA synthetase . Reverse transcription/polymerase chain reaction was used to demonstrate that this lysS gene is co-transcribed with gltX in R . meliloti . This is the first reported case of two immediately adjacent and co-transcribed genes encoding aminoacyl-tRNA synthetases. Appl Environ Microbiol, 1999 May, 65(5), 2015 - 9 Enhanced nitrogen fixation in a rhizobium etli ntrC mutant that overproduces the bradyrhizobium japonicum symbiotic terminal oxidase cbb3 Soberon M, Lopez O, Morera C, Girard ML, Tabche ML, Miranda J. The ntrC gene codes for a transcriptional activator protein that modulates gene expression in response to nitrogen . The cytochrome production pattern of a Rhizobium etli ntrC mutant (CFN2012) was studied . CO difference spectral analysis of membranes showed that CFN2012 produced a terminal oxidase similar to the symbiotic terminal oxidase of bacteroids in free-living cells under aerobic conditions, with a characteristic trough at 553 nm . CFN2012 produced two c-type cytochromes with molecular masses of 27 and 32 kDa, in contrast with the wild-type strain, which produced only a 32-kDa c-type cytochrome . The expression levels of the R . etli fixNOQP operon, which codes for terminal oxidase cbb3, were not affected by the ntrC mutation . However, the production levels of the two c-type cytochromes (27 and 32 kDa) were enhanced at least eightfold when the Bradyrhizobium japonicum fixNOQP operon was expressed in CFN2012 from the nptII promoter (pMSfixc), suggesting that these proteins are subunits FixO (27 kDa) and FixP (32 kDa) of cbb3 and that CFN2012/pMSfixc overproduced this terminal oxidase . CFN2012/pMSfixc showed a significant increase in its symbiotic performance as judged by the determination of nitrogenase activities of plants inoculated with this strain, suggesting that the overproduction of cbb3 terminal oxidase correlates with an enhancement in symbiotic nitrogen fixation. Microbiology, 1999 Mar, 145 ( Pt 3), 603 - 11 Biosynthesis of the exopolysaccharide galactoglucan in Sinorhizobium meliloti is subject to a complex control by the phosphate-dependent regulator PhoB and the proteins ExpG and MucR; Ruberg S et al.; The soil bacterium Sinorhizobium meliloti (Rhizobium meliloti) has the ability to produce the alternative exopolysaccharide galactoglucan (EPS II) in addition to succinoglycan (EPS I) . In the wild-type strain EPS II production is induced by phosphate-limiting conditions or by extra copies of the exp gene cluster . Based on similarities to transcriptional regulators of the MarR family, an additional putative regulatory gene, expG, was identified in the exp gene cluster . Using exp-lacZ transcriptional fusions, a stimulating effect of extra copies of this expG gene on the transcription of all exp complementation groups was determined . Phosphate limitation also resulted in increased expression of the exp-lacZ fusions . This increase was reduced in strains characterized by a deletion of expG . The previously reported high level of exp gene transcription in a mucR mutant was further elevated under phosphate-limiting conditions . The expA, expD, expG and expE promoters contain sequences with similarities to the PHO box known as the PhoB-binding site in phosphate-regulated promoters in Escherichia coli . The S . meliloti phoB gene was required for the activation of exp gene expression under phosphate limitation, but not for induction of exp expression by MucR or ExpG. Microbiology, 1999 Mar, 145 ( Pt 3), 593 - 601 The fhu genes of Rhizobium leguminosarum, specifying siderophore uptake proteins: fhuDCB are adjacent to a pseudogene version of fhuA; Stevens JB et al.; A mutant of Rhizobium leguminosarum was isolated which fails to take up the siderophore vicibactin . The mutation is in a homologue of fhuB, which in Escherichia coli specifies an inner-membrane protein of the ferric hydroxamate uptake system . In Rhizobium, fhuB is in an operon fhuDCB, which specifies the cytoplasmic membrane and periplasmic proteins involved in siderophore uptake . fhuDCB mutants make vicibactin when grown in Fe concentrations that inhibit its production in the wild-type . Nodules on peas induced by fhuDCB mutants were apparently normal in N2 fixation . Transcription of an fhuDCB-lacZ fusion was Fe-regulated, being approximately 10-fold higher in Fe-depleted cells . Downstream of fhuB, in the opposite orientation, is a version of fhuA whose homologues in other bacteria specify hydroxamate outer-membrane receptors . This fhuA gene appears to be a pseudogene with stop codons and undetectable expression. Mol Microbiol, 1999 Apr, 32(1), 63 - 73 The regulator gene phoB mediates phosphate stress-controlled synthesis of the membrane lipid diacylglyceryl-N,N,N-trimethylhomoserine in Rhizobium (Sinorhizobium) meliloti; Geiger O et al.; Bacteria react to phosphate starvation by activating genes involved in the transport and assimilation of phosphate as well as other phosphorous compounds . Some soil bacteria have evolved an additional mechanism for saving phosphorous . Under phosphate-limiting conditions, they replace their membrane phospholipids by lipids not containing phosphorus . Here, we show that the membrane lipid pattern of the free-living microsymbiotic bacterium Rhizobium (Sinorhizobium) meliloti is altered at low phosphate concentrations . When phosphate is growth limiting, an increase in sulpholipids, ornithine lipids and the de novo synthesis of diacylglyceryl trimethylhomoserine (DGTS) lipids is observed . Rhizobium meliloti phoCDET mutants, deficient in phosphate uptake, synthesize DGTS constitutively at low or high medium phosphate concentrations, suggesting that reduced transport of phosphorus sources to the cytoplasm causes induction of DGTS biosynthesis . Rhizobium meliloti phoU or phoB mutants are unable to form DGTS at low or high phosphate concentrations . However, the functional complementation of phoU or phoB mutants with the phoB gene demonstrates that, of the two genes, only intact phoB is required for the biosynthesis of the membrane lipid DGTS. Novartis Found Symp, 1999, 221, 112 - 26; discussion 126-30 Acid tolerance in root nodule bacteria; Glenn AR et al.; Biological nitrogen fixation, especially via the legume Rhizobium symbiosis, is important for world agriculture . The productivity of legume crops and pastures is significantly affected by soil acidity; in some cases it is the prokaryotic partner that is pH sensitive . Growth of Rhizobium is adversely affected by low pH, especially in the 'acid stress zone' . Rhizobia exhibit an adaptive acid tolerance response (ATR) that is influenced by calcium concentration . Using Tn5-mutagenesis, gusA fusions and 'proteome' analysis, we have identified a range of genes that are essential for growth at low pH (such as actA, actP, exoR, actR and actS) . At least three regulatory systems exist . The two-component sensor-regulator system, actSR, is essential for induction of the adaptive ATR . Two other regulatory circuits exist that are independent of ActR . One system involves the low pH-induced regulator gene, phrR, which may control other low pH-regulated genes . The other circuit, involving a regulator that is yet unidentified, controls the expression of a pH-regulated structural gene (lpiA) . We have used pH-responsive gusA fusions to identify acid-inducible genes (such as lpiA), and then attempted to identify the regulators of these genes . The emerging picture is of a relatively complex set of systems that respond to external pH. Proc Natl Acad Sci U S A, 1999 Apr 13, 96(8), 4704 - 9 Ligand specificity of a high-affinity binding site for lipo-chitooligosaccharidic nod factors in medicago cell suspension cultures Gressent F, Drouillard S, Mantegazza N, Samain E, Geremia RA, Canut H, Niebel A, Driguez H, Ranjeva R, Cullimore J, Bono JJ. Rhizobial lipo-chitooligosaccharides (LCOs) are signaling molecules involved in host-range recognition for the establishment of the symbiosis with leguminous plants . The major LCO of Rhizobium meliloti, the symbiont of Medicago plants contains four or five N-acetylglucosamines, O-acetylated and N-acylated with a C16:2 fatty acid on the terminal nonreducing sugar and O-sulfated on the reducing sugar . In this paper, the ligand specificity of a high-affinity binding site (Nod factor binding site 2 or NFBS2), enriched in a plasma membrane-enriched fraction of Medicago cell suspension cultures, is reported . By using chemically synthesized LCOs, the role of structural elements, important for symbiotic activities, as recognition motifs for NFBS2 was determined . The results show that the substitutions on the nonreducing sugar of the LCOs (the O-acetate group, the fatty acid, and the hydroxyl group on the C4 of the sugar) are determinants for high-affinity binding to NFBS2 . In contrast, the sulfate group, which is necessary for all biological activities on Medicago, is not discriminated by NFBS2 . However, the reducing sugar of the LCO seems to interact with NFBS2, because ligand binding is affected by the reduction of the free anomeric carbon and depends on the number of N-acetyl glucosamine residues . These results suggest that the recognition of the LCOs by NFBS2 is mediated by structural elements in both the lipid and oligosaccharidic moities, but not by the sulfate group. J Bacteriol, 1999 Apr, 181(8), 2535 - 47 Investigation of two evolutionarily unrelated halocarboxylic acid dehalogenase gene families; Hill KE et al.; Dehalogenases are key enzymes in the metabolism of halo-organic compounds . This paper describes a systematic approach to the isolation and molecular analysis of two families of bacterial alpha-halocarboxylic acid (alphaHA) dehalogenase genes, called group I and group II deh genes . The two families are evolutionarily unrelated and together represent almost all of the alphaHA deh genes described to date . We report the design and evaluation of degenerate PCR primer pairs for the separate amplification and isolation of group I and II deh genes . Amino acid sequences derived from 10 of 11 group I deh partial gene products of new and previously reported bacterial isolates showed conservation of five residues previously identified as essential for activity . The exception, DehD from a Rhizobium sp., had only two of these five residues . Group II deh gene sequences were amplified from 54 newly isolated strains, and seven of these sequences were cloned and fully characterized . Group II dehalogenases were stereoselective, dechlorinating L- but not D-2-chloropropionic acid, and derived amino acid sequences for all of the genes except dehII degrees P11 showed conservation of previously identified essential residues . Molecular analysis of the two deh families highlighted four subdivisions in each, which were supported by high bootstrap values in phylogenetic trees and by enzyme structure-function considerations . Group I deh genes included two putative cryptic or silent genes, dehI degrees PP3 and dehI degrees 17a, produced by different organisms . Group II deh genes included two cryptic genes and an active gene, dehIIPP3, that can be switched off and on . All alphaHA-degrading bacteria so far described were Proteobacteria, a result that may be explained by limitations either in the host range for deh genes or in isolation methods. J Biol Chem, 1999 Apr 16, 274(16), 11150 - 8 A deacylase in Rhizobium leguminosarum membranes that cleaves the 3-O-linked beta-hydroxymyristoyl moiety of lipid A precursors; Basu SS et al.; Lipid A from the nitrogen-fixing bacterium Rhizobium leguminosarum displays many structural differences compared with lipid A of Escherichia coli . R . leguminosarum lipid A lacks the usual 1- and 4'-phosphate groups but is derivatized with a galacturonic acid substituent at position 4' . R . leguminosarum lipid A often contains an aminogluconic acid moiety in place of the proximal glucosamine 1-phosphate unit . Striking differences also exist in the secondary acyl chains attached to E . coli versus R . leguminosarum lipid A, specifically the presence of 27-hydroxyoctacosanoate and the absence of laurate and myristate in R . leguminosarum . Recently, we have found that lipid A isolated by pH 4.5 hydrolysis of R . leguminosarum cells is more heterogeneous than previously reported (Que, N . L . S., Basu, S . S., White, K . A., and Raetz, C . R . H . (1998) FASEB J . 12, A1284 (abstr.)) . Lipid A species lacking the 3-O-linked beta-hydroxymyristoyl residue on the proximal unit contribute to this heterogeneity . We now describe a membrane-bound deacylase from R . leguminosarum that removes a single ester-linked beta-hydroxymyristoyl moiety from some lipid A precursors, including lipid X, lipid IVA, and (3-deoxy-D-manno-octulosonic acid)2-lipid IVA . The enzyme does not cleave E . coli lipid A or lipid A precursors containing an acyloxyacyl moiety on the distal glucosamine unit . The enzyme is not present in extracts of E . coli or Rhizobium meliloti, but it is readily demonstrable in membranes of Pseudomonas aeruginosa, which also contains a significant proportion of 3-O-deacylated lipid A species . Optimal reaction rates are seen between pH 5.5 and 6.5 . The enzyme requires a nonionic detergent and divalent metal ions for activity . It cleaves the monosaccharide lipid X at about 5% the rate of lipid IVA and (3-deoxy-D-manno-octulosonic acid)2-lipid IVA . 1H NMR spectroscopy of the deacylase reaction product, generated with lipid IVA as the substrate, confirms unequivocally that the enzyme cleaves only the ester-linked beta-hydroxymyristoyl residue at the 3-position of the glucosamine disaccharide. J Biol Chem, 1999 Apr 16, 274(16), 11139 - 49 A phosphotransferase that generates phosphatidylinositol 4-phosphate (PtdIns-4-P) from phosphatidylinositol and lipid A in Rhizobium leguminosarum . A membrane-bound enzyme linking lipid a and ptdins-4-p biosynthesis; Basu SS et al.; Membranes of Rhizobium leguminosarum contain a 3-deoxy-D-manno-octulosonic acid (Kdo)-activated lipid A 4'-phosphatase required for generating the unusual phosphate-deficient lipid A found in this organism . The enzyme has been solubilized with Triton X-100 and purified 80-fold . As shown by co-purification and thermal inactivation studies, the 4'-phosphatase catalyzes not only the hydrolysis of (Kdo)2-{4'-32P}lipid IVA but also the transfer the 4'-phosphate of Kdo2-{4'-32P}lipid IVA to the inositol headgroup of phosphatidylinositol (PtdIns) to generate PtdIns-4-P . Like the 4'-phosphatase, the phosphotransferase activity is not present in Escherichia coli, Rhizobium meliloti, or the nodulation-defective mutant 24AR of R . leguminosarum . The specific activity for the phosphotransferase reaction is about 2 times higher than that of the 4'-phosphatase . The phosphotransferase assay conditions are similar to those used for PtdIns kinases, except that ATP and Mg2+ are omitted . The apparent Km for PtdIns is approximately 500 microM versus 20-100 microM for most PtdIns kinases, but the phosphotransferase specific activity in crude cell extracts is higher than that of most PtdIns kinases . The phosphotransferase is absolutely specific for the 4-position of PtdIns and is highly selective for PtdIns as the acceptor . The 4'-phosphatase/phosphotransferase can be eluted from heparin- or Cibacron blue-agarose with PtdIns . A phosphoenzyme intermediate may account for the dual function of this enzyme, since a single 32P-labeled protein species (Mr approximately 68,000) can be trapped and visualized by SDS gel electrophoresis of enzyme preparations incubated with Kdo2-{4'-32P}lipid IVA . Although PtdIns is not detected in cultures of R . leguminosarum/etli (CE3), PtdIns may be synthesized during nodulation or supplied by plant membranes, given that soybean PtdIns is an excellent phosphate acceptor . A bacterial enzyme for generating PtdIns-4-P and a direct link between lipid A and PtdIns-4-P biosynthesis have not been reported previously. Biochemistry, 1999 Mar 30, 38(13), 4045 - 52 Chitin oligosaccharide synthesis by rhizobia and zebrafish embryos starts by glycosyl transfer to O4 of the reducing-terminal residue; Kamst E et al.; Lipochitin oligosaccharides are organogenesis-inducing signal molecules produced by rhizobia to establish the formation of nitrogen-fixing root nodules in leguminous plants . Chitin oligosaccharide biosynthesis by the Mesorhizobium loti nodulation protein NodC was studied in vitro using membrane fractions of an Escherichia coli strain expressing the cloned M . loti nodC gene . The results indicate that prenylpyrophosphate-linked intermediates are not involved in the chitin oligosaccharide synthesis pathway . We observed that, in addition to N-acetylglucosamine (GlcNAc) from UDP-GlcNAc, NodC also directly incorporates free GlcNAc into chitin oligosaccharides . Further analysis showed that free GlcNAc is used as a primer that is elongated at the nonreducing terminus . The synthetic glycoside p-nitrophenyl-beta-N-acetylglucosaminide (pNPGlcNAc) has a free hydroxyl group at C4 but not at C1 and could also be used as an acceptor by NodC, confirming that chain elongation by NodC takes place at the nonreducing-terminal residue . The use of artificial glycosyl acceptors such as pNPGlcNAc has not previously been described for a processive glycosyltransferase . Using this method, we show that also the DG42-directed chitin oligosaccharide synthase activity, present in extracts of zebrafish embryos, is able to initiate chitin oligosaccharide synthesis on pNPGlcNAc . Consequently, chain elongation in chitin oligosaccharide synthesis by M . loti NodC and zebrafish DG42 occurs by the transfer of GlcNAc residues from UDP-GlcNAc to O4 of the nonreducing-terminal residue, in contrast to earlier models on the mechanism of processive beta-glycosyltransferase reactions. Biotechnol Bioeng, 1998 Apr 5, 58(2-3), 250 - 3 In vivo 13C-NMR studies of polymer synthesis in rhizobium meliloti M5N1 strain Tavernier P, Besson I I, Portais JC, Courtois J, Courtois B, Barbotin JN. The use of in vivo 13C-NMR approach for the monitoring of the synthesis of various polymers within cells of Rhizobium meliloti (M5N1 strain) is reported . Significant differences in polymer biosynthesis have been shown as a function of the metabolic state of the cells and the labeled carbon source used . Consumption of carbon source and produced glycogen was complete with mid-exponential phase harvested cells . This was not the case with stationary phase harvested cells, for which polyhydroxybutyrate synthesis was higher and gluconate synthesis was lower than the former . {1-13C}fructose-grown cells produced more exopolysaccharide and polyhydroxybutyrate, but less beta-(1,2) glucan and gluconate than {1-13C}glucose-grown cells . This approach offers a suitable tool to examine the kinetics of polymer biosynthesis by Rhizobia . Mol Plant Microbe Interact, 1999 Apr, 12(4), 293 - 318 Rhizobium sp . strain NGR234 and R . fredii USDA257 share exceptionally broad, nested host ranges; Pueppke SG et al.; Genetically, Rhizobium sp . strain NGR234 and R . fredii USDA257 are closely related . Small differences in their nodulation genes result in NGR234 secreting larger amounts of more diverse lipo-oligosaccharidic Nod factors than USDA257 . What effects these differences have on nodulation were analyzed by inoculating 452 species of legumes, representing all three subfamilies of the Leguminosae, as well as the nonlegume Parasponia andersonii, with both strains . The two bacteria nodulated P . andersonii, induced ineffective outgrowths on Delonix regia, and nodulated Chamaecrista fasciculata, a member of the only nodulating genus of the Caesalpinieae tested . Both strains nodulated a range of mimosoid legumes, especially the Australian species of Acacia, and the tribe Ingeae . Highest compatibilities were found with the papilionoid tribes Phaseoleae and Desmodieae . On Vigna spp . (Phaseoleae), both bacteria formed more effective symbioses than rhizobia of the "cowpea" (V . unguiculata) miscellany . USDA257 nodulated an exact subset (79 genera) of the NGR234 hosts (112 genera) . If only one of the bacteria formed effective, nitrogen-fixing nodules it was usually NGR234 . The only exceptions were with Apios americana, Glycine max, and G . soja . Few correlations can be drawn between Nod-factor substituents and the ability to nodulate specific legumes . Relationships between the ability to nodulate and the origin of the host were not apparent . As both P . andersonii and NGR234 originate from Indonesia/Malaysia/Papua New Guinea, and NGR234's preferred hosts (Desmodiinae/Phaseoleae) are largely Asian, we suggest that broad host range originated in Southeast Asia and spread outward. Appl Environ Microbiol, 1999 Apr, 65(4), 1420 - 7 Isolation and characterization of alfalfa-nodulating rhizobia present in acidic soils of central argentina and uruguay del Papa MF, Balague LJ, Sowinski SC, Wegener C, Segundo E, Abarca FM, Toro N, Niehaus K, P hler A, Aguilar OM, Martinez-Drets G, Lagares A. We describe the isolation and characterization of alfalfa-nodulating rhizobia from acid soils of different locations in Central Argentina and Uruguay . A collection of 465 isolates was assembled, and the rhizobia were characterized for acid tolerance . Growth tests revealed the existence of 15 acid-tolerant (AT) isolates which were able to grow at pH 5.0 and formed nodules in alfalfa with a low rate of nitrogen fixation . Analysis of those isolates, including partial sequencing of the genes encoding 16S rRNA and genomic PCR-fingerprinting with MBOREP1 and BOXC1 primers, demonstrated that the new isolates share a genetic background closely related to that of the previously reported Rhizobium sp . Or191 recovered from an acid soil in Oregon (B . D . Eardly, J . P . Young, and R . K . Selander, Appl . Environ . Microbiol . 58:1809-1815, 1992) . Growth curves, melanin production, temperature tolerance, and megaplasmid profiles of the AT isolates were all coincident with these characteristics in strain Or191 . In addition to the ability of all of these strains to nodulate alfalfa (Medicago sativa) inefficiently, the AT isolates also nodulated the common bean and Leucaena leucocephala, showing an extended host range for nodulation of legumes . In alfalfa, the time course of nodule formation by the AT isolate LPU 83 showed a continued nodulation restricted to the emerging secondary roots, which was probably related to the low rate of nitrogen fixation by the largely ineffective nodules . Results demonstrate the complexity of the rhizobial populations present in the acidic soils represented by a main group of N2-fixing rhizobia and a second group of ineffective and less-predominant isolates related to the AT strain Or191. Appl Environ Microbiol, 1999 Apr, 65(4), 1491 - 500 Disaccharides as a new class of nonaccumulated osmoprotectants for Sinorhizobium meliloti; Gouffi K et al.; Sucrose and ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) are very unusual osmoprotectants for Sinorhizobium meliloti because these compounds, unlike other bacterial osmoprotectants, do not accumulate as cytosolic osmolytes in salt-stressed S . meliloti cells . Here, we show that, in fact, sucrose and ectoine belong to a new family of nonaccumulated sinorhizobial osmoprotectants which also comprises the following six disaccharides: trehalose, maltose, cellobiose, gentiobiose, turanose, and palatinose . Also, several of these disaccharides were very effective exogenous osmoprotectants for strains of Rhizobium leguminosarum biovars phaseoli and trifolii . Sucrose and trehalose are synthesized as endogenous osmolytes in various bacteria, but the other five disaccharides had never been implicated before in osmoregulation in any organism . All of the disaccharides that acted as powerful osmoprotectants in S . meliloti and R . leguminosarum also acted as very effective competitors of {14C}sucrose uptake in salt-stressed cultures of these bacteria . Conversely, disaccharides that were not osmoprotective for S . meliloti and R . leguminosarum did not inhibit sucrose uptake in these bacteria . Hence, disaccharide osmoprotectants apparently shared the same uptake routes in these bacteria . Natural-abundance 13C nuclear magnetic resonance spectroscopy and quantification of cytosolic solutes demonstrated that the novel disaccharide osmoprotectants were not accumulated to osmotically significant levels in salt-stressed S . meliloti cells; rather, these compounds, like sucrose and ectoine, were catabolized during early exponential growth, and contributed indirectly to enhance the cytosolic levels of two endogenously synthesized osmolytes, glutamate and the dipeptide N-acetylglutaminylglutamine amide . The ecological implication of the use of these disaccharides as osmoprotectants is discussed. Mol Cells, 1999 Feb 28, 9(1), 49 - 55 Isolation and characterization of the catalase gene from Rhizobium sp . SNU003, a root nodule symbiont of Canavalia lineata; Kwon SI et al.; A catalase gene from Rhizobium sp . SNU003, a root nodule symbiont of Canavalia lineata, was cloned and its nucleotide sequence was determined . The Rhizobium DNA of about 280 bp was amplified using two PCR primers synthesized from the conserved sequences of the type I catalase gene . The nucleotide sequence of the amplified fragment revealed three regions that were conserved in the catalase, showing it as being part of the catalase gene . A genomic Southern hybridization using this fragment as a probe showed that the 5.5 kb PstI, 1.8 kb EcoRI, and 0.7 kb StyI fragments hybridized strongly with the probe . The Rhizobium genomic library constructed into the EMBL3 vector was screened, and one catalase clone was selected . The nucleotide sequence of the 5.5 kb PstI fragment from the clone revealed an open reading frame of 1455 bp, encoding a polypeptide of 485 amino acids with a molecular mass of 54,958 Da and a pI of 6.54 . The predicted amino acid sequence of the catalase is 66.3% identical to that of Bacteroides fragilis, but was only 53.3% identical to the Rhizobium meliloti catalase. Biochim Biophys Acta, 1999 Mar 19, 1444(3), 451 - 6 Sequence and molecular analysis of the Rhizobium etli glsA gene, encoding a thermolabile glutaminase; Calderon J et al.; We sequenced a 2.1 kb fragment of DNA carrying the structural glsA gene, which codes for the Rhizobium etli thermolabile glutaminase (A) . The glsA gene complements the R . etli LM16 mutant that lacks glutaminase A activity, and is expressed in the heterologous host Sinorhizobium meliloti . The deduced amino acid sequence consists of 309 residues, with a calculated molecular mass of 33 kDa . The amino acid sequence shares 53% and 43% identity with two hypothetical glutaminases of E . coli; 42% identity with liver-type; 38% identity with kidney-type glutaminase; 41% and 40% identity hypothetical glutaminases of Bacillus subtilis; and 41% and 37% identity with two putative glutaminases of Caenorhabditis elegans . The glsA gene represents the first glutaminase gene cloned and sequenced in prokaryotes. Acta Crystallogr D Biol Crystallogr, 1998 Nov 1, 54(Pt 6 Pt 2), 1416 - 8 Crystallization and preliminary X-ray studies of the Rhizobium meliloti DctD two-component receiver domain; Staley M et al.; The Rhizobium meliloti DctD two-component receiver domain was expressed in Escherichia coli and purified to homogeneity . Crystals were obtained using the hanging-drop vapor-diffusion geometry with ammonium phosphate as the precipitant . The crystals diffract to 2.3 A and exhibit the symmetry of space group I222 or I212121 . The unit-cell dimensions are a = 59.0, b = 58.6 and c = 169.8 A . The asymmetric unit contains a dimer and the crystals have a Vm of 2.16 A3 Da-1. Plant Mol Biol, 1999 Jan, 39(1), 177 - 81 The soybean ENOD40(2) promoter is active in Arabidopsis thaliana and is temporally and spatially regulated; Mirabella R et al.; The ENOD40 gene is induced early during Rhizobium-legume symbiosis and has probably a primary role in the nodule organogenesis . In this paper we show that the 1.7 kb 5'-flanking region of the GmENOD40(2) is able to drive the expression of a gusA-int marker in transgenic Arabidopsis thaliana . The promoter activity is developmentally regulated and the major activity is detected in the root and in the stigma. FEMS Microbiol Lett, 1999 Mar 1, 172(1), 1 - 7 The Sinorhizobium meliloti insertion sequence (IS) elements ISRm102F34-1/ISRm7 and ISRm220-13-5 belong to a new family of insertion sequence elements; Selbitschka W et al.; The Sinorhizobium meliloti insertion sequence (IS) elements ISRm102F34-1 and ISRm220-13-5 are 1481 and 1550 base pairs (bp) in size, respectively . ISRm102F34-1 is bordered by 15 bp imperfect terminal inverted repeat sequences (two mismatches), whereas the terminal inverted repeat of ISRm220-13-5 has a length of 16 bp (two mismatches) . Both insertion sequence elements generate a 6-bp target duplication upon transposition . The putative transposase enzymes of ISRm102F34-1 and ISRm220-13-5 consist of 449 or 448 amino acid residues with predicted molecular weights of 50.7 or 51.3 kDa and theoretical isoelectric points of 10.8 or 11.1, respectively . ISRm102F34-1 is identical in 98.9% of its nucleotide sequence to an apparently inactive copy of an insertion sequence element, designated ISRm7, which flanks the left-end of the nodule formation efficiency (nfe) region of plasmid pRmeGR4b of S . meliloti strain GR4 . ISRm102F34-1 and ISRm220-13-5 are closely related since they show an overall identity of 57.0% at the nucleotide sequence level and of 47.3% at the deduced amino acid level of their putative transposases . Both insertion sequence elements displayed significant similarity to the Xanthomonas campestris ISXc6 and its homolog IS1478a . Since none of these insertion sequence elements could be allocated to existing families of insertion sequence elements, a new family is proposed . Analysis of the distribution of ISRm102F34-1/ISRm7 in various local S . meliloti populations sampled from Medicago sativa, Medicago sphaerocarpa and Melilotus alba host plants at different locations in Spain revealed its presence in 35% of the isolates with a copy number ranging from 1 to 5 . Furthermore, ISRm102F34-1/ISRm7 homologs were identified in other rhizobial species. Mikrobiol Z, 1998 Nov-Dec, 60(6), 3 - 25 {The molecular biological mechanisms of the functioning of microbial glycopolymers and carbohydrases}; Zakharova IIa; Special attention has been paid to glycopolymers which perform various functions in the microbial cell, determine its interaction with the environment, possess a broad range of biological activity (antigenicity, cytotoxicity, immunomodulating properties, participation in the bean-rhizobial symbiosis) . Composition and structure of the following polysaccharides, lipopolysaccharides and their components: O-polysaccharide, core oligosaccharide and lipid A from bacteria of genera Clavibacter, Ralstonia, Pseudomonas and Rhizobium, have been studied . Phenotypization of the studied strains has been carried out and chemotaxonomic criteria for creation of serological classification schemes have been proposed . Serogrouping of strains is determined by the availability and chemical nature of lateral substituents of the basic chain of O-specific polysaccharide . Interrelation between physico-chemical characteristics of the polysaccharide complex of rhizobia and formation of bean-rhizobial symbiotic structures has been shown . It is established that polysaccharides, lipo- and exopolysaccharides are efficient immunomodulators, manifest high antileucosis and antimetastasis activities . Physico-chemical properties, specificity, kinetics and action mechanism, structure of active and substrate-binding centres of bacterial and fungal enzymes: alpha-amylase, alpha-galactosidase, galactose oxidase . Data obtained permitted developing recommendations on enzymes application to some fields of the national economy. J Microbiol Methods, 1999 Feb, 35(1), 85 - 92 Bi-functional gfp- and gusA-containing mini-Tn5 transposon derivatives for combined gene expression and bacterial localization studies; Xi C et al.; The gfp gene, encoding the green fluorescent protein, was combined with the gusA gene, coding for the beta-glucuronidase enzyme, in mini-Tn5 transposon derivatives for use in Gram-negative bacteria . These mini-Tn5 elements allow simultaneously monitoring of gene expression and localization of the marked bacteria . Introduction of the resultant mini-Tn5 transposons into Rhizobium etli, Azospirillum brasilense and Pseudomonas stutzeri allowed us to visualise the interaction of these bacteria with their host plant . The dual-marker mini-Tn5 transposons constitute a powerful new tool for studying gene expression and ecology of bacteria in the environment and during the interaction with plants. Plant Physiol, 1999 Mar, 119(3), 951 - 60 Regulation of soybean nodulation independent of ethylene signaling Schmidt JS, Harper JE, Hoffman TK, Bent AF. Leguminous plants regulate the number of Bradyrhizobium- or Rhizobium-infected sites that develop into nitrogen-fixing root nodules . Ethylene has been implicated in the regulation of nodule formation in some species, but this role has remained in question for soybean (Glycine max) . The present study used soybean mutants with decreased responsiveness to ethylene, soybean mutants with defective regulation of nodule number, and Ag+ inhibition of ethylene perception to examine the role of ethylene in the regulation of nodule number . Nodule numbers on ethylene-insensitive mutants and plants treated with Ag+ were similar to those on wild-type plants and untreated plants, respectively . Hypernodulating mutants displayed wild-type ethylene sensitivity . Suppression of nodule numbers by high nitrate was also similar between ethylene-insensitive plants, wild-type plants, and plants treated with Ag+ . Ethylene insensitivity of the roots of etr1-1 mutants was confirmed using assays for sensitivity to 1-aminocyclopropane-1-carboxylic acid and for ethylene-stimulated root-hair formation . Additional phenotypes of etr1-1 roots were also characterized . Ethylene-dependent pathways regulate the number of nodules that form on species such as pea and Medicago truncatula, but our data indicate that ethylene is less significant in regulating the number of nodules that form on soybean. Curr Microbiol, 1999 Apr, 38(4), 205 - 9 Chemotaxis of Rhizobium sp.S2 towards Cajanus cajan root exudate and its major components; Pandya S et al.; The chemotactic response of Rhizobium sp . S2, a slow-growing Cajanus cajan isolate, towards its host root exudate was examined . Two classes of mutants, one nonchemotactic towards nutrients (amino acids and sugars) and signal compounds like flavonoids and the other, nonchemotactic towards amino acids and sugars but positive towards naringenin, the flavonoid present in Cajanus cajan root exudate, were obtained . The plasmid-cured derivative of the parent showed positive response towards amino acids and sugars but was nonchemotactic towards naringenin . A possible presence of dual chemotaxis pathways, one towards nutrients and the other for sensing signal compounds, was thus demonstrated . The possible involvement of naringenin as a chemoattractant in the preliminary stages of this Rhizobium-legume interaction was also established. Curr Opin Plant Biol, 1998 Aug, 1(4), 353 - 9 Genes and signal molecules involved in the rhizobia-leguminoseae symbiosis; Bladergroen MR et al.; The symbiosis between Rhizobium bacteria and their host plants is dependent on the specific recognition of signal molecules produced by each partner . Many players in the signal exchange have been identified . Among them are signal molecules such as flavonoids, LCOs, auxin, cytokinin, ethylene and uridine and genes such as Enod40, Enod2 and Enod12 . Their interconnection, however, is only starting to be understood . The most recent insights into their interconnection include: advances in the use of transgenic leguminous plants containing reporter gene constructs for studying the effect of the signal molecules; novel methods for delivery of signal molecules using ballistic microtargeting; and the discovery of the role of chitin oligosaccharides in animal embryogenesis. Curr Opin Plant Biol, 1998 Aug, 1(4), 360 - 5 Development of the arbuscular mycorrhizal symbiosis; Harrison MJ; The arbuscular mycorrhizal (AM) symbiosis formed between plant roots and fungi is one of the most widespread symbiotic associations found in plants, yet our understanding of events underlying its development are limited . The recent integration of biochemical, molecular and genetic approaches into analyses of the symbiosis is providing new insights into various aspects of its development . In the past year there have been advances in our understanding of the signals required for the formation of appressoria, the molecular changes in the root in response to colonisation, and components of the signal transduction pathways common to both the AM and Rhizobium symbioses. Mol Plant Microbe Interact, 1999 Mar, 12(3), 252 - 8 Comparison of characteristics of the nodX genes from various Rhizobium leguminosarum strains; Ovtsyna AO et al.; We have analyzed the nucleotide sequences of the nodX genes from two strains of Rhizobium leguminosarum bv . viciae able to nodulate Afghan peas (strains A1 and Himalaya) and from two strains of R . leguminosarum bv . trifolii (ANU843 and CSF) . The nodX genes of strains A1 and ANU843 were shown to be functional for the induction of nodules on Afghan peas . To analyze the cause of phenotypic differences of strain A1 and strain TOM we have studied the composition of the lipochitin-oligosaccharides (LCOs) produced by strain A1 after induction by the flavonoid naringenin or various pea root exudates . The structural analysis of the LCOs by mass spectrometry revealed that strain A1 synthesizes a family of at least 23 different LCOs . The use of exudates instead of naringenin resulted only in quantitative differences in the ratios of various LCOs produced. Mol Plant Microbe Interact, 1999 Mar, 12(3), 236 - 46 The nolL gene from Rhizobium etli determines nodulation efficiency by mediating the acetylation of the fucosyl residue in the nodulation factor; Corvera A et al.; The nodulation factors (Nod factors) of Rhizobium etli and R . loti carry a 4-O-acetyl-L-fucosyl group at the reducing end . It has been claimed, based on sequence analysis, that NolL from R . loti participates in the 4-O-acetylation of the fucosyl residue of the Nod factors, as an acetyl-transferase (D . B . Scott, C . A . Young, J . M . Collins-Emerson, E . A . Terzaghi, E . S . Rockman, P . A . Lewis, and C . E . Pankhurst . Mol . Plant-Microbe Interact . 9:187-197, 1996) . Further support for this hypothesis was obtained by studying the production of Nod factors in an R . etli nolL::Km mutant . Chromatographic and mass spectrometry analysis of the Nod factors produced by this strain showed that they lack the acetyl-fucosyl substituent, having a fucosyl group instead . Acetyl-fucosylation was restored upon complementation with a wild-type nolL gene . These results indicate that the nolL gene determines 4-O-acetylation of the fucosyl residue in Nod factors . Analysis of the predicted NolL polypeptide suggests a transmembranal location and that it belongs to the family of integral membrane transacylases (J . M . Slauch, A . A . Lee, M . J . Mahan, and J . J . Mekalanos . J . Bacteriol . 178:5904-5909, 1996) . NolL from R . loti was also proposed to function as a transporter; our results show that NolL does not determine a differential secretion of Nod factors from the cell . We also performed plant assays that indicate that acetylation of the fucose conditions efficient nodulation by R . etli of some Phaseolus vulgaris cultivars, as well as of an alternate host (Vigna umbellata). Mol Plant Microbe Interact, 1999 Mar, 12(3), 207 - 17 Mutation in GDP-fucose synthesis genes of Sinorhizobium fredii alters Nod factors and significantly decreases competitiveness to nodulate soybeans; Lamrabet Y et al.; We mutagenized Sinorhizobium fredii HH103-1 with Tn5-B20 and screened about 2,000 colonies for increased beta-galactosidase activity in the presence of the flavonoid naringenin . One mutant, designated SVQ287, produces lipochitooligosaccharide Nod factors (LCOs) that differ from those of the parental strain . The nonreducing N-acetylglucosamine residues of all of the LCOs of mutant SVQ287 lack fucose and 2-O-methylfucose substituents . In addition, SVQ287 synthesizes an LCO with an unusually long, C20:1 fatty acyl side chain . The transposon insertion of mutant SVQ287 lies within a 1.1-kb HindIII fragment . This and an adjacent 2.4-kb HindIII fragment were sequenced . The sequence contains the 3' end of noeK, nodZ, and noeL (the gene interrupted by Tn5-B20), and the 5' end of nolK, all in the same orientation . Although each of these genes has a similarly oriented counterpart on the symbiosis plasmid of the broad-host-range Rhizobium sp . strain NGR234, there are significant differences in the noeK/nodZ intergenic region . Based on amino acid sequence homology, noeL encodes GDP-D-mannose dehydratase, an enzyme involved in the synthesis of GDP-L-fucose, and nolK encodes a NAD-dependent nucleotide sugar epimerase/dehydrogenase . We show that expression of the noeL gene is under the control of NodD1 in S . fredii and is most probably mediated by the nod box that precedes nodZ . Transposon insertion into neoL has two impacts on symbiosis with Williams soybean: nodulation rate is reduced slightly and competitiveness for nodulation is decreased significantly . Mutant SVQ287 retains its ability to form nitrogen-fixing nodules on other legumes, but final nodule number is attenuated on Cajanus cajan. Can J Microbiol, 1998 Nov, 44(11), 1102 - 5 Group-specific differentiation of Rhizobium from clover species by PCR amplification of 23S rDNA sequences; Tesfaye M et al.; Two 20-bp primers that provide group-specific detection of Rhizobium spp . by polymerase chain reaction (PCR) have been used to differentiate strains that belong to different effectiveness groups within the Rhizobium-Trifolium cross-inoculation group . The target for DNA amplification was a 370-bp fragment of the 23S rDNA region . Analysis of additional root-nodule forming, as well as root-associated bacterial species by PCR-primer assay revealed that variability within this 20-bp segment of the 23S rDNA region may be widespread and provide an effective identification tool . Our data suggest that strains of Rhizobium isolated from the perennial clover Trifolium semipilosum may be phylogenetically more closely related to Rhizobium etli. Int J Syst Bacteriol, 1999 Jan, 49 Pt 1, 51 - 65 Diversity of rhizobia associated with Amorpha fruticosa isolated from Chinese soils and description of Mesorhizobium amorphae sp . nov; Wang ET et al.; Fifty-five Chinese isolates from nodules of Amorpha fruticosa were characterized and compared with the type strains of the species and genera of bacteria which form nitrogen-fixing symbioses with leguminous host plants . A polyphasic approach, which included RFLP of PCR-amplified 16S rRNA genes, multilocus enzyme electrophoresis (MLEE), DNA-DNA hybridization, 16S rRNA gene sequencing, electrophoretic plasmid profiles, cross-nodulation and a phenotypic study, was used in the comparative analysis . The isolates originated from several different sites in China and they varied in their phenotypic and genetic characteristics . The majority of the isolates had moderate to slow growth rates, produced acid on YMA and harboured a 930 kb symbiotic plasmid (pSym) . Five different RFLP patterns were identified among the 16S rRNA genes of all the isolates . Isolates grouped by PCR-RFLP of the 16S rRNA genes were also separated into groups by variation in MLEE profiles and by DNA-DNA hybridization . A representative isolate from each of these DNA homology groups had a separate position in a phylogenetic tree as determined from sequencing analysis of the 16S rRNA genes . A new species, Mesorhizobium amorphae, is proposed for the majority of the isolates, which belonged to a moderately slow- to slow-growing, acid-producing group based upon their distinct phylogenetic position, their unique electrophoretic type, their low DNA homology with reference strains representing the species within the genus Mesorhizobium and their distinct phenotypic features . Strain ACCC 19665 was chosen as the type strain for M . amorphae sp . nov. Proc Natl Acad Sci U S A, 1999 Feb 16, 96(4), 1738 - 43 A protein phosphatase 2C gene, LjNPP2C1, from Lotus japonicus induced during root nodule development; Kapranov P et al.; Symbiotic interactions between legumes and compatible strains of rhizobia result in root nodule formation . This new plant organ provides the unique physiological environment required for symbiotic nitrogen fixation by the bacterial endosymbiont and assimilation of this nitrogen by the plant partner . We have isolated two related genes (LjNPP2C1 and LjPP2C2) from the model legume Lotus japonicus that encode protein phosphatase type 2C (PP2C) . Expression of the LjNPP2C1 gene was found to be enhanced specifically in L . japonicus nodules, whereas the LjPP2C2 gene was expressed at a similar level in nodules and roots . A glutathione S-transferase-LjNPP2C1 fusion protein was shown to have Mg2+- or Mn2+-dependent and okadaic acid-insensitive PP2C activity in vitro . A chimeric construct containing the full-length LjNPP2C1 cDNA, under the control of the Saccharomyces cerevisiae alcohol dehydrogenase promoter, was found to be able to complement a yeast PP2C-deficient mutant (pct1Delta) . The transcript level of the LjNPP2C1 gene was found to increase significantly in mature nodules, and its highest expression level occurred after leghemoglobin (lb) gene induction, a molecular marker for late developmental events in nodule organogenesis . Expression of the LjNPP2C1 gene was found to be drastically altered in specific L . japonicus lines carrying monogenic-recessive mutations in symbiosis-related loci, suggesting that the product of the LjNPP2C1 gene may function at both early and late stages of nodule development. J Bacteriol, 1999 Feb, 181(4), 1238 - 48 Identification and characterization of a two-component sensor-kinase and response-regulator system (DcuS-DcuR) controlling gene expression in response to C4-dicarboxylates in Escherichia coli; Golby P et al.; The dcuB gene of Escherichia coli encodes an anaerobic C4-dicarboxylate transporter that is induced anaerobically by FNR, activated by the cyclic AMP receptor protein, and repressed in the presence of nitrate by NarL . In addition, dcuB expression is strongly induced by C4-dicarboxylates, suggesting the presence of a novel C4-dicarboxylate-responsive regulator in E . coli . This paper describes the isolation of a Tn10 mutant in which the 160-fold induction of dcuB expression by C4-dicarboxylates is absent . The corresponding Tn10 mutation resides in the yjdH gene, which is adjacent to the yjdG gene and close to the dcuB gene at approximately 93.5 min in the E . coli chromosome . The yjdHG genes (redesignated dcuSR) appear to constitute an operon encoding a two-component sensor-regulator system (DcuS-DcuR) . A plasmid carrying the dcuSR operon restored the C4-dicarboxylate inducibility of dcuB expression in the dcuS mutant to levels exceeding those of the dcuS+ strain by approximately 1.8-fold . The dcuS mutation affected the expression of other genes with roles in C4-dicarboxylate transport or metabolism . Expression of the fumarate reductase (frdABCD) operon and the aerobic C4-dicarboxylate transporter (dctA) gene were induced 22- and 4-fold, respectively, by the DcuS-DcuR system in the presence of C4-dicarboxylates . Surprisingly, anaerobic fumarate respiratory growth of the dcuS mutant was normal . However, under aerobic conditions with C4-dicarboxylates as sole carbon sources, the mutant exhibited a growth defect resembling that of a dctA mutant . Studies employing a dcuA dcuB dcuC triple mutant unable to transport C4-dicarboxylates anaerobically revealed that C4-dicarboxylate transport is not required for C4-dicarboxylate-responsive gene regulation . This suggests that the DcuS-DcuR system responds to external substrates . Accordingly, topology studies using 14 DcuS-BlaM fusions showed that DcuS contains two putative transmembrane helices flanking a approximately 140-residue N-terminal domain apparently located in the periplasm . This topology strongly suggests that the periplasmic loop of DcuS serves as a C4-dicarboxylate sensor . The cytosolic region of DcuS (residues 203 to 543) contains two domains: a central PAS domain possibly acting as a second sensory domain and a C-terminal transmitter domain . Database searches showed that DcuS and DcuR are closely related to a subgroup of two-component sensor-regulators that includes the citrate-responsive CitA-CitB system of Klebsiella pneumoniae . DcuS is not closely related to the C4-dicarboxylate-sensing DctS or DctB protein of Rhodobacter capsulatus or rhizobial species, respectively . Although all three proteins have similar topologies and functions, and all are members of the two-component sensor-kinase family, their periplasmic domains appear to have evolved independently. Mass Spectrom Rev, 1998 Mar-Apr, 17(2), 75 - 95 Mass spectrometric analysis of lipo-chitin oligosaccharides--signal molecules mediating the host-specific legume-rhizobium symbiosis; van der Drift KM et al.; Lipo-chitin oligosaccharides (LCOs) are novel bacterial glycolipid signal molecules that mediate the species--specific symbiosis between rhizobial bacteria and leguminous plants . Nodulation of the legume roots and nitrogen-fixation in the resulting nodules by Rhizobia is controlled by the bacterial nodulation genes that encode the LCO biosynthetic enzymes . The length of the LCO chitin backbone, the length and degree of unsaturation of the fatty acyl chain attached to it, and the combination of different chemical substituents on the reducing- and nonreducing-terminal residues all contribute to the species--specificity of the signal . LCOs are bioactive in the nanomolar and subnanomolar concentration range and are produced as heterogeneous mixtures, making determination of their structures a difficult task, most successfully approached by the application of modern mass spectrometric methods in combination with specific chemical treatments aimed at identifying specific chemical moieties . This review presents an overview of these methods as they are being used for the structural elucidation of LCOs, and discusses the role of structural diversity in mediating species-specificity. Bioorg Med Chem Lett, 1998 Dec 15, 8(24), 3549 - 54 Organometallic flavonoid derivatives as spectroscopic probes; Anson CE et al.; Derivatives of naringenin have been synthesized with organometalcarbonyl reporting groups for IR spectroscopy attached at C-2, C-3', or C-6, and the products have been tested for the induction of nod gene expression using a Rhizobium leguminosarum strain which contains the Escherichia coli lacZ (beta-galactosidase) gene fused to nodABC . Derivatives with an OMe substituent within the reporting group moiety showed residual gene induction activity. Phytochemistry, 1999 Jan, 50(2), 313 - 6 Purification of leghemoglobin from nodules of Crotalaria infected with Rhizobium; Mendonca EH et al.; The leghemoglobin from nodules of Crotalaria juncea infected with Rhizobium spp . was purified to homogeneity . The protein was purified after precipitation with 40-80% (NH4)2SO4, and chromatography by anionic exchange and gel filtration . The leghemoglobin has a single component and showed an apparent M(r) of ca . 17,300 and 23,700 determined by SDS-PAGE and gel filtration, respectively . The amino acid composition showed that asparagine/aspartic acid, glutamine/glutamic acid, alanine, lysine, serine and leucine were the main amino acids . Iron was detected only in the band corresponding to the purified protein . The N-terminal amino acid sequence for the first 19 residues showed high similarities with several other leghemoglobins from other plants. Annu Rev Genet, 1998, 32, 33 - 57 Regulation of symbiotic root nodule development; Schultze M et al.; Symbiosis between rhizobia and leguminous plants leads to the formation of N2-fixing root nodules . The interaction of rhizobia and plants shows a high degree of host specificity based on the exchange of chemical signals between the symbiotic partners . The plant signals, flavonoids exuded by the roots, activate the expression of nodulation genes, resulting in the production of the rhizobial lipochitooligosaccharide signals (Nod factors) . Nod factors act as morphogens that, under conditions of nitrogen limitation, induce cells within the root cortex to divide and to develop into nodule primordia . This review focuses on how the production of Nod factors is regulated, how these signals are perceived and transduced by the plant root, and the physiological conditions and plant factors that control the early events leading to root nodule development. Appl Environ Microbiol, 1999 Feb, 65(2), 374 - 80 Analysis of nifH gene pool complexity in soil and litter at a Douglas fir forest site in the Oregon cascade mountain range; Widmer F et al.; Nitrogen-fixing microbial populations in a Douglas fir forest on the western slope of the Oregon Cascade Mountain Range were analyzed . The complexity of the nifH gene pool (nifH is the marker gene which encodes nitrogenase reductase) was assessed by performing nested PCR with bulk DNA extracted from plant litter and soil . The restriction fragment length polymorphisms (RFLPs) of PCR products obtained from litter were reproducibly different than the RFLPs of PCR products obtained from the underlying soil . The characteristic differences were found during the entire sampling period between May and September . RFLP analyses of cloned nifH PCR products also revealed characteristic patterns for each sample type . Among 42 nifH clones obtained from a forest litter library nine different RFLP patterns were found, and among 64 nifH clones obtained from forest soil libraries 13 different patterns were found . Only two of the patterns were found in both the litter and the soil, indicating that there were major differences between the nitrogen-fixing microbial populations . A sequence analysis of clones representing the 20 distinct patterns revealed that 19 of the patterns had a proteobacterial origin . All of the nifH sequences obtained from the Douglas fir forest litter localized in a distinct phylogenetic cluster characterized by the nifH sequences of members of the genera Rhizobium, Sinorhizobium, and Azospirillum . The nifH sequences obtained from soil were found in two additional clusters, one characterized by sequences of members of the genera Bradyrhizobium, Azorhizobium, Herbaspirillum, and Thiobacillus and the other, represented by a single nifH clone, located between the gram-positive bacteria and the cyanobacteria . Our results revealed the distinctness of the nitrogen-fixing microbial populations in litter and soil in a Douglas fir forest; the differences may be related to special requirements for degradation and mineralization processes in the plant litter. J Bacteriol, 1999 Feb, 181(3), 981 - 90 Cell density-dependent starvation survival of Rhizobium leguminosarum bv . phaseoli: identification of the role of an N-acyl homoserine lactone in adaptation to stationary-phase survival; Thorne SH et al.; The cell density dependence of stationary-phase survival of Rhizobium leguminosarum has been investigated . Following starvation by exhaustion of carbon or nitrogen, but not of phosphorus, the survival of cultures was dependent on the cell density at entry into stationary phase . High-density cultures survived with little or no loss of viability over a 20-day period in stationary phase . In contrast, low-density cultures lost viability rapidly but consisted of a heterogeneous population, a small fraction of which successfully adapted and eventually formed a stable, surviving population . The threshold density above which the cultures survived successfully in stationary phase was dependent on the growth conditions and the strain used . We took advantage of the fact that R . leguminosarum survives poorly following starvation by resuspension in carbon-free medium to demonstrate that cell density-dependent survival was mediated by a component accumulating in the growth medium . The effects of this medium component on survival in resuspension assays could be mimicked by an N-acyl homoserine lactone, N-(3R-hydroxy-7-cis-tetradecanoyl)-L-homoserine lactone, previously demonstrated to have a role in controlling cell density-dependent phenomena in R . leguminosarum . The Sym plasmids pRP2JI and pRL1JI were found to be essential for the production of the extracellular factor, which could also be made in Escherichia coli carrying the cosmid clone pIJ1086 containing a specific region of pRL1JI. J Bacteriol, 1999 Feb, 181(3), 849 - 57 Poly-3-hydroxybutyrate degradation in Rhizobium (Sinorhizobium) meliloti: isolation and characterization of a gene encoding 3-hydroxybutyrate dehydrogenase; Aneja P et al.; We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (bdhA) from Rhizobium (Sinorhizobium) meliloti . The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07) . The R . meliloti Bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily . bdhA is the first gene transcribed in an operon that also includes xdhA, encoding xanthine oxidase/dehydrogenase . Transcriptional start site analysis by primer extension identified two transcription starts . S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon . Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences . Although a sigma54 consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a rpoN mutant of R . meliloti was able to utilize 3-hydroxybutyrate as a sole carbon source . The R . meliloti bdhA gene is able to confer upon Escherichia coli the ability to utilize 3-hydroxybutyrate as a sole carbon source . An R . meliloti bdhA mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects . Studies with a strain carrying a lacZ transcriptional fusion to bdhA demonstrated that gene expression is growth phase associated. FEMS Microbiol Lett, 1999 Jan 1, 170(1), 111 - 7 Characterisation of rhizobia from African acacias and other tropical woody legumes using Biolog and partial 16S rRNA sequencing; McInroy SG et al.; A Biolog (sole carbon source utilisation) user database of tropical and temperature rhizobial strains was created and used in conjunction with the partial 16S rRNA sequencing method to characterise 12 rhizobial isolates from African acacias and other tropical woody legumes . There was close agreement between the two methods but also some significant discrepancies . A high degree of diversity was shown in the relatively small sample of isolates, with 4 out of 5 of the currently proposed rhizobial genera represented . This is the first time Biolog has shown congruence with genotypic fingerprinting using a wide selection of rhizobial reference and test strains. Arch Microbiol, 1999 Jan, 171(2), 131 - 4 Accumulation of ppGpp in symbiotic and free-living nitrogen-fixing bacteria following amino acid starvation Howorth SM, England RR. Following amino acid or ammonium starvation, ppGpp is accumulated by Rhizobium meliloti strain 1021 but not by R . meliloti strain 41 or Rhizobium tropici . Azorhizobium caulinodans ORS571 produced ppGpp following amino acid deprivation; however, the free-living nitrogen-fixing bacteria Azotobacter vinelandii and Azomonas agilis did not produce ppGpp . Western blot analysis using anti-RelA antibody demonstrated that R . meliloti strain 1021, Azotobacter vinelandii and Azorhizobium caulinodans cross-reacted under conditions that detected RelA in Escherichia coli CF1648 . Cross-reaction was not observed in R . meliloti strain 41, R . tropici, or Azomonas agilis . All strains that accumulated ppGpp also produced high intracellular levels of ATP. Biochemistry, 1999 Jan 5, 38(1), 347 - 53 Purification and functional reconstitution of soybean nodulin 26 . An aquaporin with water and glycerol transport properties; Dean RM et al.; Infection of soybean roots by nitrogen-fixing Bradyrhizobium japonicum leads to expression of plant nodule-specific genes known as nodulins . Nodulin 26, a member of the major intrinsic protein/aquaporin (AQP) channel family, is a major component of the soybean symbiosome membrane (SM) that encloses the rhizobium bacteroid . To investigate the water and solute transport characteristics of nodulin 26, we purified the protein from SMs and reconstituted it into carboxyfluorescein-loaded liposomes for transport studies using stopped-flow spectrofluorimetry . Liposomes containing nodulin 26 exhibited a high osmotic permeability (Pf = 0 . 012 +/- 0.0013 cm/s), a value fivefold higher than that obtained with control liposomes . Water flux through nodulin 26 showed a low activation energy (Ea) (4.07 kcal/mol) and was reduced 70% upon addition of 1 mM HgCl2 . Reconstituted nodulin 26 exhibited a single-channel conductance of 3.8 +/- 2.5 x 10(-)15 cm3/s (n = 3), a value that is lower than other characterized AQPs . Nodulin 26 proteoliposomes also facilitate glycerol transport, showing a 43-fold higher rate of glycerol flux than control liposomes . This observation was supported by expression experiments in Xenopus oocytes that showed that nodulin 26 facilitated glycerol flux in a manner indistinguishable from the Escherichia coli GlpF glycerol facilitator . Consistent with the results of water transport, glycerol transport was inhibited by HgCl2 and showed a low Ea (4.43 kcal/mol) . These results indicate that nodulin 26 is a multifunctional AQP that confers water and glycerol transport to the SM, and likely plays a role in osmoregulation during legume/rhizobia symbioses. Adv Microb Physiol, 1998, 40, 191 - 231 Genes involved in the formation and assembly of rhizobial cytochromes and their role in symbiotic nitrogen fixation; Delgado MJ et al.; Rhizobia fix nitrogen in a symbiotic association with leguminous plants and this occurs in nodules . A low-oxygen environment is needed for nitrogen fixation, which paradoxically has a requirement for rapid respiration to produce ATP . These conflicting demands are met by control of oxygen flux and production of leghaemoglobin (an oxygen carrier) by the plant, coupled with the expression of a high-affinity oxidase by the nodule bacteria (bacteroids) . Many of the bacterial genes encoding cytochrome synthesis and assembly have been identified in a variety of rhizobial strains . Nitrogen-fixing bacteroids use a cytochrome cbb3-type oxidase encoded by the fixNOQP operon; electron transfer to this high-affinity oxidase is via the cytochrome bc1 complex . During free-living growth, electron transport from the cytochrome bc1 complex to cytochrome aa3 occurs via a transmembrane cytochrome c (CycM) . In some rhizobia (such as Bradyrhizobium japonicum) there is a second cytochrome oxidase that also requires electron transport via the cytochrome bc1 complex . In parallel with these cytochrome c oxidases there are quinol oxidases that are expressed during free-living growth . A cytochrome bb3 quinol oxidase is thought to be present in B . japonicum; in Rhizobium leguminosarum, Rhizobium etli and Azorhizobium caulinodans cytochrome d-type oxidases have been identified . Spectroscopic data suggest the presence of a cytochrome o-type oxidase in several rhizobia, although the absence of haem O in B . japonicum may indicate that the absorption attributed to cytochrome o could be due to a high-spin cytochrome b in a cytochrome bb3-type oxidase . In some rhizobia, mutation of genes involved in cytochrome c assembly does not strongly affect growth, presumably because the bacteria utilize the cytochrome c-independent quinol oxidases . In this review, we outline the work on various rhizobial mutants affected in different components of the electron transport pathways, and the effects of these mutations on symbiotic nitrogen fixation and free-living growth. Mol Plant Microbe Interact, 1999 Jan, 12(1), 35 - 44 Regulation of expression of avirulence gene avrRxv and identification of a family of host interaction factors by sequence analysis of avrBsT; Ciesiolka LD et al.; Resistance in tomato line Hawaii 7998 as well as in several nonhost plants to Xanthomonas campestris pv . vesicatoria tomato strain (XcvT) is mediated in part by the avirulence gene avrRxv . Analysis of growth of wild-type and avrRxv deletion strains indicates that avrRxv plays a crucial role in the ability of XcvT 92-14 to induce resistance on Hawaii 7998 . We used avrRxv reporter gene fusions and Northern (RNA) blot analysis to test several growth environments for inductive potential . We found that avrRxv is constitutively expressed at high levels and that growth in planta, in tobacco conditioned medium, and in hrp-inductive medium XVM2 did not affect the high levels of expression . In addition, hrp structural and regulatory mutant backgrounds had no effect . We mutated the bipartite plant inducible promoter (PIP)-box sequence and found that avrRxv activity appears to be independent of an intact PIP-box element . We present the sequence of the avrRxv homologue called avrBsT and align the six AvrRxv host interaction factor family members including mammalian pathogen virulence factors YopJ and YopP from Yersinia spp . and AvrA from Salmonella typhimurium, and open reading frame Y4LO with unknown function from the symbiont Rhizobium sp. Mol Plant Microbe Interact, 1999 Jan, 12(1), 24 - 34 The Rhizobium etli metZ gene is essential for methionine biosynthesis and nodulation of Phaseolus vulgaris; Tate R et al.; A mutant strain (CTNUX23) of Rhizobium etli carrying Tn5 unable to grow with sulfate as the sole sulfur source was isolated and characterized . Sequence analysis showed that Tn5 is inserted into a metZ (O-succinylhomoserine sulfhydrylase)-homologous gene . The CTNUX23 mutant strain had a growth dependency for methionine, although cystathionine or homocysteine, but not homoserine or O-succinylhomoserine, allowed growth of the mutant . RNase protection assays showed that the metZ-like gene had a basal level of expression in methionine- or cysteine-grown cells, which was induced when sulfate or thiosulfate was used . The metZ gene was cloned from the parent wild-type strain, CE3, and the resulting plasmid pAR204 relieved, after transformation, the methionine auxotrophy of both strains CTNUX23 of R . etli and PAO503(metZ) of Pseudomonas aeruginosa . Unlike strain CE3 or CTNUX23 (pAR204), strain CTNUX23 showed undetectable levels of O-succinylhomoserine sulfhydrylase activity . Strain CTNUX23 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) or to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris, unless methionine was added to the growth medium . These data and our previous results support the notion that cysteine or glutathione, but not methionine, is supplied by the root cells to bacteria growing inside the plant. Microbiology, 1998 Dec, 144 ( Pt 12), 3335 - 42 The transcriptional regulator gene phrR in Sinorhizobium meliloti WSM419 is regulated by low pH and other stresses; Reeve WG et al.; The phrR gene in Sinorhizobium meliloti (previously known as Rhizobium meliloti) WSM419, directly downstream from actA, is induced by low pH or certain stresses (e.g . high concentrations of Zn2+, Cu2+, H2O2 or ethanol), but not in stationary phase or by other stresses (e.g . phosphate limitation, elevated temperature, high concentrations of sucrose or iron) . A DNA fragment containing the wild-type phrR gene could not be cloned and inverse PCR was therefore used to amplify a 3.5 kb BamHI fragment containing phrR from the mutant S . meliloti TG2-6 (actA::Tn5) . DNA fragments from a BamHI/SalI digest of the amplified product were cloned into pUK21 and sequenced . The phrR open reading frame contiguous to actA appears to code for a 15.2 kDa protein showing significant identity with the proteins encoded by y4wC and y4aM in Rhizobium sp . NGR234 . All three proteins resemble transcriptional regulators in containing a DNA-binding helix-turn-helix motif similar to that reported for URF4 in Rhodospirillum rubrum and repressors in coliphage. J Bacteriol, 1999 Jan, 181(2), 389 - 95 Rhizobium (Sinorhizobium) meliloti phn genes: characterization and identification of their protein products; Parker GF et al.; In Escherichia coli, the phn operon encodes proteins responsible for the uptake and breakdown of phosphonates . The C-P (carbon-phosphorus) lyase enzyme encoded by this operon which catalyzes the cleavage of C-P bonds in phosphonates has been recalcitrant to biochemical characterization . To advance the understanding of this enzyme, we have cloned DNA from Rhizobium (Sinorhizobium) meliloti that contains homologues of the E . coli phnG, -H, -I, -J, and -K genes . We demonstrated by insertional mutagenesis that the operon from which this DNA is derived encodes the R . meliloti C-P lyase . Furthermore, the phenotype of this phn mutant shows that the C-P lyase has a broad substrate specificity and that the organism has another enzyme that degrades aminoethylphosphonate . A comparison of the R . meliloti and E . coli phn genes and their predicted products gave new information about C-P lyase . The putative R . meliloti PhnG, PhnH, and PhnK proteins were overexpressed and used to make polyclonal antibodies . Proteins of the correct molecular weight that react with these antibodies are expressed by R . meliloti grown with phosphonates as sole phosphorus sources . This is the first in vivo demonstration of the existence of these hitherto hypothetical Phn proteins. Plant Mol Biol, 1998 Dec, 38(6), 1161 - 8 Jasmonic acid stimulates the expression of nod genes in Rhizobium; Rosas S et al.; Jasmonates and salicylic acid are considered to be signal molecules that induce a variety of plant genes involved in wound or defence response, as well as affecting nos promoter activity . In this paper we examined whether these chemicals could also affect nod genes from isogenic rhizobia strains . Isogenic strains contain the Rhizobium leguminosarum nodA promoter fused to the lacZ gene of Escherichia coli and differ only in the source of the regulatory nodD gene . Naringenin, jasmonic acid and methyl jasmonate induced expression of nod genes in strain RBL1284 and salicylic acid showed no activity alone or when used in combination with other compounds; addition of naringenin + jasmonic acid produced a synergistic effect . Results obtained with strain RBL5284 were similar to those for RBL1284 albeit the combination of naringenin with the other compounds markedly inhibited nod gene expression . Whereas RBL5283 responded to naringenin with a strong induction, jasmonic acid, methyl jasmonate or salicylic acid showed no significant responses . The inhibitory effect of salicylic acid on nod gene expression indicates that the induction mechanism of jasmonic acid, methyl jasmonate, N-propyldihydrojasmonate and naringenin is probably different from that of salicylic acid. FEMS Microbiol Lett, 1998 Dec 15, 169(2), 295 - 302 Rhizobium leguminosarum bv . viciae hypA gene is specifically expressed in pea (Pisum sativum) bacteroids and required for hydrogenase activity and processing; Hernando Y et al.; Rhizobium leguminosarum bv . viciae strain UPM791 induces in symbiosis with peas the synthesis of a nickel-containing hydrogenase which recycles the hydrogen evolved by nitrogenase . The genes required for synthesis of this hydrogenase, hupSLCDEFGHIJKhypABFCDEX, are clustered in the symbiotic plasmid . Analysis of a hypA-deficient mutant showed that HypA is essential for symbiotic hydrogenase activity and required for correct processing of the hydrogenase large subunit . Unlike other microoxically induced hyp genes, the hypA gene was only expressed in pea bacteroids from its own promoter . The hypA mRNA 5' end was mapped 109 bp upstream of the translational start codon . This distinct pattern of expression suggests a different role for HypA and the remaining Hyp proteins in hydrogenase synthesis. FEMS Microbiol Lett, 1998 Dec 15, 169(2), 227 - 33 Feasibility of using prokaryote biosensors to assess acute toxicity of polycyclic aromatic hydrocarbons; Reid BJ et al.; The aim of this study was to assess the acute toxicity of polycyclic aromatic hydrocarbons using lux-marked bacterial biosensors . Standard solutions of phenanthrene, pyrene and benzo{a}pyrene were produced using 50 mM hydroxpropyl-beta-cyclodextrin solution which contained each respective polycyclic aromatic hydrocarbon at 6.25 times the aqueous solubility limit of the compound . The polycyclic aromatic hydrocarbon solutions were incubated with each of the biosensors for 280 min and the bioluminescence monitored every 20 min . Over the incubation time period, there was no significant decrease in bioluminescence in any of the biosensors tested with the exception of Rhizobium leguminosarum biovar trifolii TA1 luxAB . In this series of incubations, there was a dramatic increase in bioluminescence in the presence of phenanthrene (2.5 times) and benzo{a}pyrene (3 times) above that of the background control (biosensor without polycyclic aromatic hydrocarbon) after 20 min . Over the next 3 h, bioluminescence decreased to that of the control . An ATP assay was carried out on the biosensors to assess if uncoupling of the oxidative phosphorylation mechanisms in the respiratory chain of the cells had occurred . However, it was found that the polycyclic aromatic hydrocarbons had no effect on the organisms indicating that there was no uncoupling . Additionally, mineralisation studies using 14C-labelled polycyclic aromatic hydrocarbons showed that the biosensors could not mineralise the compounds . This study has shown that the three polycyclic aromatic hydrocarbons tested are not acutely toxic to the prokaryotic biosensors tested, although acute toxicity has been shown in other bioassays . These results question the rationale for using prokaryote biosensors to assess the toxicity of hydrophobic chemicals, such as polycyclic aromatic hydrocarbons. J Bacteriol, 1999 Jan, 181(1), 83 - 90 Multiple small heat shock proteins in rhizobia; Munchbach M et al.; Seven genes coding for small heat shock proteins (sHsps) in Bradyrhizobium japonicum have been identified . They are organized in five operons that are coordinately regulated by ROSE, a negatively cis-acting DNA element . The deduced sHsps can be divided into two separate classes: class A, consisting of proteins that show similarity to Escherichia coli IbpA and IbpB, and class B, whose members display significant similarity to other sHsps from prokaryotes and eukaryotes . Two-dimensional gel electrophoresis and Edman sequencing revealed the presence of at least 12 sHsps in B . japonicum, indicating a remarkable abundance of sHsps in this organism . Three additional members of class A and two potentially novel heat shock proteins were identified on the basis of their amino termini . The presence of multiple sHsps was also demonstrated for a variety of Rhizobium and Bradyrhizobium species by immunoblot analysis and two-dimensional gel electrophoresis . An extensive database survey revealed that, in contrast to the rhizobia, other bacteria contain maximally two sHsps whereas many plants have been reported to possess a sHsp superfamily. Gene, 1998 Nov 26, 223(1-2), 283 - 90 Mapping of 41 chemotaxis, flagellar and motility genes to a single region of the Sinorhizobium meliloti chromosome; Sourjik V et al.; Three previously identified gene clusters that contain chemotaxis (che), flagellar (fla) and motility (mot) genes of Sinorhizobium meliloti (formerly Rhizobium meliloti) were mapped to a contiguous 45-kb region of the S . meliloti RU11/001 genome by pulsed-field gel electrophoresis (PFGE) in combination with Southern hybridization . The entire region was cloned and sequenced . The map combines 32 che, fla (flg, flh, fli) and mot genes and nine new open reading frames that probably encode taxis-related functions as well . It is concluded that between 80 and 90% of the genes responsible for chemotaxis and motility are located in a single region of the S . meliloti chromosome near the his-39 marker. Appl Environ Microbiol, 1998 Dec, 64(12), 5020 - 2 Effects of bacterial antibiotic production on rh