Mol Microbiol, 1993 Jul, 9(1), 145 - 54 Identification of nodSUIJ genes in Nod locus 1 of Azorhizobium caulinodans: evidence that nodS encodes a methyltransferase involved in Nod factor modification; Geelen D et al.; The Azorhizobium caulinodans strain ORS571 nodulation genes nodSUIJ were located downstream from nodABC . Complementation data and transcriptional analysis suggest that nodABCSUIJ form a single operon . Mutants with Tn5 insertions in the genes nodS, nodU, and nodJ were delayed in nodulation of Sesbania rostrata roots and stems . The NodS amino acid sequences of ORS571, Bradyrhizobium japonicum, and Rhizobium sp . strain NGR234, contain a consensus with similarity to S-adenosylmethionine (SAM)-utilizing methyltransferases . A naringenin-inducible nodS-dependent protein of approximately 25 kDa could be cross-linked to radiolabelled SAM . By applying L-{methyl-3H}-methionine in vivo, Nod factors of ORS571, known to be N-methylated, could be labelled in wild type and nodU mutants but not in nodS mutants . Therefore, we propose that NodS is a SAM-utilizing methyltransferase involved in Nod factor synthesis. Can J Microbiol, 1993 Jul, 39(7), 665 - 73 Phylogenetic grouping and identification of Rhizobium isolates on the basis of random amplified polymorphic DNA profiles; Dooley JJ et al.; Through the use of a single, random 15mer as a primer, between 1 and 12 DNA amplification products were obtained per strain from a selection of 84 Rhizobium and Bradyrhizobium isolates . A principal-coordinate analysis was used to analyse the resulting amplified DNA profiles and it was possible to assign isolates to specific groupings . Within the species Rhizobium leguminosarum, the biovar phaseoli formed a distinct group from the other biovars of the species, viciae and trifolii, which grouped together . Isolates of Rhizobium meliloti and Bradyrhizobium species formed their own clear, specific groups . Although it was possible to identify individual isolates on the basis of differences in their amplified DNA profiles, there was evidence that some amplified segments were conserved among individuals at the biovar and species levels. J Bacteriol, 1993 Jul, 175(13), 4186 - 96 Isolation and characterization of a gene coding for a novel aspartate aminotransferase from Rhizobium meliloti; Alfano JR et al.; Aspartate aminotransferase (AAT) is an important enzyme in aspartate catabolism and biosynthesis and, by converting tricarboxylic acid cycle intermediates to amino acids, AAT is also significant in linking carbon metabolism with nitrogen metabolism . To examine the role of AAT in symbiotic nitrogen fixation further, plasmids encoding three different aminotransferases from Rhizobium meliloti 104A14 were isolated by complementation of an Escherichia coli auxotroph that lacks three aminotransferases . pJA10 contained a gene, aatB, that coded for a previously undescribed AAT, AatB . pJA30 encoded an aromatic aminotransferase, TatA, that had significant AAT activity, and pJA20 encoded a branched-chain aminotransferase designated BatA . Genes for the latter two enzymes, tatA and batA, were previously isolated from R . meliloti . aatB is distinct from but hybridizes to aatA, which codes for AatA, a protein required for symbiotic nitrogen fixation . The DNA sequence of aatB contained an open reading frame that could encode a protein 410 amino acids long and with a monomer molecular mass of 45,100 Da . The amino acid sequence of aatB is unusual, and AatB appears to be a member of a newly described class of AATs . AatB expressed in E . coli has a Km for aspartate of 5.3 mM and a Km for 2-oxoglutarate of 0.87 mM . Its pH optimum is between 8.0 and 8.5 . Mutations were constructed in aatB and tatA and transferred to the genome of R . meliloti 104A14 . Both mutants were prototrophs and were able to carry out symbiotic nitrogen fixation. Toxicol Lett, 1993 Jun, 68(3), 267 - 73 Effects of 2,4-dichlorophenoxyacetic acid on Rhizobium sp . growth and characterization of its transport; Arias RN et al.; The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) produced a deleterious effect on the growth of Rhizobium sp . M4, that was reversed by transferring the cells to a fresh control medium . The uptake of 2,4-D by Rhizobium sp . was not affected by chloramphenicol, indicating that it is constitutive rather than inducible . The mechanism of transport also appears to be energy independent, since uptake was not inhibited by azide or arsenate. J Bacteriol, 1993 Jun, 175(12), 3693 - 702 DNA sequence and mutational analysis of genes involved in the production and resistance of the antibiotic peptide trifolitoxin; Breil BT et al.; The 7.1-kb fragment of Rhizobium leguminosarum bv . trifolii T24 DNA which confers trifolitoxin production and resistance to nonproducing, sensitive Rhizobium strains (E . W . Triplett, M . J . Schink, and K . L . Noeldner, Mol . Plant-Microbe Interact . 2:202-208, 1989) was subcloned, sequenced, and mutagenized with a transcriptional fusion cassette . The sequence of this fragment revealed seven complete open reading frames, tfxABCDEFG, all transcribed in the same direction . TfxA has an 11-amino-acid carboxy terminus identical to the known amino acid sequence of the trifolitoxin backbone, DIGGSRXGCVA, where X is an UV-absorbing chromophore . This is evidence that trifolitoxin is synthesized ribosomally as a prepeptide that is posttranslationally modified to yield the active peptide . TfxB shows 27.6% identity with McbC, a protein required for the production of the ribosomally synthesized antibiotic microcin B17 . Tn3GUS transcriptional fusion insertions in tfxA, tfxB, tfxD, or tfxF caused a nonproducing, trifolitoxin-resistant phenotype and confirmed the direction of transcription of these frames . No insertion mutations were found in tfxE or tfxG . Sequence analysis along with insertion and deletion mutation analysis suggest that (i) trifolitoxin is synthesized ribosomally from tfxA; (ii) tfxA, tfxE, and tfxG have their own promoters; (iii) TfxG is required for immunity; (iv) TfxB, TfxD, and TfxF are required for trifolitoxin production; and (v) the UV-absorbing chromophore is derived from glutamine. Proc Natl Acad Sci U S A, 1993 Jun 1, 90(11), 4932 - 6 Gene amplification in Rhizobium: identification and in vivo cloning of discrete amplifiable DNA regions (amplicons) from Rhizobium leguminosarum biovar phaseoli; Flores M et al.; A genetic element that allows the positive selection of different genomic rearrangements was used to analyze DNA amplification in Rhizobium leguminosarum biovar phaseoli . Discrete amplifiable DNA regions (amplicons) were detected in different regions of the genome of the model strain CFN42, including the chromosome and several large plasmids . Amplicons were mobilized into Escherichia coli using a genetic approach that involves the introduction of an origin of replication active in E . coli and an origin of conjugal transfer into the amplifiable DNA regions of the Rhizobium genome . The strategy can be a valuable tool for studies on genome organization and function . We propose that amplicons define a structural characteristic of the genome that may play an important biological role. J Bacteriol, 1993 Jun, 175(11), 3653 - 5 The acetyl substituent of succinoglycan is not necessary for alfalfa nodule invasion by Rhizobium meliloti Rm1021; Reuber TL et al.; Rhizobium meliloti Rm1021 requires a Calcofluor-binding exopolysaccharide, termed succinoglycan or EPS I, to invade alfalfa nodules . We have determined that a strain carrying a mutation in the exoZ locus produces succinoglycan that lacks the acetyl substituent . The exoZ mutant nodules alfalfa normally. J Bacteriol, 1993 Jun, 175(11), 3570 - 80 Rhizobium fredii and Rhizobium meliloti produce 3-deoxy-D-manno-2-octulosonic acid-containing polysaccharides that are structurally analogous to group II K antigens (capsular polysaccharides) found in Escherichia coli; Reuhs BL et al.; The polysaccharide components from cultured cells of Rhizobium fredii USDA205 and Rhizobium meliloti AK631 were extracted with hot phenol-water and separated by repetitive gel filtration chromatography . Polyacrylamide gel electrophoresis, nuclear magnetic resonance spectrometry, and gas chromatography analyses showed that both of these bacterial species produce unique polysaccharides that contain a high proportion of 3-deoxy-D-manno-2-octulosonic acid (Kdo) . These polysaccharides, which constituted a major portion of the extracted carbohydrate, are not excreted into the growth media (i.e., they are not extracellular polysaccharides) and are structurally distinct from the lipopolysaccharides . The primary structure of the preponderant polysaccharide from R . fredii USDA205 was determined by high-performance anion-exchange liquid chromatography, nuclear magnetic resonance spectrometry, fast atom bombardment-mass spectrometry, and gas chromatography-mass spectrometry; it consists of repeating units of {-->3)-alpha-D-Galp-(1-->5)-beta-D-Kdop-(2-->}n . This molecule is structurally analogous to the constituents of one subgroup of K antigens (capsular polysaccharides) produced by Escherichia coli . Polysaccharides of this type have not previously been identified as components of rhizobial cells . The Kdo-containing polysaccharide from R . meliloti, which has not been completely characterized, appears to be structurally related to that of R . fredii. Mol Microbiol, 1993 Jun, 8(6), 1083 - 94 The presence of a novel type of surface polysaccharide in Rhizobium meliloti requires a new fatty acid synthase-like gene cluster involved in symbiotic nodule development; Petrovics G et al.; Bacterial exopolysaccharide (EPS) and lipopolysaccharide (LPS) molecules have been shown to play important roles in plant-bacterium interactions . Here we have demonstrated that the fix-23 loci, which compensate for exo mutations during symbiotic nodule development, are involved in the production of a novel polysaccharide that is rich in 3-deoxy-D-manno-2-octulosonic acid (Kdo) but is not the classical LPS . This molecule is likely to be a surface antigen since antiserum to whole Rhizobium meliloti cells reacts strongly with it, and since mutations in fix-23 result in an inability to produce this polysaccharide and to bind bacteriophage 16-3 . It is likely that this Kdo-rich polysaccharide is analogous to certain Escherichia coli K-antigens which are anchored to the membrane via a phospholipid moiety . DNA sequence analysis of one gene cluster of this region revealed that the predicted protein products of six genes exhibit a high degree of homology and similar organization to those of the rat fatty acid synthase multifunctional enzyme domains. Genetics, 1993 Jun, 134(2), 435 - 44 Regulation of syrM and nodD3 in Rhizobium meliloti; Swanson JA et al.; The early steps of symbiotic nodule formation by Rhizobium on plants require coordinate expression of several nod gene operons, which is accomplished by the activating protein NodD . Three different NodD proteins are encoded by Sym plasmid genes in Rhizobium meliloti, the alfalfa symbiont . NodD1 and NodD2 activate nod operons when Rhizobium is exposed to host plant inducers . The third, NodD3, is an inducer-independent activator of nod operons . We previously observed that nodD3 carried on a multicopy plasmid required another closely linked gene, syrM, for constitutive nod operon expression . Here, we show that syrM activates expression of the nodD3 gene, and that nodD3 activates expression of syrM . The two genes constitute a self-amplifying positive regulatory circuit in both cultured Rhizobium and cells within the symbiotic nodule . We find little effect of plant inducers on the circuit or on expression of nodD3 carried on pSyma . This regulatory circuit may be important for regulation of nod genes within the developing nodule. C R Acad Sci III, 1993 Jun, 316(6), 553 - 9 Rhizobium meliloti adenylate cyclase: probing of a NTP-binding site common to cyclases and cation transporters; Beuve A et al.; Alignments between amino acid sequences of eukaryotic adenylate (ACase) and guanylate (GCase) cyclases and the prokaryotic Rhizobium meliloti ACase revealed four conserved regions . Two were the target of site-directed mutagenesis . A positive charge at position 44 converted the enzyme to GCase, a negative charge at this position had no effect . A second modification indicated that residues 107 and 124 contribute to the nucleoside triphosphate binding pocket's conformation . This latter region was used to scan protein sequences data banks . A similar region was detected in the family of E1-E2 type ATPases . Topographical resemblance between these ATPases, eukaryotic ACases and several transporters suggest that they evolved from a common ancestor. Genetics, 1993 May, 134(1), 341 - 50 Toward an integrated linkage map of common bean . III . Mapping genetic factors controlling host-bacteria interactions; Nodari RO et al.; Restriction fragment length polymorphism (RFLP)-based genetic linkage maps allow us to dissect the genetic control of quantitative traits (QT) by locating individual quantitative trait loci (QTLs) on the linkage map and determining their type of gene action and the magnitude of their contribution to the phenotype of the QT . We have performed such an analysis for two traits in common bean, involving interactions between the plant host and bacteria, namely Rhizobium nodule number (NN) and resistance to common bacterial blight (CBB) caused by Xanthomonas campestris pv . phaseoli . Analyses were conducted in the progeny of a cross between BAT93 (fewer nodules; moderately resistant to CBB) and Jalo EEP558 (more nodules; susceptible to CBB) . An RFLP-based linkage map for common bean based on 152 markers had previously been derived in the F2 of this cross . Seventy F2-derived F3 families were inoculated in separate greenhouse experiments with Rhizobium tropici strain UMR1899 or X . c . pv . phaseoli isolate isolate W18 . Regression and interval mapping analyses were used to identify genomic regions involved in the genetic control of these traits . These two methods identified the same genomic regions for each trait, with a few exceptions . For each trait, at least four putative QTLs were identified, which accounted for approximately 50% and 75% of the phenotypic variation in NN and CBB resistance, respectively . A chromosome region on linkage group D7 carried factor(s) influencing both traits . In all other cases, the putative QTLs affecting NN and CBB were located in different linkage groups or in the same linkage group, but far apart (more than 50 cM) . Both BAT93 and Jalo EEP558 contributed alleles associated with higher NN, whereas CBB resistance was always associated with BAT93 alleles . Further investigations are needed to determine whether the QTLs for NN and CBB on linkage group D7 represent linked genes or the same gene with pleiotropic effects . Identification of the QTLs raises the possibility of initiating map-based cloning and marker-assisted selection for these traits. J Bacteriol, 1993 May, 175(10), 2826 - 32 Characterization and symbiotic importance of acidic extracellular polysaccharides of Rhizobium sp . strain GRH2 isolated from acacia nodules; Lopez-Lara IM et al.; Rhizobium sp . wild-type strain GRH2 was originally isolated from root nodules of the leguminous tree Acacia cyanophylla and has a broad host range which includes herbaceous legumes, e.g., Trifolium spp . We examined the extracellular exopolysaccharides (EPSs) produced by strain GRH2 and found three independent glycosidic structures: a high-molecular-weight acidic heteropolysaccharide which is very similar to the acidic EPS produced by Rhizobium leguminosarum biovar trifolii ANU843, a low-molecular-weight native heterooligosaccharide resembling a dimer of the repeat unit of the high-molecular-weight EPS, and low-molecular-weight neutral beta (1,2)-glucans . A Tn5 insertion mutant derivative of GRH2 (exo-57) that fails to form acidic heteropolysaccharides was obtained . This Exo- mutant formed nitrogen-fixing nodules on Acacia plants but infected a smaller proportion of cells in the central zone of the nodules than did wild-type GRH2 . In addition, the exo-57 mutant failed to nodulate several herbaceous legume hosts that are nodulated by wild-type strain GRH2. J Bacteriol, 1993 May, 175(9), 2674 - 81 Negative regulation of sigma 54-dependent dctA expression by the transcriptional activator DctD; Labes M et al.; In Rhizobium meliloti, the presence of the C4-dicarboxylate transport protein DctA is required for symbiotic N2 fixation in alfalfa root nodules . Expression of dctA is inducible and is mediated by a sensor and activator gene pair encoded by dctB and dctD . In the presence of C4-dicarboxylates, the DCTB sensor protein is believed to phosphorylate and activate DCTD, which in turn activates transcription at the sigma 54-dependent dctA promoter . Here, we present evidence that in addition to activating dctA transcription, DCTD can also repress expression of dctA . By employing an ntrC allele, ntrC283, whose product appears to activate dctA transcription independently of DCTD, we found that while ntrC283 leads to constitutive dctA expression in the absence of dctB and dctD, in a dctB+ dctD+ ntrC283 background high-level expression of dctA occurred in succinate but not in glucose-grown cells . This result suggested that in uninduced cells, inactive DCTD binds to the dctA promoter and prevents its activation by NTRC283 . Consistent with the latter interpretation was the observation that overexpression of DCTD from a plasmid promoter prevents dctA expression and results in a Dct- phenotype . Moreover the Dct- phenotype resulting from the overexpression of dctD was dominant to ntrC283 . Results from studies of the ability of ntrC283 to suppress the Dct- phenotype of dctB alleles, together with the finding that the Fix- phenotype of a particular dctB allele was dctD dependent, suggest that in particular dctB alleles, sufficient dctD transcription occurs such that the resulting inactive DCTD prevents activation of dctA transcription by NtrC283 or alternate symbiotic regulators . The latter suggestion is supported by the observation that in symbiosis, R . meliloti strains in which DCTD was overexpressed formed nodules which failed to fix nitrogen. J Bacteriol, 1993 May, 175(9), 2662 - 73 Symbiotic nitrogen fixation by a nifA deletion mutant of Rhizobium meliloti: the role of an unusual ntrC allele; Labes M et al.; In the N2-fixing alfalfa symbiont Rhizobium meliloti, the three sigma 54 (NTRA)-dependent positively acting regulatory proteins NIFA, NTRC, and DCTD are required for activation of promoters involved in N2 fixation (pnifHDKE and pfixABCX), nitrogen assimilation (pglnII), and C4-dicarboxylate transport (pdctA), respectively . Here, we describe an allele of ntrC which results in the constitutive activation of the above NTRC-, NIFA-, and DCTD-regulated promoters . The expression and activation of wild-type NTRC occur in response to nitrogen availability, whereas in cells carrying the ntrC283 allele, the NTRC283 protein appears constitutively active and is constitutively expressed . The ntrC283 allele was shown to carry a single mutation resulting in the replacement of an Asp by a Tyr residue in the helix-turn-helix motif of ntrC283 . Introduction of the ntrC283 allele into a nifA deletion mutant restores the N2-fixation ability to 70 to 80% of the wild-type level . Thus, the nifA gene is dispensable for symbiotic N2 fixation. Mol Microbiol, 1993 May, 8(3), 471 - 81 Molecular analysis of a microaerobically induced operon required for hydrogenase synthesis in Rhizobium leguminosarum biovar viciae; Rey L et al.; The nucleotide sequence (6138 bp) of a microaerobically inducible region (hupV/VI) from the Rhizobium leguminosarum bv . viciae hydrogenase gene cluster has been determined . Six genes, arranged as a single operon, were identified, and designated hypA, B, F, C, D and E based on the sequence similarities of all of them, except hypF, to genes from the hydrogenase pleiotropic operon (hyp) from Escherichia coli . The gene products from hypBFCDE were identified by in vivo expression analysis in E . coli, and their molecular sizes were consistent with those predicted from the nucleotide sequence . Transposon Tn5 insertions into hypB, hypF, hypD and hypE resulted in R . leguminosarum mutants that lacked any hydrogenase activity in symbiosis with peas, but still were able to synthesize the polypeptide for the hydrogenase large subunit . The gene products HypA, HypB, HypF and HypD contained CX2C motifs characteristic of metal-binding proteins . In addition, HypB bore a long histidine-rich stretch of amino acids near the N-terminus, suggesting a possible role in nickel binding for this protein . The gene product HypF, which was translationally coupled to HypB, presented two cysteine motifs (CX2CX18CX2C) with a capacity to form zinc finger-like structures in the N-terminal third of the protein . A role in nickel metabolism in relation to hydrogenase synthesis is postulated for proteins HypB and HypF. Biochim Biophys Acta, 1993 Apr 21, 1163(1), 31 - 41 The nitrogenase proteins of Rhizobium meliloti: purification and properties of the MoFe and Fe components; Miller RW et al.; The alfalfa-Rhizobium meliloti symbiosis contributes a major portion of biologically fixed nitrogen to temperate zone forage crop production . Highly-purified molybdenum-iron (MoFe) and iron (Fe) nitrogenase components were obtained for the first time from extracts of R . meliloti bacteroids . Intact bacteroid cells were isolated anaerobically from 100 g quantities of alfalfa nodules following storage in liquid nitrogen . Centrifuged bacteroid extracts showed a marked reduction in specific activity when assayed at protein concentrations less than 1 mg/ml . Both nitrogenase proteins were resolved and purified to homogeneity as determined spectroscopically and by SDS-PAGE . The purified MoFe protein differed in several respects from previously characterized nitrogenase proteins . Saturation of the acetylene-reducing and proton-reducing activities of the R . meliloti MoFe protein required higher relative concentrations of Fe protein than nitrogenase proteins purified from free living diazotrophs . Electron allocation to dinitrogen reduction was sustained at component ratios similar to those present in bacteroid extracts, suggesting that while the observed saturation effects were not detrimental to physiological function in the symbiotic system, overall activity could be enhanced by higher levels of iron protein . Analyses of the MoFe protein gave 22 Fe, 22 labile sulfide and 1.7 Mo atoms per molecular unit of 215 kDa . Dithionite-reduced MoFe protein contained a spin 3/2 iron centre but had a lower visible absorbance at 360 nm than the equivalent Azotobacter chroococcum component . Amino-acid composition indicated a notably lesser tryptophan content, and cysteine content greater than that of the equivalent tetrameric protein of free living diazotrophs . Ratios of acidic and basic residues were similar to other MoFe proteins . Calculation of hydrophobicity and discriminant parameters gave values midway between those expected for soluble cytoplasmic proteins and peripheral membrane associated proteins . ADP was tightly bound by the dithionite-free MoFe protein containing reduced iron-molybdenum cofactor . The R . meliloti iron protein was found to be a 64 kDa homodimer containing a single 4Fe-4S metal centre. Proc Natl Acad Sci U S A, 1993 Apr 15, 90(8), 3506 - 10 Oxygen regulation of nifA transcription in vitro; Agron PG et al.; In Rhizobium meliloti, transcription of the key nitrogen-fixation regulatory genes nifA and fixK is induced in response to microaerobiosis through the action of the FixL and FixJ proteins . These two proteins are sensor and regulator homologues, respectively, of a large family of bacterial two-component systems involved in sensing and responding to environmental changes . A soluble, truncated form of the membrane protein FixL, FixL*, has been shown to be a hemoprotein that phosphorylates and dephosphorylates FixJ in response to oxygen tension . Here we use an in vitro transcription system to prove that FixJ is a transcriptional activator of both nifA and fixK and that phosphorylation of FixJ markedly increases its activity . Phosphorylation was achieved either by preincubating FixJ with FixL* and ATP or by exposing FixJ to the inorganic phospho donor ammonium hydrogen phosphoramidate . Both FixJ and FixJ-phosphate formed heparin-resistant complexes under the assay conditions used . Lastly, we were able to show that anaerobiosis, in the presence of FixL* and ATP, greatly stimulates FixJ activity at the nifA promoter with either Escherichia coli or R . meliloti RNA polymerase . This use of atmospheric oxygen to control nifA transcription in vitro represents a reconstitution of a bacterial two-component signal transduction system in its entirety, from effector to ultimate target, by the use of purified components. Gene, 1993 Apr 15, 126(1), 67 - 75 Cloning and characterization of multiple groEL chaperonin-encoding genes in Rhizobium meliloti; Rusanganwa E et al.; Heat-shock treatment of Rhizobium meliloti cells causes major enhancement in the synthesis of several proteins with apparent molecular weights in the range of 58-60 kDa . Using the polymerase chain reaction and degenerate oligodeoxyribonucleotide primers for conserved regions of the 60-kDa heat-shock protein (HSP60) or GroEL protein family, a 0.6-kb probe for the R . meliloti hsp60 gene was prepared . Southern blot analysis of R . meliloti DNA digested with different restriction enzymes and hybridized to R . meliloti hsp60 probes indicated the presence of between four and five hsp60 or groEL in this species . From the cloning and sequencing of several of these fragments, we have been able to deduce the complete nucleotide sequences of three groEL in R . meliloti . The deduced amino acid (aa) sequences of these proteins show extensive similarity to each other (78-85% aa identity) and to other GroEL homologues . In the upstream regions of two of the groEL, but not the third, open reading frames corresponding to GroES proteins were also identified . Analysis of various prokaryotic GroEL sequences suggests that the multiple groEL of R . meliloti have evolved by means of gene duplication events within this or a related group of organisms . Results presented in this paper also show that some of the groEL in R . meliloti are located on the two megaplasmids present in these cells . The presence of multiple GroEL homologues in R . meliloti suggests a possible role of the GroEL or HSP60 chaperonins in the nodulation (symbiosis) and nitrogen fixation processes. Int J Syst Bacteriol, 1993 Apr, 43(2), 374 - 7 Reclassification of American Rhizobium leguminosarum biovar phaseoli type I strains as Rhizobium etli sp . nov; Segovia L et al.; A new Rhizobium species that nodulates Phaseolus vulgaris L . is proposed on the basis of a sequence analysis of 16S ribosomal DNA . This taxon, Rhizobium etli sp . nov., was previously named Rhizobium leguminosarum biovar phaseoli (type I strains) and is characterized by the capacity to establish an effective symbiosis with bean plants, the reiteration of the nitrogenase structural genes, the organization of the common nodulation genes into two separate transcriptional units bearing nodA and nodBC, the presence of the polysaccharide inhibition gene, psi, and the 16S ribosomal DNA sequence . An analysis of the sequence of a fragment of the 16S rRNA gene shows that this gene is quite different from the gene of R . leguminosarum . In addition, all R . etli strains have identical sequences . We describe these analyses and discuss additional evidence supporting our proposal. J Bacteriol, 1993 Apr, 175(8), 2284 - 91 Relationships between C4 dicarboxylic acid transport and chemotaxis in Rhizobium meliloti; Robinson JB et al.; The relationship between chemotaxis and transport of C4 dicarboxylic acids was analyzed with Rhizobium meliloti dct mutants defective in one or all of the genes required for dicarboxylic acid transport . Succinate, malate, and fumarate were moderately potent chemoattractants for wild-type R . meliloti and appeared to share a common chemoreceptor . While dicarboxylate transport is inducible, taxis to succinate was shown to be constitutive . Mutations in the dctA and dctB genes both resulted in the reduction, but not elimination, of chemotactic responses to succinate, indicating that transport via DctA or chemosensing via DctB is not essential for C4 dicarboxylate taxis, although they appear to contribute to it . Mutations in dctD and rpoN genes did not affect taxis to succinate . Aspartate, which is also transported by the dicarboxylate transport system, elicited strong chemotactic responses via a chemoreceptor distinct from the succinate-malate-fumarate receptor . Taxis to aspartate was unaltered in dctA and dctB mutants but was considerably reduced in both dctD and rpoN mutants, indicating that aspartate taxis is strongly dependent on elements responsible for transcriptional activation of dctA . Methylation and methanol release experiments failed to show a significant increase in methyl esterification of R . meliloti proteins in response to any of the attractants tested. Proc Natl Acad Sci U S A, 1993 Apr 1, 90(7), 2641 - 5 Bradyrhizobium japonicum rhizobitoxine genes and putative enzyme functions: expression requires a translational frameshift; Ruan X et al.; Some strains of Bradyrhizobium japonicum produce rhizobitoxine, a phytotoxin that causes foliar chlorosis on susceptible host plants . We have previously obtained Tn5-induced rhizobitoxine null mutants of B . japonicum . DNA sequence analysis of the region surrounding two Tn5 insertions identifies two overlapping open reading frames . The first open reading frame (rtxA) predicts a 54-kDa protein for which the N-terminal 280 residues have sequence similarity to serine: pyruvate aminotransferase . The sequence homology to aminotransferase is consistent with the involvement of this gene in serinol production, a likely intermediate in rhizobitoxine biosynthesis . Previously, a mutant in this open reading frame was shown not to make serinol . The predicted amino acid sequence of the second open reading frame (rtxB) has similarity to yeast O-acetylhomoserine sulfhydrolase . This enzyme function is similar to that required for dihydrorhizobitoxine synthase . The DNA sequence shows that the rtxB open reading frame overlaps rtxA, suggesting that expression of rtxB requires a -1 translational frameshift . Protein expression experiments demonstrate production of an RtxAB fusion protein . The ability of the overlapping rtxA and rtxB sequences to promote a translational frameshift was confirmed in a heterologous expression system . In Escherichia coli, this frameshift appears to be unusually efficient, occurring at a frequency of 80-90%. Mol Microbiol, 1993 Apr, 8(2), 253 - 9 The Escherichia coli cAMP receptor protein (CRP) represses the Rhizobium meliloti dctA promoter in a cAMP-dependent fashion; Wang YP et al.; The expression of the Rhizobium meliloti C4-dicarboxylic acid permease gene (dctA) is controlled by the sensor DctB and the transcriptional regulator, DctD . The R . meliloti Dct system has been reconstituted in Escherichia coli . Expression of the dctA promoter is DctBD dependent and is induced in the presence of C4-dicarboxylic acids (dCA) . Other carbon sources also influence dctA expression . We demonstrate that the cAMP receptor protein (CRP) has a repressive effect on the dctA promoter . A mutated CRP molecule (CRP-H159L), unable to activate catabolic promoters (but still proficient in DNA binding), gives similar results . This suggests that the CRP-cAMP complex represses the dctA promoter activity by direct interaction with the DNA . Direct binding of the CRP-cAMP complex to the dctA promoter was confirmed in vitro by gel mobility-shift assays . Sequence analysis of the dctA promoter indicates that the most likely binding sites for CRP are the two confirmed DctD-binding sites . It is proposed that the CRP-cAMP complex competes with DctD for occupancy of these sites . Since in the presence of CRP-cAMP complex the uninduced levels of dctA expression are reduced, whereas induced levels are largely unaffected, such competition appears to be an essential regulatory feature of dctA expression. Mol Microbiol, 1993 Apr, 8(1), 159 - 66 Rhizobium phaseoli cytochrome c-deficient mutant induces empty nodules on Phaseolus vulgaris L; Soberon M et al.; A Rhizobium phaseoli cytochrome mutant, unable to oxidize N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), was isolated after Mu-dl (Kan lac) mutagenesis of the wild-type strain CE-3 . Mutant strain CFN4202 had sixfold less haem-c but similar levels of b type, o and aa3 cytochromes than the wild-type strain . CFN4202 strain also showed reduced NADH- and TMPD-oxidase activity than the wild-type strain . Succinate-oxidase activities were very similar . Western blot experiments, using antiserum against bovine c1 and c cytochromes, revealed that both proteins were present in CFN4202 membranes, suggesting a defect of haem binding to cytochrome c . Nodules formed by this strain in Phaseolus vulgaris did not contain bacteroids . These data suggest that the cytochrome c-aa3 chain or some other respiratory chain, containing c-type cytochromes in R . phaseoli, is essential for bacterial division during the early steps of the symbiotic interaction with the legume-host. Semin Cell Biol, 1993 Apr, 4(2), 149 - 56 The Rhizobium-legume symbiosis: plant morphogenesis in a nodule; Brewin NJ; Development of the legume root nodule can be divided conceptually into two parallel processes . On the one hand, there is the induction of a nodule meristem and the progressive differentiation of specialised cells and tissues . On the other hand, there is cell and tissue invasion by Rhizobium, which leads ultimately to the differentiation of intracellular bacteroids as specialised nitrogen-fixing endosymbionts . The early stages of plant-microbe communication seem to be mostly mediated by the exchange of soluble, diffusible signal molecules: flavonoid compounds are secreted by plant roots, and chitin-like lipooligosaccharides are secreted by rhizobia . Further development of the nodule structure may involve the interplay of fairly conventional plant growth regulators . Direct physical contact between the cell surfaces of the symbionts also plays a prominent role in the process of tissue and cell invasion. Biomed Environ Sci, 1993 Mar, 6(1), 89 - 94 Effect of pesticides on growth of rhizobia and their host plants during symbiosis; Madhavi B et al.; Effect of various pesticides (insecticides, fungicides and herbicides) has been studied on growth and efficiency of symbiotic properties of 3 fast growing Rhizobium sp . under green house conditions . The results revealed adverse effects on plant growth and nitrogen fixing capacity as measured by dry weight and total nitrogen content of plants infected with pesticide treated Rhizobium . Of the pesticides tested, herbicides were found to be more effective on the above parameters than the insecticides and fungicides. J Gen Microbiol, 1993 Mar, 139 ( Pt 3), 529 - 38 Evaluation of a strategy for identifying nodulation competitiveness genes in Rhizobium leguminosarum biovar phaseoli; Beattie GA et al.; Rhizobium leguminosarum biovar phaseoli strain KIM5s is consistently much more competitive than strain CE3 in nodulation of beans (Phaseolus vulgaris L.) in the laboratory and in the field . To identify genes that contribute to the competitiveness of KIM5s, we transferred a cosmid library containing KIM5s DNA into CE3 and applied the transconjugants to bean plants to allow the plants to enrich for those with enhanced nodulation competitiveness . The nodule isolates were then applied to plants for further enrichment . Of 75 isolates from nodules sampled after the two enrichments, 9 were more competitive than CE3 . For example, when outnumbered in the inocula 40-fold by a reference strain, these nine strains typically occupied 15-40% of the nodules compared with 0-3% for CE3 . However, when these strains were cured of the cosmids, they remained highly competitive, demonstrating that the enhanced competitiveness of the strains was not associated with the cosmids . We found no evidence for cosmid insertion into the chromosome or for cosmid-induced genetic changes in these cured strains . We found some evidence suggesting that their altered competitiveness was due to spontaneous genetic changes that did not involve the cosmids . Although these highly competitive variants remain genetically uncharacterized, they may provide insight into bacterial traits that contribute to, or detract from, successful nodulation competitiveness. J Gen Microbiol, 1993 Mar, 139 ( Pt 3), 403 - 16 Molecular and enzymological evidence for two classes of fumarase in Bacillus stearothermophilus (var . non-diastaticus); Reaney SK et al.; The gene (fumABst) encoding an oxygen-labile fumarase of Bacillus stearothermophilus has been cloned and sequenced . The structural gene (1542 bp) encodes a product (FumABst) of M(r) 56,788 containing 514 amino acid residues . The amino acid sequence is 23% identical (37% similar) to FumA and FumB, the labile {4Fe-4S}-containing fumarases (Class I enzymes) of Escherichia coli . It exhibits no significant similarity to FumC and CitG, the stable fumarases (Class II enzymes) of E . coli and Bacillus subtilis (respectively) . Enzymological studies indicated that FumABst resembles the iron-sulphur-containing fumarases in being dimeric (M(r) 2 x 58,500), oxygen labile and partially reactivated by Fe2+ plus DTT . The fumABst gene is the first gene encoding a Class I fumarase to be characterized in any organism other than E . coli . Enzymological and DNA-hybridization studies further indicated that B . stearothermophilus resembles E . coli in containing an oxygen-stable fumarase (Class II enzyme) . Sequence comparisons revealed significant similarities between the Class I fumarases and the products of adjacent open-reading frames (orfZ1 and orfZ2) located upstream of the macromolecular synthesis operon (rpsU-dnaG-rpoD) at 67 min in the E.coli linkage map . Located downstream of fumABst, there is an unidentified gene (orf2), which is homologous to the rhizobial nodB genes involved in the initiation of root nodule formation. Mol Microbiol, 1993 Mar, 7(6), 865 - 73 NAD(+)-dependent malic enzyme of Rhizobium meliloti is required for symbiotic nitrogen fixation; Driscoll BT et al.; DEAE-cellulose chromatography of extracts of free-living Rhizobium meliloti cells revealed separate NAD(+)-dependent and NADP(+)-dependent malic enzyme activities . The NAD+ malic enzyme exhibited more activity with NAD+ as cofactor, but also showed some activity with NADP+ . The NADP+ malic enzyme only showed activity when NADP+ was supplied as cofactor . Three independent transposon-induced mutants of R . meliloti which lacked NAD+ malic enzyme activity (dme-) but retained NADP+ malic enzyme activity were isolated . In an otherwise wild-type background, the dme mutations did not alter the carbon utilization phenotype; however, nodules induced by these mutants failed to fix N2 . Structurally, these nodules appeared to develop like wild-type nodules up to the stage where N2-fixation would normally begin . These results support the proposal that NAD+ malic enzyme, together with pyruvate dehydrogenase, functions in the generation of acetyl-CoA required for TCA cycle function in N2-fixing bacteroids which metabolize C4-dicarboxylic acids supplied by the plant. J Biol Chem, 1993 Feb 25, 268(6), 4370 - 5 Autophosphorylation and phosphatase activities of the oxygen-sensing protein FixL of Rhizobium meliloti are coordinately regulated by oxygen; Lois AF et al.; The FixL and FixJ proteins of Rhizobium meliloti control the expression of other nif and fix genes in response to oxygen levels . FixL is a hemoprotein kinase that senses oxygen availability and responds to the absence of oxygen by activation of its autophosphorylating activity followed by transfer of the phosphate to FixJ . FixJ in turn activates the nifA and fixK promoters . In vitro studies reported here with a soluble truncated version of FixL (FixL*) indicate that, while low oxygen tension specifically increases the autophosphorylating activity of FixL*, the ability of phospho-FixL* to act as a phosphate donor to FixJ is not affected by the presence or absence of oxygen . FixL* is also shown to possess a phosphatase activity that is repressed under anaerobic conditions only when the protein is in the phosphorylated form . A fixL mutant that induces a higher level of nifA promoter activity in the presence of fixJ in vivo displayed both an increased autophosphorylating activity and a decreased phosphatase activity in vitro . These data provide evidence for a role for both autophosphorylation and phosphatase activities of FixL in the mechanism by which oxygen tension within the alfalfa nodule induces expression of bacterial nitrogen fixation genes during symbiosis. Carbohydr Res, 1993 Feb 24, 240, 71 - 8 Structural characterization and rheological properties of an extracellular glucuronan produced by a Rhizobium meliloti M5N1 mutant strain; Heyraud A et al.; The mutant strain M5N1 C.S . (NCIMB 40472) of Rhizobium meliloti M5N1 is able to produce during fermentation a partially acetylated extracellular (1-->4)-beta-D-glucuronan . At low concentration (1 g.l-1), in the presence of monovalent cations, this new glucuronate behaves as a thickening agent, whereas at higher concentration a thermoreversible gel is obtained . With such divalent cations as Ca2+, a thermally stable gel can be formed. Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1551 - 5 Three unusual modifications, a D-arabinosyl, an N-methyl, and a carbamoyl group, are present on the Nod factors of Azorhizobium caulinodans strain ORS571; Mergaert P et al.; Azorhizobium caulinodans strain ORS571 is a symbiont of the tropical legume Sesbania rostrata . Upon nod gene induction with naringenin, strain ORS571 secretes into the culture medium Nod factors that morphologically change the host plant--in particular, deformed root hairs (Hai/Had) and meristematic foci are formed at the basis of lateral roots . The latter infrequently develop further into nodule-like structures . The azorhizobial Nod factors are chitin tetramers or pentamers, N-acylated at the nonreducing-end glucosamine with either vaccenic acid (C18:1) or stearic acid (C18:0) . They, thus, resemble the previously described Nod factors from (brady)rhizobia . The backbone lipooligosaccharide is substituted with unusual modifications, presumably involved in host-specificity determination . There is a D-arabinose branch on the reducing end and an N-methyl and O-carbamoyl substitution on the nonreducing end of the oligosaccharide chain . The previously identified nod gene nolK may be involved in the synthesis of a D-arabinose derivative . The nodS gene product is probably responsible for the N-methylation of Nod factors. Genome, 1993 Feb, 36(1), 43 - 9 Genetic relationships and variation in the Stylosanthes guianensis species complex assessed by random amplified polymorphic DNA; Kazan K et al.; Genetic variation in the five taxonomic groups of the Stylosanthes guianensis (Aubl.) Sw . complex was investigated using random amplified polymorphic DNA markers (RAPDs) . DNA samples from four plants of each of 45 accessions within the S . guianensis species complex were analyzed using 20 oligonucleotides of random sequence . Little variation was found within each of the 18 accessions (1-7% of total RAPD bands in pairwise comparisons) and none within each of the other 27 accessions . However, higher levels of polymorphisms were observed both within (index of genetic distance = 1 - F = 0.16-0.248) and between (1 - F = 0.254-0.408) the five taxa . This level of differentiation at the DNA level supported an earlier classification of the taxa as distinct species . A phenogram based on band sharing was constructed to show genetic relationships among the taxa studied . This phenogram corroborated the description of relationships based on morphological-agronomic characteristics, seed protein patterns, rhizobial affinities, crossability, and pollen stainability of the hybrids . In this phenogram, the most similar species were S . grandiflora and S . hippocampoides (1 - F = 0.264), with S . acuminata also showing closest similarity to these two species (1 - F = 0.277 and 0.283, respectively) . Stylosanthes gracilis accessions showed the closest similarity (1 - F = 0.296) to S . guianensis ssp . guianensis accessions . Lowest similarity values (1 - F = 0.335-0.411) were found between these two species and S . grandiflora, S . acuminata, and S . hippocampoides. Plant Cell, 1993 Feb, 5(2), 215 - 26 Molecular characterization of NADH-dependent glutamate synthase from alfalfa nodules; Gregerson RG et al.; Alfalfa NADH-dependent glutamate synthase (NADH-GOGAT), together with glutamine synthetase, plays a central role in the assimilation of symbiotically fixed nitrogen into amino acids in root nodules . Antibodies previously raised against purified NADH-GOGAT were employed to screen a cDNA library prepared using RNA isolated from nodules of 20-day-old alfalfa plants . A 7.2-kb cDNA clone was obtained that contained the entire protein coding region of NADH-GOGAT . Analysis of this cDNA and determination of the amino-terminal amino acids of the purified protein revealed that NADH-GOGAT is synthesized as a 2194-amino acid protein that includes a 101-amino acid presequence . The deduced amino acid sequence shares significant identity with maize ferredoxin-dependent GOGAT, and with both large and small subunits of Escherichia coli NADPH-GOGAT . DNA gel blot analysis of alfalfa genomic DNA suggests the presence of a single NADH-GOGAT gene or a small gene family . The expression of NADH-GOGAT mRNA, enzyme protein, and enzyme activity was developmentally regulated in root nodules . A dramatic increase in gene expression occurred coincidentally with the onset of nitrogen fixation in the bacteroid, and was absent in both ineffective plants that were nodulated with effective Rhizobium meliloti and effective plants that had been nodulated with ineffective R . meliloti strains . Maximum NADH-GOGAT expression, therefore, appears to require an effective, nitrogen-fixing symbiosis. Anal Biochem, 1993 Feb 1, 208(2), 363 - 71 N,N-(2,4-dinitrophenyl)octylamine derivatives for the isolation, purification, and mass spectrometric characterization of oligosaccharides; Zhang Y et al.; A simple, sensitive method for the structural characterization of oligosaccharides by fast atom bombardment-mass spectrometry (FAB-MS) has been designed . Oligosaccharides are labeled with a uv chromophore (which also serves as a charge stabilizing group) and with a hydrophobic alkyl tail . The chromophore, a 2,4-dinitrophenyl group, aids uv detection during HPLC and stabilizes negative ion species formed during analysis by FAB-MS . The hydrophobic tail, provided by an octyl group, enhances the surface activity of the analytes and makes them amenable to separation by reverse-phase chromatography using a C18 bonded phase . This method was applied to the structural analysis of the components of a mixture of starch maltodextrins with a degree of polymerization 1-16, to the analysis of the structure of pure maltohexaose, and to a previously characterized oligosaccharide from a Rhizobium capsular polysaccharide . The method gave a good yield of {M-H}- anions for the derivatized compounds, which in most cases were detectable at a level of about 1 pmol . In the case of maltohexaose, four series of sequence anions corresponding to sequential loss of glycosyl residues from the reducing and nonreducing end by different mechanisms were observed . The mixture of derivatized malto-oligosaccharides could easily be separated by HPLC . Based on the relative proportions of the individual oligomers in the mixture calculated from HPLC analysis, even though the higher oligomers were present in amounts of about 0.1%, they could still be easily detected in mass spectra of the entire mixture.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1993 Feb, 175(4), 1103 - 9 The oxygen sensor FixL of Rhizobium meliloti is a membrane protein containing four possible transmembrane segments; Lois AF et al.; Regulation of nitrogen fixation genes in Rhizobium meliloti is mediated by two proteins, FixL and FixJ, in response to oxygen availability . FixL is an oxygen-binding hemoprotein with kinase and phosphatase activities that is thought to sense oxygen levels directly and to transmit this signal to FixJ via phosphorylation-dephosphorylation reactions . FixJ controls the expression of other regulatory genes, including nifA, that regulate the transcription of genes required for symbiotic nitrogen fixation . We have been studying the structural and functional features of FixL that are required for oxygen sensing . We constructed mutant derivatives and confirmed that FixL consists of 505 amino acids instead of 464, as originally reported . Hydropathy plots of the full-length protein, together with TnphoA insertional analysis, lead us to propose that FixL is likely to be a polytopic integral membrane protein containing four membrane-spanning segments . We have also constructed an N-terminal deletion of the FixL protein whose in vivo activity indicates that the hydrophobic membrane-spanning regions are not absolutely required for oxygen sensing in vivo . We also report that FixL shares homology in its N terminus with other sensor proteins, including KinA from Bacillus subtilis and NtrB from Bradyrhizobium parasponia . The region of homology comprises a 70-amino-acid residue stretch that is also conserved in two oxygenases, P-450 and isopenicillin synthase. Proc Natl Acad Sci U S A, 1993 Feb 1, 90(3), 943 - 7 Two functional soybean genes encoding p34cdc2 protein kinases are regulated by different plant developmental pathways; Miao GH et al.; We have isolated two cDNA clones (cdc2-S5 and cdc2-S6) encoding p34cdc2 protein kinases, homologs of yeast cdc2/CDC28 genes, from a soybean nodule cDNA library . The two sequences share 90% sequence homology in the coding regions . The 5' and 3' noncoding regions are distinct from each other, however, indicating that at least two genes encode p34cdc2 protein kinases in soybean . Both sequences can rescue the cdc28 mutation in Saccharomyces cerevisiae but rescue it with different efficiency . Genomic Southern analysis showed the existence of two copies for each of these genes, which are not closely linked and are nonallelic . The relative expression level of the two soybean p34cdc2 genes varies in different tissues . Expression of cdc2-S5 is higher in roots and root nodules, whereas cdc2-S6 is more actively expressed in aerial tissues, indicating that regulation of these two p34cdc2 genes is coupled with plant developmental pathways . Expression of cdc2-S5 is, furthermore, enhanced after Rhizobium infection, whereas cdc2-S6 fails to respond, suggesting that cdc2-S5 plays a role in nodule initiation and organogenesis . This latter gene preferentially responds to auxin (alpha-naphthaleneacetic acid) treatment, indicating that phytohormones may be involved in the control of cell division mediated by Rhizobium infection . Thus, different p34cdc2 protein kinases may control cell division in different tissues in a multicellular organism and respond to different signals--e.g., phytohormones. J Bacteriol, 1993 Feb, 175(3), 750 - 7 Polysaccharide synthesis in relation to nodulation behavior of Rhizobium leguminosarum; Breedveld MW et al.; In this study, we characterized four Tn5 mutants derived from Rhizobium leguminosarum RBL5515 with respect to synthesis and secretion of cellulose fibrils, extracellular polysaccharides (EPS), capsular polysaccharides, and cyclic beta-(1,2)-glucans . One mutant, strain RBL5515 exo-344::Tn5, synthesizes residual amounts of EPS, the repeating unit of which lacks the terminal galactose molecule and the substituents attached to it . On basis of the polysaccharide production pattern of strain RBL5515 exo-344::Tn5, the structural features of the polysaccharides synthesized, and the results of an analysis of the enzyme activities involved, we hypothesize that this strain is affected in a galactose transferase involved in the synthesis of EPS only . All four mutants failed to nodulate plants belonging to the pea cross-inoculation group; on Vicia sativa they induced root hair deformation and rare abortive infection threads . All of the mutants appeared to be pleiotropic, since in addition to defects in the synthesis of EPS, lipopolysaccharide, and/or capsular polysaccharides significant increases in the synthesis and secretion of cyclic beta-(1,2)-glucans were observed . We concluded that it is impossible to correlate a defect in the synthesis of a particular polysaccharide with nodulation characteristics. Sci China B, 1993 Feb, 36(2), 225 - 36 Evidence that the nodulation regulatory gene nodD3 of Rhizobium meliloti is transcribed from two separate promoters; Yu GQ et al.; The Bgl II fragment carried in plasmid pMH903, which covers the nodD3 region of Rhizobium meliloti, has been sequenced . By using both S1 nuclease mapping and primer extension, two transcription start sites were demonstrated in the sequence . After the first transcription start site, there were two open reading frames (ORF) followed by the nodD3 coding sequence which was also preceded by the second promoter . The nodD3 gene under the first promoter mediated high, constitutive expression of nodC-lacZ fusion, and the gene under the second promoter required the product of nodD1 and alfalfa (Medicago sativa) seed exudate for the activation of fusion . Nodulation experiments showed that the nodD3 gene under either promoter was functional in eliciting nodules on alfalfa . The deletion of part of the two ORFs after the first promoter or deletion of the second promoter did not block the constitutive expression of nodC-lacZ fusion, whereas the deletion of the first promoter region or a polar insertion mutation between the two promoters did cause nodD3 to activate nodC only in the presence of the inducer . It indicates that nodD3 can be transcribed from the first promoter as well as from a separate second promoter. J Mol Biol, 1993 Jan 20, 229(2), 570 - 6 Nucleotide sequence and characterization of Rhizobium meliloti nodulation competitiveness genes nfe; Soto MJ et al.; Rhizobium meliloti large plasmid pRmeGR4b carries the nodulation competitiveness locus nfe responsible for the nodulation efficiency and competitive ability of strain GR4 on alfalfa roots . We report here the nucleotide sequence and characterization of a 3345 base-pair DNA section of the nfe region . Sequence analysis revealed four open reading frames (ORFs), two of them with rightward polarity, termed nfe1 and nfe2, are preceded by functional nif consensus sequences and NifA-binding motifs . An additional, NifA-independent, transcriptional start site for nfe1 was also found . Two other ORFs with leftward polarity, designated ORFA and ORFB, were identified upstream from nfe1 and nfe2 but no nif consensus sequences were found . However, expression of ORFA might be indirectly coupled to the NifA-NtrA regulatory network . The gene products of nfe1 and nfe2 were identified using in vitro transcription/translation and bacteriophage T7 RNA polymerase/promoter system, respectively . A high degree of homology between the amino terminal domain of Nfe1 and the nifH gene product was found . In addition, nfe1 shows homology with the upstream non-coding DNA region of the fixABCX operon . Furthermore, the putative ORFB encoded protein contains a helix-turn-helix motif that resembles the DNA-binding consensus sequence proposed for many prokaryotic regulatory proteins. Proc Natl Acad Sci U S A, 1993 Jan 15, 90(2), 625 - 9 Rhizobium NodB protein involved in nodulation signal synthesis is a chitooligosaccharide deacetylase; John M et al.; The common nodulation genes nodABC are conserved in all rhizobia and are involved in synthesis of a lipooligosaccharide signal molecule . This bacterial signal consists of a chitooligosaccharide backbone, which carries at the nonreducing end a fatty acyl chain . The modified chitooligosaccharide molecule triggers development of nodules on the roots of the leguminous host plant . To elucidate the specific role of the NodB protein in nodulation factor synthesis, we have purified recombinant NodB and determined its biochemical role by direct assays . Our data show that the NodB protein of Rhizobium meliloti deacetylates the nonreducing N-acetylglucosamine residue of chitooligosaccharides . The monosaccharide N-acetylglucosamine is not deacetylated by NodB . In the pathway of Nod factor synthesis, deacetylation at the nonreducing end of the oligosaccharide backbone may be a necessary requirement for attachment of the fatty acyl chain. J Biol Chem, 1993 Jan 5, 268(1), 469 - 75 Isolation and characterization of a novel glutamine synthetase from Rhizobium meliloti; Shatters RG et al.; Two glutamine synthetases, GSI and GSII, are found in most rhizobia . However, WSU650, a Rhizobium meliloti glnA glnII mutant that lacks both enzymes, can grow without a glutamine supplement in minimal medium that contains both ammonium and glutamate . The bacteria contained a third glutamine synthetase, GSIII, which has been purified and partially characterized . GSIII had considerable glutamine synthetase activity when assayed using a semibiosynthetic (glutamate- and hydroxylamine-dependent) assay, but had no detectable transferase (glutamine- and hydroxyl-amine-dependent) activity . GSIII was inhibited by ADP and pyrophosphate but not by various nitrogen-containing metabolites that inhibit other GS enzymes . Activity was also inhibited by methionine sulfoximine, a transition state analog, but the concentration needed to inhibit GSIII was 50 to 100 times higher than that needed to inhibit GSI or GSII . GSIII had a Km for glutamate of 13.3 mM, for ammonium of 33 mM, and for hydroxylamine of 5.3 mM with a pH optimum of 6.8 and a temperature optimum of 50 degrees C . The purified protein had related subunits of 46.5 and 49 kDa and a native molecular mass of 355 kDa, indicating the native enzyme was an octamer . Polyclonal antibodies specific for GSIII reacted with a protein of similar molecular weight in Escherichia coli strains that carry R . meliloti glnT on a plasmid . GSIII activity was detected in some of these strains that contained glnT . Extracts of root nodules formed by WSU650 also react with the antibodies. Plasmid, 1993 Jan, 29(1), 10 - 8 An ultraviolet-sensitive mutant of Zymomonas mobilis affecting the stability of its natural plasmid pZMO2; Vartholomatos G et al.; Plasmid pZMO2, a 1.9-kb natural plasmid of Zymomonas mobilis strain ATCC 10988, was found to be absent from the extract of a mutant isolate sensitive to ultraviolet irradiation, methyl methanesulfonic acid, and mitomycin C . This mutant, named uvs51, also exhibited extremely high segregational instability of the recombinant plasmid pDS212, whose maintenance in the parental strain is known to be due to pZMO2 sequences . Helped conjugations, employing uvs51 as recipient with Escherichia coli donors containing the conjugative plasmid pRK2013 and a mobilizable recombinant plasmid carrying the Rhizobium meliloti recA gene, showed complementation of uvs51 with the recA functions . Moreover, uvs51/recA stable transconjugants, containing the recA sequence integrated into their chromosomes, showed an increased stability of recombinant plasmid pDS212 . The data presented here indicate that the stable maintenance of the natural plasmid pZMO2 in Z . mobilis is affected by a putative recA function. Plant Cell, 1993 Jan, 5(1), 87 - 96 MsERK1: a mitogen-activated protein kinase from a flowering plant; Duerr B et al.; The induction of proliferation and differentiation in cultured mammalian cells is mediated by a cascade of protein phosphorylations . A key enzyme in this signaling pathway is mitogen-activated protein (MAP) kinase (or ERK, extracellular signal-regulated kinase) . We report the recovery of a full-length cDNA clone encoding a MAP kinase from alfalfa . We have named the 44-kD protein encoded by this clone MsERK1 . Recombinant MsERK1 (rMsERK1), when overexpressed in Escherichia coli, is recognized by antibodies raised against MAP kinases from rat, Xenopus, and sea star and by anti-phosphotyrosine antibodies . Site-directed mutagenesis of MsERK1 demonstrated that Tyr-215 is either directly or indirectly responsible for recognition of the protein by anti-phosphotyrosine antibodies . Semipurified rMsERK1 phosphorylated itself and a model substrate, myelin basic protein, in vitro, but the Tyr-215 mutant did neither . Genomic DNA gel blot analysis suggested that the gene that encodes MsERK1 is either a member of a small multigene family or a member of a polymorphic allelic series in alfalfa . Because MAP kinase activation has been associated with mitotic stimulation in animal systems, such an enzyme may play a role in the mitogenic induction of symbiotic root nodules on alfalfa by Rhizobium signal molecules. Mol Plant Microbe Interact, 1993 Jan-Feb, 6(1), 55 - 65 Genetic analysis of the Rhizobium meliloti exoYFQ operon: ExoY is homologous to sugar transferases and ExoQ represents a transmembrane protein; Muller P et al.; The nucleotide sequence of a 4.8-kb ClaI-EcoRI DNA fragment of megaplasmid 2 of Rhizobium meliloti Rm2011 involved in succinoglucan (EPS I) synthesis and nodule infection was determined . Four open reading frames (ORFs) were identified on this fragment . A mutational analysis revealed that these ORFs represent genes that were termed exoX, exoY, exoF, and exoQ . The locations of transposon insertions in these exo genes were determined at the nucleotide level . Plasmid integration mutagenesis revealed that the genes exoY, exoF, and exoQ are organized in an operon . The exoX gene running in opposite direction forms a monocistronic transcriptional unit . The exoX gene was shown to negatively influence the amount of EPS I synthesized . The exoY gene is coding for a membrane associated protein homologous to the C-terminal part of the Xanthomonas campestris glucosyltransferase GumD and the Salmonella typhimurium galactose transferase RfbP . ExoF, a probable periplasmatic protein, is nearly identical to the protein encoded by ORF1 of Rhizobium sp . strain NGR234 . ExoQ is most probably a membrane associated protein as deduced by its hydrophobic structural features . All three genes of the exoYFQ operon were shown to be essential for succinoglucan synthesis and nodule infection. Mol Plant Microbe Interact, 1993 Jan-Feb, 6(1), 127 - 34 pSym nod gene influence on elicitation of peroxidase activity from white clover and pea roots by rhizobia and their cell-free supernatants; Salzwedel JL et al.; The activities of salt-elutable peroxidases from roots of white clover and pea were examined during the early interaction of these legume hosts with strains of Rhizobium leguminosarum in homologous and heterologous combination . Peroxidase-specific activity from clover root hairs began to increase 6 hr after inoculation with R . l . bv . viciae RL300 and was localized over the entire area of their deformations . In contrast, the onset of elicitation of peroxidase activity from root hairs was delayed after inoculation with R . l . bv . trifolii ANU843 and was localized only at the site of infection thread initiation . Three wild-type strains (R . l . bv . trifolii ANU843, R . l . bv . viciae RL300 and 1003) and one hybrid transconjugant strain of R . leguminosarum containing pSym from R . l . bv . viciae 248 (RBL5715) elicited increased specific activity of peroxidases eluted from pea and clover roots in heterologous combination . A comparison of peroxidase activity eluted from pea roots inoculated with ANU843 or its pSym-cured derivative indicated that pSym is required for elicitation of peroxidase on this heterologous host . The level of peroxidase activity elicited by nodE mutants (which have extended host range) is decreased on their new host . An extracellular fraction of RL300 contained flavonoid-dependent, heat-stable, and ethanol-soluble elicitor(s) of peroxidase activity . Treatment of clover seedlings with this cell-free fraction decreased the number of root hairs infected by ANU843 . We propose that elicitation of root hair peroxidase may contribute to the infection process in this Rhizobium-legume symbiosis by altering root hair wall structure at sites of incipient penetration. FEMS Microbiol Rev, 1993 Jan, 10(1-2), 39 - 63 Regulation and function of rhizobial nodulation genes; Gottfert M; This review focuses on the functions of nodulation (nod) genes in the interaction between rhizobia and legumes . The nod genes are the key bacterial determinants of the signal exchange between the two symbiotic partners . The product of the nodD gene is a transcriptional activator protein that functions as receptor for a flavonoid plant compound . This signaling induces the expression of a set of nod genes that produces several related Nod factors, substituted lipooligosaccharides . The Nod factors are then excreted and serve as signals sent from the bacterium to the plant . The plant responds with the development of a root nodule . The plant-derived flavonoid, as well as the rhizobial signal, must have distinct chemical structures which guarantee that only matching partners are brought together. J Bacteriol, 1993 Jan, 175(2), 438 - 47 Multiple copies of nodD in Rhizobium tropici CIAT899 and BR816; van Rhijn PJ et al.; Rhizobium tropici strains are able to nodulate a wide range of host plants: Phaseolus vulgaris, Leucaena spp., and Macroptilium atropurpureum . We studied the nodD regulatory gene for nodulation of two R . tropici strains: CIAT899, the reference R . tropici type IIb strain, and BR816, a heat-tolerant strain isolated from Leucaena leucocephala . A survey revealed several nodD-hybridizing DNA regions in both strains: five distinct regions in CIAT899 and four distinct regions in BR816 . Induction experiments of a nodABC-uidA fusion in combination with different nodD-hybridizing fragments in the presence of root exudates of the different hosts indicate that one particular nodD copy contributes to nodulation gene induction far more than any other nodD copy present . The nucleotide sequences of both nodD genes are reported here and show significant homology to those of the nodD genes of other rhizobia and a Bradyrhizobium strain . A dendrogram based on the protein sequences of 15 different NodD proteins shows that the R . tropici NodD proteins are linked most closely to each other and then to the NodD of Rhizobium phaseoli 8002. J Bacteriol, 1993 Jan, 175(1), 94 - 102 Siderophore-mediated iron transport correlates with the presence of specific iron-regulated proteins in the outer membrane of Rhizobium meliloti; Reigh G et al.; A universal chemical assay used to detect the production of siderophores in a range of Rhizobium strains showed that production is strain specific . Iron nutrition bioassays carried out on Rhizobium meliloti strains to determine cross-utilization of their siderophores showed that R . meliloti 2011, 220-5, and 220-3 could each use the siderophores produced by the other two but not the siderophore produced by R . meliloti DM4 (and vice versa) . Mutants of R . meliloti 2011 and 220-5 defective in siderophore production were isolated by Tn5-mob mutagenesis . The Tn5-mob-containing EcoRI fragment of mutant R . meliloti 220-5-1 was cloned into pUC19 . By using this fragment as a probe, the presence of a homologous region was observed in R . meliloti 2011 and 220-3 but not in R . meliloti DM4 . A complementing cosmid from a gene bank of R . meliloti 2011 was identified by using the same probe . Introduction of this cosmid into R . meliloti 102F34, a strain not producing a siderophore, resulted in the ability of this strain to produce a siderophore and also in the ability to utilize the siderophores produced by R . meliloti 2011, 220-5, and 220-3 but not the siderophore produced by R . meliloti DM4 . A comparative analysis of the outer membrane proteins prepared from iron-deficient cultures of R . meliloti 102F34 and 102F34 harboring the cosmid revealed the presence, in the latter, of a low-iron-induced outer membrane protein corresponding to a low-iron-induced protein in R . meliloti 2011, 220-5, and 220-3 . This protein is not present in R . meliloti DM4 . The results suggest that R . meliloti 2011, 220-5, and 220-3 produce siderophores that are identical or sufficiently similar in structure to be transported by the membrane transport system of each strain while also indicating that utilization of a particular siderophore is correlated with the presence of specific outer membrane proteins. Res Microbiol, 1993 Jan, 144(1), 55 - 67 Enzyme polymorphism of Azorhizobium strains and other stem- and root-nodulating bacteria isolated from Sesbania rostrata; Rinaudo G et al.; Relationships between bacterial groups nodulating Sesbania rostrata were evaluated through examination of electrophoretic polymorphism of esterases and metabolic enzymes . The following conclusions were drawn: (i) the differentiation of two genomic species within Azorhizobium strains and a group of non-identified strains (probably Rhizobium) was strongly supported by enzyme electrophoresis; (ii) esterases were more electrophoretically polymorphic than metabolic enzymes, since 35 and 11 electrophoretic types, respectively, were detected within the 57 strains studied; (iii) strains isolated from stem or root nodules were genetically very similar and could not be differentiated; (iv) six Azorhizobium strains isolated from plants growing in saline soils could not be grouped separately from the other strains, which might be attributed to the adaptation of azorhizobia to epiphytic conditions; and (v) a comparative study of esterase patterns of azorhizobia showed that strains isolated in the Philippines probably originated in northern Senegal, but did not reveal a clear separation between strains originating from northern and central Senegal. Antonie Van Leeuwenhoek, 1993, 64(1), 1 - 8 Influence of growth conditions on production of capsular and extracellular polysaccharides by Rhizobium leguminosarum; Breedveld MW et al.; The influence of growth rate and medium composition on exopolymer production by Rhizobium leguminosarum was studied . When grown in medium containing 10 g/l mannitol and 1 g/l glutamic acid, Rhizobium leguminosarum biovar trifolii TA-1 synthesized up to 2.0 g/l of extracellular polysaccharide (EPS), and up to 1.6 g/l of capsular polysaccharide (CPS) . Under non-growing cell conditions in medium without glutamic acid, CPS synthesis by strain TA-1 could proceed to 2.1 g/l, while EPS-production remained relatively low (0.8 g/l) . Maximal CPS-yield was 2.9 g CPS/l medium in a medium containing 20 g/l mannitol and 2 g/l glutamic acid . The EPS-deficient strain R . leguminosarum RBL5515, exo4::Tn5 was able to produce CPS to similar levels as strain TA-1, but CPS-recovery was easier because of the low viscosity of the medium and growth of the cells in pellets . With strain TA-1 in nitrogen-limited continuous cultures with a constant biomass of 500 mg cell protein/l, EPS was the most abundant polysaccharide present at every dilution rate D (between 0.12 and 0.02 h-1) . The production rates were 50-100 mg/g protein/h for EPS and 15-20 mg/g protein/h for CPS . Only low amounts of cyclic beta-(1,2)-glucans were excreted (10-30 mg/l) over the entire range of growth rates. Acta Biochim Pol, 1993, 40(4), 477 - 82 Homology of genes for exopolysaccharide synthesis in Rhizobium leguminosarum and effect of cloned exo genes on nodule formation; Skorupska A et al.; A 5.4 kb BamHI fragment of R . leguminosarum bv . trifolii TA1 was found to carry genes involved in exopolysaccharide synthesis (exo genes) . This fragment was strongly hybridized to the total DNA from R . l . bv . viciae and bv . phaseoli digested with EcoRI . No homology was found with total DNA of R . meliloti and Rhizobium sp . NGR 234 . The exo genes from R . l . bv . trifolii TA1 conjugally introduced into R . l . bv . viciae 1302 considerably affected the symbiosis: the nodules induced on vetch were abortive and did not fix nitrogen . On the other hand, Phaseolus beans infected with R . l . bv . phaseoli harbouring R . l . bv . trifolii exo genes formed the nitrogen-fixing nodules . It can be concluded that additional copies of exo genes introduced into wild type Rhizobium leguminosarum strains can disturb the synthesis of acidic exopolysaccharides and affect symbiosis of the plants forming indeterminate nodules, but do not affect symbiosis of the plants forming the determinate nodules. Mol Plant Microbe Interact, 1993 Jan-Feb, 6(1), 135 - 43 Rhizobium meliloti mutants with decreased DAHP synthase activity are sensitive to exogenous tryptophan and phenylalanine and form ineffective nodules; Jelesko JG et al.; We isolated two Tn5-generated mutants of Rhizobium meliloti whose growth was inhibited by rich medium or by exogenous tryptophan or phenylalanine . These mutants, Rm7479 and Rm7480, belonged to the same genetic complementation group . The mutant locus could not be found on either indigenous megaplasmid but was localized on the chromosome . The mutants formed ineffective nodules on alfalfa plants . They invaded nodules within infection threads and were released into plant cells enclosed within peribacteroid membranes, but once released into the plant cells they failed to differentiate into mature bacteroids . The mutants demonstrated a decrease in total 2-keto-3-deoxy-D-arabino-heptonic acid 7-phosphate synthase (DAHP synthase) activity, which is the first committed step in aromatic biosynthesis . Wild-type genes were isolated that complemented in one case or suppressed in another case, all three mutant phenotypes: growth on rich medium, symbiotic effectiveness, and DAHP synthase activity . Each mutant strain gave rise to linked second-site suppressor mutations that restored growth on rich medium . The suppressor mutants showed restoration of near wild-type DAHP synthase levels . One of the suppressor strains restored effective symbiosis while the other did not . Genetic complementation experiments showed that growth on rich medium, DAHP synthase activity, and effective symbiosis were all affected by the same genetic lesion . These results suggest that normal flux of metabolites through the aromatic biosynthesis pathway is essential for bacteroid development. Chin J Biotechnol, 1993, 9(3), 137 - 41 Tn5-Mob transposon mediated transfer of salt tolerance and symbiotic characteristics between Rhizobia genera; Yang S et al.; Rhizobium meliloti 042B is a fast-growing, salt-tolerant and high efficiency nitrogen-fixing symbiont with alfalfa . Bradyrhizobium japonicum USDA110 grows slowly, and cannot grow in YMA medium containing 0.1M NaCl, but nodulates and fixed nitrogen efficiently with soybean . Eighty-six transconjugants, called SR, were obtained by inserting Tn5-Mob randomly into genomes of 042B using pSUP5011 and helper plasmid RP4 . Selecting 4 SR strains randomly and introducing DNA fragment of SR into USDA110 with helper plasmid R68.45 by triparental mating, 106 transconjugants, called BSR, were constructed . Most of BSR strains had the fast-growing phenotype and could tolerate 0.3-0.5M NaCl generally . Some of them produced melanine . When soybean and alfalfa were inoculated with these transconjugants BSR, 47 out of 90 BSR were found to nodulate in both of these plants, but no nitrogenase activity was observed with alfalfa; 26 strains could only nodulate and fix nitrogen in soybean; 13 strains could nodulate in alfalfa but did not fix nitrogen; 4 strains failed to nodulate in either soybean or alfalfa . Among them, 4 transconjugants which tolerated and fixed nitrogen efficiently in soybean were constructed. Plant Mol Biol, 1993 Jan, 21(2), 375 - 80 Identification of two alfalfa early nodulin genes with homology to members of the pea Enod12 gene family; Allison LA et al.; In a search for plant genes expressed during early symbiotic interactions between Medicago sativa and Rhizobium meliloti, we have isolated and characterized two alfalfa genes which have strong sequence similarity to members of the Enod12 gene family of Pisum sativum . The M . sativa genes, MsEnod12A and B, encode putative protein products of 8066 Da and 12849 Da, respectively, each with a signal sequence at the N-terminus followed by a repetitive proline-rich region . Based on their expression during the initial period of nodule development, MsEnod12A and B are alfalfa early nodulin genes. J Mol Biol, 1992 Dec 5, 228(3), 998 - 1002 Nucleotide sequence and organization of an H2-uptake gene cluster from Rhizobium leguminosarum bv . viciae containing a rubredoxin-like gene and four additional open reading frames; Rey L et al.; The nucleotide sequence of a 3.2 kb region following the hydrogenase structural operon (hupSLCDEF) in the H2-uptake gene cluster from Rhizobium leguminosarum by viciae strain 128C53 has been determined . Five closely linked genes encoding products of 16.3 (HupG), 30.5 (HupH), 8.0 (HupI), 18.4 (HupJ) and 38.7 (HupK) kDa were identified 166 bp downstream from hupF . Transposon insertions into hupG, hupH, hupJ and hupK suppress the H2-oxidizing capability of the wild-type strain . The amino acid sequence deduced from hupI contains two Cys-X-X-Cys motifs, characteristic of rubredoxins, separated by 29 amino acid residues showing strong sequence homology with other bacterial rubredoxins . The amino acid-derived sequence from hupG and hupH showed homology to products from genes hyaE and hyaF of the operon encoding hydrogenase 1 from Escherichia coli, and hupJ and hupK were related to open reading frames identified in Rhodobacter capsulatus and Azotobacter vinelandii hydrogenase gene clusters . An involvement of the hupGHIJK gene cluster in redox reactions related to hydrogenase synthesis or activity is predicted on the basis of the function as electron carrier attributed to rubredoxin. Mol Microbiol, 1992 Dec, 6(23), 3575 - 84 Broad-host-range Rhizobium species strain NGR234 secretes a family of carbamoylated, and fucosylated, nodulation signals that are O-acetylated or sulphated; Price NP et al.; Rhizobium species strain NGR234 is the most promiscuous known rhizobium . In addition to the non-legume Parasponia andersonii, it nodulates at least 70 genera of legumes . Here we show that the nodulation genes of this bacterium determine the production of a large family of Nod-factors which are N-acylated chitin pentamers carrying a variety of substituents . The terminal non-reducing glucosamine is N-acylated with vaccenic or palmitic acids, is N-methylated, and carries varying numbers of carbamoyl groups . The reducing N-acetyl-glucosamine residue is substituted on position 6 with 2-O-methyl-L-fucose which may be acetylated or sulphated or non-substituted . All three internal residues are N-acetylated . At pico- to nanomolar concentrations, these signal molecules exhibit biological activities on the tropical legumes Macroptilium and Vigna (Phaseoleae), as well as on both the temperate genera Medicago (Trifoliae) and Vicia (Viciae) . These data strongly suggest that the uniquely broad host range of NGR234 is mediated by the synthesis of a family of varied sulphated and non-sulphated lipo-oligosaccharide signals. Mol Microbiol, 1992 Dec, 6(23), 3479 - 92 Tandem DctD-binding sites of the Rhizobium meliloti dctA upstream activating sequence are essential for optimal function despite a 50- to 100-fold difference in affinity for DctD; Ledebur H et al.; The Rhizobium meliloti genes dctB and dctD positively regulate the expression of dctA, which encodes a C4-dicarboxylate transport protein . Here we characterize an element (UAS) located upstream of dctA that has tandem binding sites for the dctD gene product (DctD) . At relatively low concentrations of active DctD, the element activated dctA transcription, but at relatively high concentrations of DctD it was inhibitory . The UAS failed to function when placed further upstream of dctA . Both DctD-binding sites were required for optimal UAS function, despite a 50- to 100-fold difference in binding affinities . Moving the promoter distal binding site 5 bp further upstream was functionally equivalent to its deletion . Based on these data, we hypothesize that the sigma 54-dependent activator DctD binds co-operatively to the R . meliloti dctA UAS, and that occupancy of both sites is required for maximal activation of dctA. Glycobiology, 1992 Dec, 2(6), 535 - 9 A new method for the analysis of amide-linked hydroxy fatty acids in lipid-As from gram-negative bacteria; Bhat UR et al.; Lipid-A represents the ubiquitous, covalently bound hydrophobic component of bacterial lipopolysaccharides (endotoxins) . Lipid-As isolated and characterized from rhizobial species have large variations in their backbone sugars, as well as in their hydroxy fatty acid substituents . The sugar backbones consist of either glucosamine and galacturonic acid or glucosamine and 2,3-diaminoglucose . The published procedures for characterizing amide-linked fatty acids do not release all these fatty acids, hence a new method was developed to characterize the amide-linked hydroxy fatty acids . This method involves a mild methanolysis procedure to release glucosamine methyl glycosides which still contain the amide-bound hydroxy fatty acids . The products were analysed by fast atom bombardment mass spectrometry (FAB-MS) and, after trimethylsilylation, by electron impact (E.I.) and chemical ionization (C.I.) gas chromatography-mass spectrometry (GC-MS) . The procedure was applied to lipid-A preparations from several gram-negative bacteria . This method allows the unequivocal identification of amide-linked hydroxy fatty acids and also allows determination of the microheterogeneity of the N-acyl substituents in lipid-As from gram-negative bacteria. Plant Mol Biol, 1992 Dec, 20(6), 1089 - 96 Nucleosomal structure and histone H1 subfractional composition of pea (Pisum sativum) root nodules, radicles and callus chromatin; Bers EP et al.; Higher-order packaging of DNA in chromatin structures could be an essential step in the complex chain of events leading to activation/repression of eukaryotic gene expression . With the goal to investigate this aspect of transcriptional regulation of plant genes involved in symbiotic interactions between legumes and rhizobia we analyze here the molecular parameters of chromatin structure in functioning root nodules, callus and radicles of pea . Morphological intactness and the typical nucleosomal organization are preserved in purified nuclei isolated from all three sources . The calculated values of nucleosomal repeat changed from 185 +/- 5 bp in the nuclei of radicles to 168 +/- 5 bp and 195 +/- 6 bp in nodules and callus respectively . The observed changes are due to alterations in linker DNA lengths . The core histones are identical in all cases, but the subfractional composition of H1 linker histone is subjected to quantitative alterations . The most pronounced is the several-fold increase in content of the lowest-molecular-weight subfraction H1-6 which takes place during nodule development. Plant Mol Biol, 1992 Dec, 20(6), 1049 - 58 Mutational analysis of pea lectin . Substitution of Asn125 for Asp in the monosaccharide-binding site eliminates mannose/glucose-binding activity; van Eijsden RR et al.; As part of a strategy to determine the precise role of pea (Pisum sativum) lectin, Psl, in nodulation of pea by Rhizobium leguminosarum, mutations were introduced into the genetic determinant for pea lectin by site-directed mutagenesis using PCR . Introduction of a specific mutation, N125D, into a central area of the sugar-binding site resulted in complete loss of binding of Psl to dextran as well as of mannose/glucose-sensitive haemagglutination activity . As a control, substitution of an adjacent residue, A126V, did not have any detectable influence on sugar-binding activity . Both mutants appeared to represent normal Psl dimers with a molecular mass of about 55 kDa, in which binding of Ca2+ and Mn2+ ions was not affected . These results demonstrate that the NHD2 group of Asn125 is essential in sugar binding by Psl . To our knowledge, Psl N125D is the first mutant legume lectin which is unable to bind sugar residues . This mutant could be useful in the identification of the potential role of the lectin in the recognition of homologous symbionts. Plant Mol Biol, 1992 Dec, 20(5), 977 - 86 Rhizobial lipo-oligosaccharides: answers and questions; Spaink HP; Rhizobium bacteria produce certain lipo-oligosaccharides (modified chitin oligomers) after induction of nodulation (nod) gene transcription by the host plant . The function of the rhizobial nod genes in the biosynthesis of these lipo-oligosaccharides, focusing on their host specific aspects, is discussed . The lipo-oligosaccharides can elicit various responses in the host plants, like the formation of pre-infection threads and nodule meristems . Speculating on their function in plant morphogenesis the question is raised: do the rhizobial lipo-oligosaccharides resemble unknown plant signal molecules? J Bacteriol, 1992 Dec, 174(24), 7910 - 8 Structure and expression of a cyanobacterial ilvC gene encoding acetohydroxyacid isomeroreductase; Rieble S et al.; Acetohydroxyacid isomeroreductase (AHAIR) is the shared second enzyme in the biosynthetic pathways leading to isoleucine and valine . AHAIR is encoded by the ilvC gene in bacteria . A 1,544-bp fragment of genomic DNA containing the ilvC gene was cloned from the cyanobacterium Synechocystis sp . strain PCC 6803, and the complete nucleotide sequence was determined . The identity of the gene was established by comparison of the nucleotide and derived peptide sequences with those of other ilvC genes . The highest degree of sequence similarity was found with the ilvC gene from Rhizobium meliloti . The isolated Synechocystis ilvC gene complemented an Escherichia coli ilvC mutant lacking AHAIR activity . The expressed Synechocystis gene encodes a protein that has a molecular mass of 35.7 kDa and that has AHAIR activity in an in vitro assay . Polyclonal antibodies raised against purified Synechocystis AHAIR produced a single band on a Western blot (immunoblot) of a Synechocystis cell extract and detected the protein in an extract of an E . coli ilvC mutant strain that was transformed with a plasmid containing the Synechocystis ilvC gene . The antibody did not react with an extract of an E . coli ilvC mutant strain that was transformed with a control plasmid lacking the Synechocystis ilvC gene or with an extract of an E . coli IlvC+ control strain. Genetics, 1992 Dec, 132(4), 899 - 909 Rhizobium meliloti genes involved in sulfate activation: the two copies of nodPQ and a new locus, saa; Schwedock JS et al.; The nitrogen-fixing symbiont Rhizobium meliloti establishes nodules on leguminous host plants . Nodulation (nod) genes used for this process are located in a cluster on the pSym-a megaplasmid of R . meliloti . These genes include nodP and nodQ (here termed nodPQ), which encode ATP sulfurylase and APS kinase, enzymes that catalyze the conversion of ATP and SO(4)2- into the activated sulfate form 3'-phosphoadenosine 5'-phosphosulfate (PAPS), an intermediate in cysteine synthesis . In Rhizobium, PAPS is also a precursor for sulfated and N-acylated oligosaccharide Nod-factor signals that cause symbiotic responses on specific host plants such as alfalfa . We previously found a highly conserved second copy of nodPQ in R . meliloti . We report here the mapping and cloning of this second copy, and its location on the second megaplasmid, pSym-b . The function of nodP2Q2 is equivalent to that of nodP1Q1 in complementation tests of R . meliloti and Escherichia coli mutants in ATP sulfurylase and adenosine 5'-phosphosulfate (APS) kinase . Mutations in nodP2Q2 do not have as severe an effect on symbiosis or plant host range as do those in nodP1Q1, however, possibly reflecting differences in expression and/or channeling of metabolites to specific enzymes involved in sulfate transfer . Strains mutated or deleted for both copies of nodQ are severely defective in symbiotic phenotypes, but remain prototrophic . This suggests the existence in R . meliloti of a third locus for ATP sulfurylase and APS kinase activities . We have found a new locus saa (sulfur amino acid), which may also encode these activities. Gene, 1992 Dec 1, 122(1), 35 - 43 The Rhizobium meliloti pmi gene encodes a new type of phosphomannose isomerase; Schmidt M et al.; Interspecific complementation of a Xanthomonas campestris pv . campestris phosphomannose isomerase (PMI) mutant was used to isolate a cosmid from a genomic library of Rhizobium meliloti 2011 carrying the pmi gene of this strain . Subcloning experiments localized the coding region to a 2.0-kb SalI-ClaI fragment . Nucleotide sequence analysis of this fragment indicated the presence of two open reading frames (ORFs), coding for 18- and 43-kDa polypeptides . The analysis of the gene function by gene disruption experiments showed that ORF2 codes for pmi . A comparison of the deduced amino acid sequence with the corresponding sequences of the Pseudomonas aeruginosa and Escherichia coli PMIs revealed no significant homology, indicating that the isolated gene encodes a new type of PMI . The construction of a pmi-deficient mutant of R . meliloti using the sacB-sacR cassette technique showed that the loss of PMI activity does not affect the symbiotic properties of this strain. J Bacteriol, 1992 Dec, 174(23), 7555 - 65 Rhizobium nodM and nodN genes are common nod genes: nodM encodes functions for efficiency of nod signal production and bacteroid maturation; Baev N et al.; Earlier, we showed that Rhizobium meliloti nodM codes for glucosamine synthase and that nodM and nodN mutants produce strongly reduced root hair deformation activity and display delayed nodulation of Medicago sativa (Baev et al., Mol . Gen . Genet . 228:113-124, 1991) . Here, we demonstrate that nodM and nodN genes from Rhizobium leguminosarum biovar viciae restore the root hair deformation activity of exudates of the corresponding R . meliloti mutant strains . Partial restoration of the nodulation phenotypes of these two strains was also observed . In nodulation assays, galactosamine and N-acetylglucosamine could substitute for glucosamine in the suppression of the R . meliloti nodM mutation, although N-acetylglucosamine was less efficient . We observed that in nodules induced by nodM mutants, the bacteroids did not show complete development or were deteriorated, resulting in decreased nitrogen fixation and, consequently, lower dry weights of the plants . This mutant phenotype could also be suppressed by exogenously supplied glucosamine, N-acetylglucosamine, and galactosamine and to a lesser extent by glucosamine-6-phosphate, indicating that the nodM mutant bacteroids are limited for glucosamine . In addition, by using derivatives of the wild type and a nodM mutant in which the nod genes are expressed at a high constitutive level, it was shown that the nodM mutant produces significantly fewer Nod factors than the wild-type strain but that their chemical structures are unchanged . However, the relative amounts of analogs of the cognate Nod signals were elevated, and this may explain the observed host range effects of the nodM mutation . Our data indicate that both the nodM and nodN genes of the two species have common functions and confirm that NodM is a glucosamine synthase with the biochemical role of providing sufficient amounts of the sugar moiety for the synthesis of the glucosamine oligosaccharide signal molecules. Biochimie, 1992 Dec, 74(12), 1121 - 3 Protein IIIa of Rhizobium leguminosarum is probably a porin; Chevalier G et al.; The cloning, sequencing and expression of the gene encoding the 36-kilodalton (kDa) outer membrane protein of Rhizobium leguminosarum has been recently described in the literature (De Maagd RA et al (1992) J Bacteriol 174, 214-221) . We present evidence that this protein is a porin from a sub-type covalently bound to the peptidoglycan. Mol Microbiol, 1992 Nov, 6(22), 3395 - 404 The Rhizobium leguminosarum FnrN protein is functionally similar to Escherichia coli Fnr and promotes heterologous oxygen-dependent activation of transcription; Schluter A et al.; An open reading frame from Rhizobium leguminosarum bv . viciae strain VF39, previously identified and found to be similar to Escherichia coli fnr and Rhizobium meliloti fixK (orf240, thereafter called fnrN), was further analysed . Analysis of the expression of an fnrN-lacZ transcriptional fusion revealed that fnrN is preferentially expressed under oxygen limitation . Using R . meliloti fixN-lacZ fusions it was shown that the fnrN gene product only mediates transcriptional activation under microaerobiosis, indicating that the FnrN protein responds, directly or indirectly, to oxygen . Plasmids which expressed fnrN under the control of an E . coli promoter were able to complement an E . coli fnr mutant with respect to anaerobic growth on nitrate but not fumarate, and to promote anaerobic but not aerobic activation of the Fnr-dependent E . coli genes narGHJI, nirB and fdnGHI coding for nitrate reductase, NADH-dependent nitrite reductase and formate dehydrogenase-N, respectively . Fumarate and DMSO reductase activities were not induced by FnrN . The E . coli fnr gene substituted for fnrN in oxygen-regulated transcription of nirB- and fixN-lacZ fusions in R . leguminosarum . The results indicate that Fnr and FnrN are functionally very similar and share a common mode of oxygen-dependent transcriptional activation . From hybridization studies, it appeared that fnrN-like genes are present in a number of different R . leguminosarum strains. Mol Microbiol, 1992 Nov, 6(22), 3321 - 30 Differential expression of nodS accounts for the varied abilities of Rhizobium fredii USDA257 and Rhizobium sp . strain NGR234 to nodulate Leucaena spp; Krishnan HB et al.; Transfer of a cosmid containing nodSU from Rhizobium sp . NGR234 to Rhizobium fredii USDA257 expands the host range for nodulation to include the perennial tropical legumes, Leucaena leucocephala and Leucaena diversifolia . Complementation experiments with a series of subclones established that nodS and its associated nod-box promoter from NGR234 are sufficient to confer this extended host-range phenotype to L . leucocephala . Strain USDA257 contains its own copy of nodSU, including upstream nod-box sequences . Although both nucleotide and deduced amino acid sequences of the reading frames are homologous between the two strains, there are gaps within the promoter region and the 5'-end of nodS of USDA257 . Consequently, the deduced NodS protein of USDA257 is shorter than its counterpart from NGR234, and the distance between the nod-box and the initiation codon is greater . A 36 bp deletion encompasses the extreme right border of the USDA257 nod-box and extends into the upstream leader sequence . Transcriptional fusions with both nod-boxes confirmed that the promoter from NGR234 is flavonoid-inducible, and that the nod-box from USDA257 is not . These observations were corroborated by Northern analysis with a nodS-containing Xhol fragment as hybridization probe . Flavonoid-induced cells of NGR234 gave an intense signal, but those of USDA257 yielded only a weak trace of hybridization . EcoRI fragments with homology to nodSU of USDA257 are present in 17 of 35 tested strains, including several representatives of Bradyrhizobium japonicum, Rhizobium sp., R . loti, and R . fredii . Two wild-type, leucaena-nodulating strains of Rhizobium sp . lack this homology . We conclude that a genetic defect in expression of nodS accounts for the inability of USDA257 to nodulate leucaena and that diverse rhizobia may have evolved alternative mechanisms to nodulate this legume species. Mol Plant Microbe Interact, 1992 Nov-Dec, 5(6), 484 - 8 Evaluation of acidic heteropolysaccharide structures in Rhizobium leguminosarum biovars altered in nodulation genes and host range; Orgambide G et al.; 1H-NMR spectroscopy showed that the extracellular heterpolysaccharides (EPS) from derivatives of Rhizobium leguminosarum bv . trifolii ANU843 altered in pSym nod composition or function (transposon insertions, deletion of pSym, induction by flavone, and introduction of cloned pSym nod regions from ANU843 and R . l . bv . viciae 248 on recombinant plasmids into the pSym-cured background of ANU843) differed only in 3-hydroxybutyrate stoichiometry per octaglycosyl unit . This change in EPS was likely to be an indirect effect of altered growth during expression of pSym nod genes in the presence of the flavone . No modifications were found in EPS made by R . l . bv . phaseoli 8002 when its resident pSym was deleted or replaced with pSym from R . l . bv . viciae 248, or with a derivative of this pSym lacking the host-specific nodulation genes nodFELMNTO . Thus, although certain O-acyl noncarbohydrate substitutions in EPS are affected by pSym nod genes (including the ones that determine host range) in certain backgrounds of R . leguminosarum, this change does not occur universally among all strains of R . leguminosarum . We conclude that the structure of the acidic EPS does not control host-specific nodulation of white clover, hairy vetch, and beans for the strains of R . leguminosarum tested here. Mol Microbiol, 1992 Nov, 6(21), 3137 - 47 Molecular and symbiotic characterization of exopolysaccharide-deficient mutants of Rhizobium tropici strain CIAT899; Milner JL et al.; We studied the symbiotic behaviour of 20 independent Tn5 mutants of Rhizobium tropici strain CIAT899 that were deficient in exopolysaccharide (EPS) production . The mutants produced non-mucoid colonies, were motile, grew in broth cultures at rates similar to those of the parent, and produced significantly less EPS than did CIAT899 in broth culture . A genomic library of strain CIAT899, constructed in pLA2917, was mobilized into all of the mutants, and cosmids that restored EPS production were identified . EcoRI restriction digests of the cosmids revealed nine unique inserts . Mutant complementation and hybridization analysis showed that the mutations affecting EPS production fell into six functional and physical linkage groups . On bean, the mutants were as efficient in nodulation and as effective in acetylene reduction as strain CIAT899, induced a severe interveinal chlorosis, and all but one were less competitive than CIAT899 . On siratro, CIAT899 induced nodules that were ineffective in acetylene reduction, whereas the EPS-deficient mutants induced effective nodules . Microscopic examination of thin sections showed that nodules from both siratro and bean plants inoculated with either CIAT899 or an EPS-deficient mutant contained infected cells . These data indicate that EPS is not required for normal nodulation of bean by R . tropici, that it may contribute to competitiveness of R . tropici on bean, and that the loss of EPS production is accompanied by acquisition of the ability to reduce acetylene on siratro. J Bacteriol, 1992 Nov, 174(22), 7486 - 9 A region of the broad-host-range plasmid RK2 causes stable in planta inheritance of plasmids in Rhizobium meliloti cells isolated from alfalfa root nodules; Weinstein M et al.; We demonstrate for the first time that the broad-host-range stabilization loci from plasmid RK2 cause total retention of plasmids in cells of Rhizobium meliloti during symbiosis with alfalfa . Two derivatives of plasmid RK2, pRK290 and a 7.3-kb mini-RK2 plasmid, were stabilized in R . meliloti cells isolated from root nodules by the insertion of a 3.2-kb DNA fragment or a smaller 0.8-kb DNA fragment derived from the RK2 stabilization region. Infect Immun, 1992 Nov, 60(11), 4496 - 503 Identification of 2,3-dihydroxybenzoic acid as a Brucella abortus siderophore; Lopez-Goni I et al.; Brucella abortus grown in low-iron medium or in the presence of iron chelators {ethylenediamine-di(o-hydroxyphenylacetic acid) and 2,2-dipyridyl} showed reduced cell yields and released a material positive in chemical and biological assays for catechols . This material was purified from culture fluids of B . abortus 2308 by chromatography on agarose-iminodiacetic acid-Fe3+ and identified as 2,3-dihydroxybenzoic acid (2,3-DHBA) by thin-layer chromatography, paper electrophoresis, and UV-visible nuclear magnetic resonance and mass spectroscopy . No other major catechols were observed at different stages of growth, and 2,3-DHBA was also produced upon iron limitation by representative strains of B . abortus biotypes 1, 5, 6, and 9 . Both synthetic 2,3-DHBA and the natural catechol relieved the growth inhibition of B . abortus 2308 by ethylenediamine-di(o-hydroxyphenylacetic acid), and 2,3-DHBA promoted 55Fe uptake by B . abortus 2308 by an energy-dependent mechanism . Two other monocatechols tested, 2,3-dihydroxybenzoyl-Ser and 2,3-dihydroxybenzoyl-Gly, also promoted 55Fe uptake . More complex catechol siderophores (agrobactin and enterobactin), hydroxamate siderophores (aerobactin, ferrichrome, and deferriferrioxamine mesylate {Desferal}), and an EDTA-related siderophore (rhizobactin) failed to mediate 55Fe uptake . B . abortus cells grown in low-iron medium or in medium with iron had similar rates of iron uptake when supplied with 55Fe-2,3-DHBA, and the release of 2,3-DHBA under iron starvation was not associated with the expression of new outer membrane proteins . These results suggest an uptake system in which only the synthesis of the siderophore is regulated by the iron available for growth. Cell, 1992 Oct 16, 71(2), 191 - 9 Molecular signals in the interactions between plants and microbes; Clarke HR et al.; The field of plant-microbe interactions has witnessed several recent breakthroughs, such as the molecular details of vir gene induction, identification of Nod factors, and the cloning and characterization of avr genes . Other breakthroughs, such as the cloning and characterization of R genes, appear imminent . Parallels to mammalian systems are emerging in the world of plant-microbe interactions, for example, ion channels formed by Rhizobium proteins, similarities of hrp genes to pathogenicity genes of mammalian pathogens, and plant signal transduction via calcium and protein phosphorylation . We remain, however, largely ignorant of many facets of signaling in plant-microbe interactions . We know little about how microbial signals are perceived by plants or how subsequent signal transduction occurs within plant cells and are probably unaware of many of the microbe-generated signals to which plants respond or of plant-generated signals to which bacteria and fungi respond . Contributions from those working on the genetics, molecular biology, and physiology of bacteria, fungi, and plants will be required to address these questions . The many nonpathogenic plant-microbe interactions in addition to the Rhizobium-plant interaction remain relatively unexplored . Genetic and molecular approaches are being initiated to investigate the signaling that is likely to underlie interactions such as those between mycorrhizal fungi and plant roots and between epiphytic bacteria and plant leaf surfaces . The importance of these interactions to plant growth and development makes it likely that they will figure more prominently at future symposia.(ABSTRACT TRUNCATED AT 250 WORDS) FEMS Microbiol Lett, 1992 Oct 15, 76(3), 249 - 54 Eight bacterial proteins, including UDP-N-acetylglucosamine acyltransferase (LpxA) and three other transferases of Escherichia coli, consist of a six-residue periodicity theme; Vaara M; Only a few prokaryotic or eukaryotic enzymes are known to consist of a tandem-repeat structure . This report describes a common hexapeptide-repeat theme in four Escherichia coli transferases and in four less-characterized bacterial proteins . The proteins are the Ssc protein of Salmonella typhimurium (25), UDP-N-acetylglucosamine acyltransferase of E . coli (24), the hypothetical proteins Tms of Bacillus subtilis (23) and Yglm of E . coli (22), succinyldiaminopimelate aminotransferase of E . coli (14), serine acetyltransferase of E . coli (13), NodL of Rhizobium leguminasorum (13), and thiogalactoside acetyltransferase of E . coli (8) (number of repeats indicated in parentheses) . In UDP-N-acetylglucosamine acyltransferase, the repeats constitute 55% of the total protein . Each hexapeptide repeat of the eight proteins starts with Ile, Leu, or Val . Position b is occupied by Gly, position d by Gly, Asn, or Asp, and position e by Val or Ala in 52%, 54%, and 56% of the hexapeptide repeats, respectively. Gene, 1992 Oct 12, 120(1), 125 - 6 Sequence of ISRm4 from Rhizobium meliloti strain GR4; Soto MJ et al.; ISRm4, an IS-like sequence structurally similar to Pseudomonas cepacia insertion element IS402, was identified by sequence analysis . This 933-bp element carries 17-bp putative terminal inverted repeats with five mismatches and a putative direct target duplication of 3 bp. J Mol Biol, 1992 Oct 5, 227(3), 602 - 20 Role of integration host factor in stimulating transcription from the sigma 54-dependent nifH promoter; Santero E et al.; In a wide variety of nitrogen-fixing organisms among the Purple Bacteria (large division of Gram-negative bacteria) the nitrogen fixation (nif) operons are transcribed by an alternative holoenzyme form of RNA polymerase, sigma 54-holoenzyme . Transcription depends on the activator protein NIFA (nitrogen fixation protein A), which catalyzes isomerization of closed complexes between this polymerase and a promoter to transcriptionally productive open complexes . NIFA-mediated activation of transcription from the nifH promoter of Klebsiella pneumoniae is greatly stimulated by the integration host factor IHF, which binds to a site between the upstream binding site for NIFA and the promoter, and bends the DNA . IHF fails to stimulate activation of transcription from this promoter by another activator of sigma 54-holoenzyme, NTRC (nitrogen regulatory protein C), which lacks a specific binding site in the nifH promoter region . As predicted, if the IHF-induced bend facilitates interaction between NIFA and sigma 54-holoenzyme, substitution of an NTRC-binding site for the NIFA-binding site allowed IHF to stimulate NTRC-mediated activation of transcription from the nifH promoter . The stimulation was of the same order of magnitude as that for NIFA in the native configuration of the promoter-regulatory region (up to 20-fold) . With purified NTRC and the substitution construct we could demonstrate that stimulation by IHF in a purified transcription system was comparable to that in a crude coupled transcription-translation system, indicating that the stimulation in the crude system could be accounted for by IHF . The IHF stimulation was observed on linear as well as supercoiled templates, indicating that the geometric requirements are relatively simple . We have attempted to visualize the arrangement of proteins on DNA fragments carrying the nifH promoter-regulatory region of K . pneumoniae by electron microscopy . IHF stimulated NIFA-mediated activation of transcription from the nifH and nifD promoters of Bradyrhizobium japonicum and less so from the nifH promoters of Rhizobium meliloti and Thiobacillus ferrooxidans, consistent with previous observations that stimulation is greatest at promoters that are weak binding sites for sigma 54-holoenzyme in closed complexes. Mol Microbiol, 1992 Oct, 6(20), 2897 - 903 Molecular mechanisms of attachment of Rhizobium bacteria to plant roots; Smit G et al.; Attachment of bacteria to plant cells is one of the earliest steps in many plant-bacterium interactions . This review covers the current knowledge on one of the best-studied examples of bacterium-plant attachment, namely the molecular mechanism by which Rhizobium bacteria adhere to plant roots . Despite differences in several studies with regard to growth conditions of bacteria and plants and to methods used for measuring attachment, an overall consensus can be drawn from the available data . Rhizobial attachment to plant root hairs appears to be a two-step process . A bacterial Ca(2+)-binding protein, designated as rhicadhesin, is involved in direct attachment of bacteria to the surface of the root hair cell . Besides this step, there is another step which results mainly in accumulation and anchoring of the bacteria to the surface of the root hair . This leads to so-called firm attachment . Depending on the growth conditions of the bacteria, the latter step is mediated by plant lectins and/or by bacterial appendages such as cellulose fibrils and fimbriae . The possible role of these adhesions in root nodule formation is discussed. Can J Microbiol, 1992 Oct, 38(10), 1009 - 15 Characterisation of Rhizobium isolates by amplification of DNA polymorphisms using random primers; Harrison SP et al.; The use of single random primers, selected in the absence of target sequence information, has been shown to be effective in producing DNA amplifications that provide fingerprints which are unique to individual organisms . DNA amplification by random priming was applied to the DNA from isolates of Rhizobium leguminosarum biovar trifolii . Amplification products were produced using a number of primers, and the resulting fingerprints allowed strain differentiation . However, the effectiveness of primers was dependent upon length and GC content . It was also possible to amplify DNA directly from cells in culture and in nodule tissue . Lysis of these cells was achieved simply through heat applied in the initial DNA denaturation stage of the thermal reaction . The ability to produce varied amplification patterns from different Rhizobium isolates, especially directly from nodules, gives this method potential for use in examining genetic structures and relationships in Rhizobium populations. Arch Environ Contam Toxicol, 1992 Oct, 23(3), 301 - 6 Effects of 2,4-dichlorophenoxyacetic acid on Rhizobium sp . membrane fluidity; Fabra de Peretti A et al.; The effects of 1 mM 2,4-Dichlorophenoxyacetic acid on Rhizobium sp . in pure culture was studied . In a previous work it was demonstrated that this herbicide produces an alteration on the saturated to unsaturated phospholipid acyl chains ratio . In the present paper, it was found that this alteration produces a modification on the membrane fluidity in bacteria that had achieved the stationary phase . The same results were obtained when determinations were done in liposomes from sonicated lipids of treated bacteria, indicating that changes in phospholipid acyl chains may be the main cause of membrane fluidity alteration . In addition, it was determined that leucine transport as well as Ca(++)-ATPase activity (a membrane enzyme) was affected under the experimental conditions. Plant Cell, 1992 Oct, 4(10), 1199 - 211 Rhizobium meliloti elicits transient expression of the early nodulin gene ENOD12 in the differentiating root epidermis of transgenic alfalfa; Pichon M et al.; To study the molecular responses of the host legume during early stages of the symbiotic interaction with Rhizobium, we have cloned and characterized the infection-related early nodulin gene MtENOD12 from Medicago truncatula . In situ hybridization experiments have shown that, within the indeterminate Medicago nodule, transcription of the MtENOD12 gene begins in cell layers of meristematic origin that lie ahead of the infection zone, suggesting that these cells are undergoing preparation for bacterial infection . Histochemical analysis of transgenic alfalfa plants that express an MtENOD12 promoter-beta-glucuronidase gene fusion has confirmed this result and further revealed that MtENOD12 gene transcription occurs as early as 3 to 6 hr following inoculation with R . meliloti in a zone of differentiating root epidermal cells which lies close to the growing root tip . It is likely that this transient, nodulation (nod) gene-dependent activation of the ENOD12 gene also corresponds to the preparation of the plant for bacterial infection . We anticipate that this extremely precocious response to Rhizobium will provide a valuable molecular marker for studying early signal exchange between the two symbiotic organisms. Mol Gen Genet, 1992 Oct, 235(1), 49 - 54 Modular structure of the FixL protein of Rhizobium meliloti; de Philip P et al.; FixL protein of Rhizobium meliloti is a haemo-protein kinase which activates the transcription of nifA and fixK genes via the transcriptional activator protein FixJ under microaerobic conditions . FixL and FixJ proteins belong to the family of two-component regulatory systems for which primary sequence data predicts a modular structure . We showed, using Escherichia coli as heterologous host, that FixL indeed has a modular structure . The amino-terminal hydrophobic domain is dispensable for the oxygen-regulated activity of FixL in vivo . The central cytoplasmic non-conserved domain is necessary for the oxygen-sensing function of FixL whereas it is not necessary for the activation of FixJ by FixL . We propose that, under aerobic conditions, the central domain represses the activating function associated with the carboxy-terminal conserved domain. J Bacteriol, 1992 Oct, 174(20), 6444 - 54 Rhodobacter sphaeroides rdxA, a homolog of Rhizobium meliloti fixG, encodes a membrane protein which may bind cytoplasmic {4Fe-4S} clusters; Neidle EL et al.; In the photosynthetic bacterium Rhodobacter sphaeroides, a chromosomal gene, rdxA, which encodes a 52-kDa protein, was found to be homologous to fixG, the first gene of a Rhizobium meliloti nitrogen fixation operon on the pSym plasmid (D . Kahn, M . David, O . Domergue, M.-L . Daveran, J . Ghai, P . R . Hirsch, and J . Batut, J . Bacteriol . 171:929-939, 1989) . The deduced amino acid sequences of RdxA and FixG are 53% identical and 73% similar; sequence analyses suggested that each has five transmembrane helices and a central region resembling bacterial-type ferredoxins . Translational fusion proteins with an alkaline phosphatase reporter group were expressed in both R . sphaeroides and Escherichia coli and were used to assess the membrane topology of RdxA . Its ferredoxinlike sequence, which may bind two {4Fe-4S} centers, was found to be cytoplasmically located . Genetic disruptions showed that rdxA is not essential for nitrogen fixation in R . sphaeroides . Immediately downstream of rdxA, an open reading frame (ORFT2) that encoded a 48-kDa protein was found . This DNA sequence was not homologous to any region of the R . meliloti fixG operon . The N-terminal sequence of the ORFT2 gene product resembled amino acid sequences found in members of the GntR family of regulatory proteins (D . J . Haydon and J . R . Guest, FEMS Microbiol . Lett . 79:291-296, 1991) . The rdxA gene was localized to the smaller of two R . sphaeroides chromosomes, upstream of and divergently transcribed from hemT, which encodes one of two 5-aminolevulinate synthase isozymes . The rdxA and hemT genes may share a transcriptional regulatory region . Southern hybridization analysis de