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In this paper, we describe the regulatory roles of VisN, VisR and Rem in the expression of flagellar, motility and chemotaxis genes in Rhizobium leguminosarum biovar viciae strains VF39SM and 3841. Individual mutations in the genes encoding these proteins resulted in a loss of motility and an absence of flagella, indicating that these regulatory genes are essential for flagellar synthesis and function. Transcriptional experiments involving gusA--gene fusions in wild-type and mutant backgrounds were performed to identify the genes under VisN/R and Rem regulation. Results showed that the chemotaxis and motility genes of R. leguminosarum could be separated into two groups: one group under VisN/R-Rem regulation and another group that is independent of this regulation. VisN and VisR regulate the expression of rem, while Rem positively regulates the expression of flaA, flaB, flaC, flaD, motA, motB, che1 and mcpD. All of these genes except mcpD are located within the main motility and chemotaxis gene cluster of R. leguminosarum. Other chemotaxis and motility genes, which are found outside of the main motility gene cluster (che2 operon, flaH for VF39SM, and flaG) or are plasmid-borne (flaE and mcpC), are not part of the VisN/R-Rem regulatory cascade. In addition, all genes exhibited the same regulation pattern in 3841 and in VF39SM, except flaE and flail, flaE is not regulated by VisN/R-Rem in 3841 but it is repressed by Rem in VF39SM. flail is under VisN/R-Rem regulation in 3841, but not in VF39SM. A kinetics experiment demonstrated that a subset of the flagellar genes is continuously expressed in all growth phases, indicating the importance of continuous motility for R. leguminosarum under free-living conditions. On the other hand, motility is repressed under symbiotic conditions. Nodulation experiments showed that the transcriptional activators VisN and Rem are dramatically downregulated in the nodules, suggesting that the symbiotic downregulation of motility-related genes could be mediated by repressing the expression of VisN/R and Rem. DOI 10.1099/mic.0.035386-0

While establishing a nitrogen-fixing symbiosis with leguminous plants, rhizobia are faced with the problem of penetrating the plant cell wall at several stages of the infection process. One of the major components of this barrier is pectin, a heteropolysaccharide composed mainly of galacturonic acid subunits. So far, no enzymes capable of degrading pectin have been isolated from rhizobia. Here, we make an inventory of rhizobial candidate pectinolytic enzymes based on available genome sequence data and present an initial biochemical and functional characterization of a protein selected from this list. Rhizobium etli hrpW is associated with genes encoding a type III secretion system, a macromolecular structure that allows bacteria to directly inject so-called effector proteins into a eukaryotic host's cell cytosol and an essential virulence determinant of many Gram-negative pathogenic bacteria. In contrast to harpin HrpW from phytopathogens, R. etli HrpW possesses pectate lyase activity and is most active on highly methylated substrates. Through comparative sequence analysis, three amino acid residues crucial for the observed enzymic activity were identified: Trp192, Gly212 and Gly213. Their importance was confirmed by site-directed mutagenesis and biochemical characterization of the resulting proteins, with the tryptophan mutant showing no detectable pectate lyase activity and the double-glycine mutant's activity reduced by about 80%. Surprisingly, despite hrpW expression being induced specifically on the plant root surface, a knockout mutation of the gene does not appear to affect symbiosis with the common bean Phaseolus vulgaris.

The lipopolysaccharide (LPS) of the Gram-negative legume symbiont Rhizobium leguminosarum biovar viciae 3841 contains several unique modifications, including the addition of a 27-hydroxyoctacosanoic acid (27OHC28:0), also termed the very long chain fatty acid (VLCFA), attached at the 2' position of lipid A. A transposon mutant that lacks expression of two putative 3-oxo-acyl [acyl-carrier protein] synthase II genes, fabF1 and fabF2, from the VLCFA biosynthetic cluster, was isolated and characterized. MS indicated that the lipid A of the mutant lacked the VLCFA modification, and sodium deoxycholate (DOC)-PAGE of the LPS indicated further structural alterations. The mutant was characteristically sensitive to several stresses that would be experienced in the soil environment, such as desiccation and osmotic stresses. An increase in the excretion of neutral surface polysaccharides was observed in the mutant. This mutant was also altered in its attachment to solid surfaces, and was non-motile, with most of the mutant cells lacking flagella. Despite the pleiotropic effects of the mutation, these mutants were still able to nodulate legumes and fix atmospheric nitrogen. This report emphasizes that a structurally intact VLCFA-containing lipid A is critical to cellular traits that are important for survival in the rhizosphere.

The physiological role and transcriptional expression of Rhizobium etli sigma factors rpoH1 and rpoH2 are reported in this work. Both rpoH1 and rpoH2 were able to complement the temperature-sensitive phenotype of an Escherichia coli rpoH mutant. The R. etli rpoH1 mutant was sensitive to heat shock, sodium hypochlorite and hydrogen peroxide, whereas the rpoH2 mutant was sensitive to NaCl and sucrose. The rpoH2 rpoH1 double mutant had increased sensitivity to heat shock and oxidative stress when compared with the rpoH1 single mutant. This suggests that in R. etli, RpoH1 is the main heat-shock sigma factor, but a more complete protective response could be achieved with the participation of RpoH2. Conversely, RpoH2 is involved in osmotic tolerance. In symbiosis with bean plants, the R. etli rpoH1 and rpoH2 rpoH1 mutants still elicited nodule formation, but exhibited reduced nitrogenase activity and bacterial viability in early and late symbiosis compared with nodules produced by rpoH2 mutants and wild-type strains. In addition, nodules formed by R. etli rpoH1 and rpoH2 rpoH1 mutants showed premature senescence. It was also determined that fixNf and fixKf expression was affected in rpoH1 mutants. Both rpoH genes were induced under microaerobic conditions and in the stationary growth phase, but not in response to heat shock. Analysis of the upstream region of rpoH1 revealed a [[sigma].sup.70] and a probable [[sigma].sup.E] promoter, whereas in rpoH2, one probable [[sigma].sup.E]-dependent promoter was detected. In conclusion, the two RpoH proteins operate under different stress conditions, RpoH1 in heat-shock and oxidative responses, and RpoH2 in osmotic tolerance.

Acyl carrier proteins (ACPs) are small acidic proteins that carry growing acyl chains during fatty acid or polyketide synthesis. In rhizobia, there are four different and well-characterized ACPs: AcpP, NodF, AcpXL and RkpF. The genome sequence of Sinorhizobium meliloti 1021 reveals two additional ORFs that possibly encode additional ACPs. One of these, smb20651, is located on the plasmid pSymB as part of an operon. The genes of the operon encode a putative asparagine synthetase (AsnB), the predicted ACP (SMb20651), a putative long-chain fatty acyl-CoA ligase (SMb20650) and a putative ammonium-dependent [NAD.sup.+] synthetase (NadE1). When SMb20651 was overexpressed in Escherichia coil, [[sup.3]H][beta]-alanine, a biosynthetic building block of 4'-phosphopantetheine, was incorporated into the protein in vivo. The purified SMb20651 was modified with 4'-phosphopantetheine in the presence of S. meliloti holo-ACP synthase (AcpS). Also, holo-SMb20651 was modified in vitro with a malonyl group by malonyl CoA-ACP transacylase. In E. coil, coexpression of SMb20651 together with other proteins such as AcpS and SMb20650 led to the formation of additional forms of SMb20651. In this bacterium, acytation of SMb20651 with C12 : 0 or C18 : 0 fatty acids was detected, demonstrating that this protein is involved in fatty acid biosynthesis or transfer. Expression of SMb20651 was detected in S. meliloti as holo-SMb20651 and acyl-SMb20651.

Azospirillum strains have been used as plant-growth-promoting rhizobacteria (PGPR) of cereal crops, but their adaptation to the root remains poorly understood. Here, we used a global approach based on differential fluorescence induction (DFI) promoter trapping to identify genes of the wheat isolate Azospirillum brasilense Sp245 that are induced in the presence of spring wheat seed extracts. Fluorescence-based flow cytometry sorting of Sp245 cells was validated using PlacZ, PsbpA and PnifH promoters and egfp. A random promoter library was constructed by cloning 1-3 kb Sp245 fragments upstream of a promoterless version of egfp in the promoter-trap plasmid pOT1e (genome coverage estimated at threefold). Exposure to spring wheat seed extracts obtained using a methanol solution led to the detection of 300 induced DFI clones, and upregulation by seed extracts was confirmed in vitro for 46 clones. Sequencing of 21 clones enabled identification of seven promoter regions. Five of them displayed upregulation once inoculated onto spring wheat seedlings. Their downstream sequence was similar to (i) a predicted transcriptional regulator, (ii) a serine/threonine protein kinase, (iii) two conserved hypothetical proteins, or (iv) the copper-containing dissimilatory nitrite reductase NirK. Two of them were also upregulated when inoculated on winter wheat and pea but not on maize, whereas the three others (including PnirK) were upregulated on the three hosts. The amounts of nitrate and/or nitrite present in spring wheat seed extracts were sufficient for PnirK upregulation. Overall, DFI promoter trapping was useful to reveal Azospirillum genes involved in the interaction with the plant.

Transfer of the Rhizobium leguminosarum biovar viciae symbiosis plasmid pRL1JI is regulated by a cascade of gene induction involving three LuxR-type quorum-sensing regulators, TraR, BisR and CinR. TraR induces the plasmid transfer tral-trb operon in a population-density-dependent manner in response to N-acylhomoserine lactones (AHLs) made by Tral. Expression of the traR gene is primarily induced by BisR in response to AHLs made by Cinl, and expression of cinl is induced by CinR and repressed by BisR. Analysis of transcription initiation of cinl, traR and tral identified potential regulatory domains recognized by the CinR, BisR and TraR regulators. Deletion and mutation of the cinl promoter identified potential recognition motifs for activation by CinR and repression by BisR. Analysis of the DNA sequence upstream of tral and expression of transcriptional gene fusions revealed a predicted TraR-binding (tra-box) domain. Two transcript initiation sites were identified upstream of the plasmid replication gene repA, which is divergently transcribed from tral; one of these repA transcripts requires the quorum-sensing cascade mediated via BisR and TraR, showing that the pRL1JI plasmid replication genes are co-regulated with the plasmid transfer genes.

Plant-interacting micro-organisms can establish either mutualistic or pathogenic associations. Although the outcome is completely different, common molecular mechanisms that mediate communication between the interacting partners seem to be involved. Specifically, nitrogen-fixing bacterial symbionts of legume plants, collectively termed rhizobia, and phytopathogenic bacteria have adopted similar strategies and genetic traits to colonize, invade and establish a chronic infection in the plant host. Quorum-sensing signals and identical two-component regulatory systems are used by these bacteria to coordinate, in a cell density-dependent manner or in response to changing environmental conditions, the expression of important factors for host colonization and infection. The success of invasion and survival within the host also requires that rhizobia and pathogens suppress and/or overcome plant defence responses triggered after microbial recognition, a process in which surface polysaccharides, antioxidant systems, ethylene biosynthesis inhibitors and virulence genes are involved.

Sinorhizobium medicae WR101 was identified as a mutant of WSM419 that contained a minitransposon-induced transcriptional gusA fusion activated at least 20-fold at pH 5.7. The expression of this fusion in moderately acid conditions was dependent on the calcium concentration; increasing the calcium concentration to enhance cell growth and survival in acid conditions decreased the expression of the fusion. A gene region containing the gusA fusion was sequenced, revealing five S. medicae genes: tcsA, tcrA, fsrR, lpiA and acvB. The gusA reporter in WR101 was fused to IpiA, which encodes a putative transmembrane protein also found in other Alphaproteobacteria such as Sinorhizobium meliloti, Rhizobium tropici and Agrobacterium tumefaciens. As lpiA has partial sequence similarity to the lysyl-phosphatidylglycerol (LPG) synthetase FmtC/MprF from Staphylococcus aureus, membrane lipid compositions of S. medicae strains were analysed. Cells cultured under neutral or acidic growth conditions did not induce any detectable LPG and therefore this lipid cannot be a major constituent of S. medicae membranes. Expression studies in S. medicae localized the acid-activated lpiA promoter within a 372 bp region upstream of the start codon. The acid-activated transcription of IpiA required the fused sensor--regulator product of the fsrR gene, because expression of lpiA was severely reduced in an S. medicae fsrR mutant. S. melilotistrain 1021 does not contain fsrR and acid-activated expression of the lpiA-gusA fusion did not occur in this species. Although acid-activated lpiA transcription was not required for cell growth, its expression was crucial in enhancing the viability of cells subsequently exposed to lethal acid (pH 4-5) conditions.

A genetic locus encoding erythritol uptake and catabolism genes was identified in Rhizobium leguminosarum bv. viciae, and shown to be plasmid encoded in a wide range of R. leguminosarum strains. A Tn5-B22 mutant (19B-3) unable to grow on erythritol was isolated from a mutant library of R. leguminosarum strain VF39SM. The mutated gene eryFwas cloned and partially sequenced, and determined to have a high homology to permease genes of ABC transporters. A cosmid complementing the mutation (pCos42) was identified and was shown to carry all the genes necessary to restore the ability to grow on erythritol to a VF39SM strain cured of pRleVF39f. In the genomic DNA sequence of strain 3841, the gene linked to the mutation in 19B-3 is flanked by a cluster of genes with high homology to the known erythritol catabolic genes from Brucella spp. Through mutagenesis studies, three distinct operons on pCos42 that are required for growth on erythritol were identified: an ABC-transporter operon (eryEFG), a catabolic operon (eryABCD) and an operon (deoR-tpiA2-rpiB) that encodes a gene with significant homology to triosephosphate isomerase (tpiA2). These genes all share high sequence identity to genes in the erythritol catabolism region of Brucella spp., and CLUSTALW alignments suggest that horizontal transfer of the erythritol locus may have occurred between R. leguminosarum and Brucella. Transcription of the eryABCD operon is repressed by EryD and is induced by the presence of erythritol. Mutant 19B-3 was impaired in its ability to compete against wild-type for nodulation of pea plants but was still capable of forming nitrogen-fixing nodules.

The ability to recognize and predict non-[[sigma].sup.54] promoters in the alphaproteobacteria is not well developed. In this study, 25 experimentally verified Sinorhizobium meliloti promoter sequences were compiled and used to predict the location of other related promoters in the S. meliloti genome. Fourteen candidate predictions were targeted for verification and of these at least 12 proved to be genuine promoters. As a result, the experimental identification of 12 novel promoters linked to genes rpoD, topA, rpmJ, trpS, ropB1, metC, rpsT, secE, trkH and three tRNA genes is reported. In all, 99 predicted and verified promoters are reported, including those linked with 13 tRNA genes, eight ribosomal protein genes and a number of other physiologically important or essential genes. On the basis of sequence conservation and a mutational analysis of promoter activity, the -35 and -10 consensus for these promoters is 5-CTTGAC-[N.sub.17]-CTATAT. This promoter structure, which seems to be widely conserved amongst several other genera in the alphaproteobacteria, shares significant similarity with, but is skewed by a 1 nt step from, the canonical Escherichia coli [[sigma].sup.70] promoter. Perhaps this difference is responsible for the observation that S. meliloti promoters are often poorly expressed in E. coli. In this regard, expression data from plasmid-borne gfp-reporter fusions to eight of the S. meliloti promoters verified in this work revealed that while these promoters were very active in S. meliloti and Agrobacterium tumefaciens only very low, near-background activity was detected in E. coli.

The predicted chromosomal origin of replication (oriC) from the alfalfa symbiont Sinorhizobium meliloti is shown to allow autonomous replication of a normally non-replicating plasmid within S. meliloti cells. This is the first chromosomal replication origin to be experimentally localized in the Rhizobiaceae and its location, adjacent to hemE, is the same as for oriC in Caulobacter crescentus, the only experimentally characterized alphaproteobacterial oriC. Using an electrophoretic mobility shift assay and purified S. meliloti DnaA replication initiation protein, binding sites for DnaA were mapped in the S. meliloti oriC region. Mutations in these sites eliminated autonomous replication. S. meliloti that expressed DnaA from a plasmid lac promoter was observed to form pleomorphic filamentous cells, suggesting that cell division was perturbed. Interestingly, this cell phenotype is reminiscent of differentiated bacteroids found inside plant cells in alfalfa root nodules.

A modABC gene cluster that encodes an ABC-type, high-affinity molybdate transporter from Bradyrhizobium japonicum has been isolated and characterized. B. japonicum modA and modB mutant strains were unable to grow aerobically or anaerobically with nitrate as nitrogen source or as respiratory substrate, respectively, and lacked nitrate reductase activity. The nitrogen-fixing ability of the mod mutants in symbiotic association with soybean plants grown in a Mo-deficient mineral solution was severely impaired. Addition of molybdate to the bacterial growth medium or to the plant mineral solution fully restored the wild-type phenotype. Because the amount of molybdate required for suppression of the mutant phenotype either under free-living or under symbiotic conditions was dependent on sulphate concentration, it is likely that a sulphate transporter is also involved in Mo uptake in B. japonicum. The promoter region of the modABC genes has been characterized by primer extension. Reverse transcription and expression of a transcriptional fusion, [P.sub.modA]-lacZ, was detected only in a B. japonicum modA mutant grown in a medium without molybdate supplementation. These findings indicate that transcription of the B. japonicum modABC genes is repressed by molybdate.

The manganese uptake regulator Mur of Rhizobium leguminosarum is a close homologue of the global iron regulatory protein Fur. Mur represses the sitABCD operon, which encodes a [Mn.sup.2+] transport system, specifically in response to [Mn.sup.2+] but not [Fe.sup.2+]. In previous work the authors mapped the 5' ends of two sit operon transcripts, termed TS1 and TS2, which were co-ordinately regulated by [Mn.sup.2+]-Mur, but this paper now shows that only TS1 is a primary transcript. DNase I protection analyses showed that purified Mur bound, with similar affinity, to two sites in the regulatory region of sitABCD, but only when [Mn.sup.2+] was present in the reaction buffer. These [Mn.sup.2+]-Mur-binding sites, termed MRS1 and MRS2 (Mur-responsive sequence), were closely related in sequence to each other and were separated by 16 bp, spanning the transcription initiation site TS1. The extent of the protected DNA was 34 and 31 bp for MRS1 and MRS2, respectively, which is in accord with other members of the Fur family. The DNA sequences recognized by [Mn.sup.2+]-Mur are wholly different from conventional Fur boxes, but some similarities to a recognition sequence for the Fur regulator from Bradyrhizobium japonicum were noted. Transcription analysis of the R. leguminosarum mur gene showed its expression to be independent of [Mn.sup.2+]-Mur. Thus, Mur is a sequence-specific DNA-binding protein that responds in vitro to manganese, and thus can occlude RNA polymerase access to the sitABCD promoter. Moreover, Mur recognizes a DNA sequence atypical for the Fur superfamily and, like Fur from B. japonicum, defines a new subclass of Fur-like transcriptional regulators.

Mutations in rirA of Rhizobium have been shown to deregulate expression of several genes that are normally repressed by iron. A conserved sequence, the iron-responsive operator (IRO), was identified near promoters of vbsC (involved in the synthesis of the siderophore vicibactin), rpol (specifies an ECF [sigma] factor needed for vicibactin synthesis) and the two fhuA genes (encoding vicibactin receptor). Removal of these IRO sequences abolished Fe-responsive repression. Most of these genes were constitutively expressed in the heterologous host, Paracoccus denitrificans, but introduction of the cloned rirA gene repressed expression of these Rhizobium genes in this heterologous host if the corresponding IRO sequences were also intact. These observations are the first to examine the mechanisms of RirA, which has no sequence similarity to well-known iron-responsive regulators such as Fur or DtxR. They provide strong circumstantial evidence that RirA is a transcriptional regulator that binds to cis-acting regulatory sequences near the promoters of at least some of the genes whose expression it controls in response to Fe availability.

The Rhizobium leguminosarum by. viciae VF39 FixL protein belongs to a distinct group of hybrid regulatory sensor proteins that bear a covalently linked C-terminal receiver domain. FixL has an unorthodox histidine kinase domain, which is shared with many other hybrid regulators. The purified FixL protein had autophosphorylation activity. A truncated protein, lacking the receiver domain, had a much-reduced autophosphorylation activity. However, this truncated protein still efficiently phosphorylated the purified receiver domain in trans. This indicates that, in the full-length FixL protein, the conserved histidine residue in the kinase domain is phosphorylated only transiently and that most of the phosphoryl label accumulates in the C-terminal receiver domain. Gone-fusion studies showed that the fixL gene is required for free-living microaerobic induction of the fnrN promoter. The presence of a functional fixK gene is not required. An R. leguminosarum strain lacking fixL could not be complemented with a truncated copy of the gone lacking the receiver domain. This indicates that the C-terminal receiver domain is an intermediate in the signal transduction pathway that links oxygen 1imitation to induction of the fnrN promoter in R. leguminosarum by. viciae VF39.

Exopolysaccharide (EPS) produced by Rhizobium sp. strain TAL1145 has been shown to be essential for effective nodulation on Leucaena/eucocephala (leucaena). This paper reports the isolation and characterization of an alternative sigma factor gene, rpoH2, involved in the regulation of EPS synthesis in TAL1145. Disruption of this gene in TAL1145 resulted in a Calcofluor-dim mutant RUH102 that produced approximately 18% of the amount of EPS made by TAL1145. This mutation did not affect the normal growth of RUH102 in free-living state. RUH102 induced few nitrogen-fixing nodules, resulting in a significant reduction in total nitrogen content in leucaena. It was complemented for EPS production and nodulation by a 2*0 kb HindIII fragment of TAL1145. Sequence analysis of this fragment revealed the rpoH2 ORF of 870 bp that encoded a protein of 32 kDa. Expression of the rpoH2 ORF in Escherichia coli also revealed a 32 kDa protein. A PCR-constructed clone of 1263 bp, containing the rpoH2 ORF and its upstream putative regulatory region, complemented RUH102 for EPS defects. Comparison of the RpoH2 sequence to proteins in the databases showed significant similarity to RpoH-like sigma factors of other Gram-negative bacteria. By constructing several exo::Tn3Hogus fusions and transferring them to the backgrounds of TAL1145 and RUH102, it was demonstrated that RpoH2 positively regulates the transcription of some exo genes.

In wild-type Rhizobium leguminosarum, the sitABCD operon specifies a [Mn.sup.2+] transporter whose expression is severely reduced in cells grown in the presence of this metal. Mutations in the R. leguminosarum gene, mur (manganese uptake regulator), whose product resembles the Fur transcriptional regulator, cause high-level expression of sitABCD in the presence of [Mn.sup.2+]. In gel-shift mobility assays, purified R. leguminosarum Mur protein bound to at Least two regions near the sitABOD promoter region, although this DNA has no conventional consensus Fur-binding sequences (fur boxes). Thus, in contrast to [gamma]-proteobacteria, where Fur binds [Fe.sup.2+], the R. leguminosarum Fur homologue, Mur, act as a [Mn.sup.2]-responsive transcriptional regulator.

Synthesis of the Rhizobium leguminesarum [NiFe] hydrogenase requires the participation of 16 accessory genes (hupCDEFGHIJKhypABFCDEX) besides the genes encoding the structural proteins (hupSL). Transcription of hupSL is controlled by a -24/-1 2-type promoter ([P.sub.1]), located upstream of hupS and regulated by NifA. In this work, a second -24/-1 2-type promoter ([P.sub.3]), located upstream of the hupG gene and transcribing hupGHU genes in R. leguminosarum pea (Pisum sativum L.) bacteroids, has been identified in the hup gene cluster. Promoter [P.sub.3] was also active in R. leguminesarum free-living cells, as evidenced by genetic complementation of hydrogenase mutants. Both NifA and NtrC activated Ps expression in the heterologous host Klebsiella pneumoniae. Also, [P.sub.3] activity was highly stimulated by K. pneumoniae NifA in Escherichia coil. This NifA activation of [P.sub.3] expression only required the [[sigma].sup.54]-binding site, and it was independent of any cis-acting element upstream of the 54 box, which suggests a direct interaction of free NifA with the RNA polymerase holoenzyme. [P.sub.3]-dependent hupGHIJ expression in pea nodules started in interzone II/III, spanned through nitrogen-fixing zone III, and was coincident with the NifA-dependent nifH expression pattern. However, [P.sub.3] was dispensable for hupGHIJ transcription and hydrogenase activity in pea bacteroids due to transcription initiated at [P.sub.1]. This fact and the lack of an activator recruitment system suggest that [P.sub.3] plays a secondary role in symbiotic hupGHIJ expression.

Diverse types of genomic DNA organization have been found in Rhizobiaceae, especially among Agrobacterium species. Previous studies of Agrobactereum concentrated mainly on biovar 1 strains. Little attention has been given to biovar 2 strains. The biovar 2 genome consists of a large, circular chromosome and second megabase-sized replicon, as well as several plasmids. In this study two biovar 2 strains were analysed, A. rhizogenes (A. radiobacter) K84 and A. rhizogenes A4, by constructing physical maps of their chromosomes and mega-replicons. The maps revealed that in both strains their chromosomes consist of approximately 3.7 Mbp, while the mega-replicons are 2.6 Mbp circular DNAs. Gene mapping and comparative genomic analysis were performed based on the physical maps using Southern hybridization. It was found that rDNA, as well as analysed virulence and virulence-related genes, are present only on the chromosomes. The inter-chromosomal relationship between biovar 1 and biovar 2 strains was also analysed. Interestingly, there was a high similarity between the chromosomes of biovar 2 and the circular chromosomes of biovar 1, whereas similarity among the smaller megabase-sized replicons was restricted to each biovar. Based on these observations the possible relationship among large replicons in Agrobacterium biovars 1 and 2 is discussed.

Research has been conducted on the phytase activity of Bacillus amyloliquefaciens FZB45. The use of the artificial sterile system which consists of culture filtrates of plant-growth-promoting rhizobacteria and maize seedlings in establishing secreted phytase contribution to the plant growth promotion by B. amyloliquefaciens is described.

Research has been conducted on the Rhizobium leguminosarum bv. viciae VF39 gabT gene which encodes 2-oxoglutarate-dependent gamma-aminobutyrate aminotransferase. The identification, cloning and characterization of gabT have been carried out and the results indicate the importance of this gene in bacteroid metabolism.

The CH34 RpoN gene of Ralstonia metallidurans has been sequenced, as has the flanking DNA. A CH34 rpoN-deficient strain has been isolated. CH34 RNA polymerase sigma(super.54) was found to positively control autotrophy as well as nitrogen metabolism, with Tn4371-directed biphenyl utilization affected indirectly.

Results points out that lateral genetic transfer of nodC and nifH genes across rhizobial species lead to diversification and structuring in rhizobial populations. Genetic analysis reveals a close relationship of rhizobial genes with those of Phaseolus vulgaris symbionts' genes.

Research analyzes the phenotypic and 16S rDNA characterization in 17 rhizobial strains associated with the black locust tree of Europe and 18 reference strains from Maryland. Genomic data indicate that majority of the former strains belong to the genus Mesorhizobium and display a high phenotypic and phylogenetic diversity.

Results show that fhuA gene codes for a receptor for the siderophore and the gene itself is regulated by iron. Data also reveal that a pseudogene version is detected in some strains of the Rhizonium bacterium.

A mutant of Rhizobium leguminosarum, which cannot take up the siderophore vicibactin, was isolated. The mutation is in a homolog of fhuB, which in Escherichia coli, encodes an inner-membrane protein of the ferric hydroxamate uptake system. In Rhizobium, fhuB is an operon fhuDCB, which encodes the cytoplasmic membrane and periplasmic proteins required in siderophore uptake. The fhuDCB mutants produce vicibactin when grown in iron concentrations that suppress its production in the wild-type.

The existence of potential methyl-accepting chemotaxis protein (MCP)-encoding genes in Rhizobium leguminosarum were determined using oligonucleotide primers. In this study, five putative MCP-encoding genes were isolated and identified. Results confirmed the existence of MCP-like genes in the bacteria. Some of these genes were found to play a role in the early stages of plant-microbe interaction.

A study was conducted on the isolation of the nodPQ genes from Rhizobium sp BR816, a broad-host-range bacterium related to Sinorhizobium meliloti. Plant inoculation analysis did not indicate differences between the mutant strain and the wild-type. Moreover, it appears that a third, functionally homologous locus complements mutations in the Nod factor sulphation genes. Southern blot analysis also indicates that this locus has genes necessary for the sulphation of amino acids.

Mutations that mediated the ability of Pck-deficient Rhizobium meliloti mutants to utilize succinate and tricarboxylic acid (TCA) intermediates were analyzed to characterize the biosynthesis of phosphoenolpyruvate (PEP). The activity of pyruvate orthophosphate dikinase (PPDK) in Rhizobium (Sinorhizobium) meliloti was enhanced by pckA suppressor mutations. Gluconeogenesis from malate in the pckA suppressor strain was also mediated by the combined activities of malic enzyme and PPDK.

Nucleotide sequences of prsD, prsE (protein secretion) and orf3 from Rhizobium leguminosarum bv. trifolii TA1 were presented. The three genes were identified adjacent to the exo 133 mutation in a cosmid carrying the genomic DNA of the Rhizobium. PrsD and prsE were found homologous to the ATP-binding proteins, while orf3, which has a typical terminal sequence, was not homologous to these proteins.

The effect of inactivated nolX on symbiotic properties was studied on the three Rhizobium fredii strains, namely, USDA191, USDA257 and HH103. Inactivation of nolX lead retarded initial nodulation rates on the soybean cultivars. Nodulation of two Erythrina species, however, was blocked if nolX was present but was allowed to proceed when the gene was inactivated. NolX revealed to have dual function and was concluded to be of symbiotic significance in all the three strains.

The biochemical characteristics of the gst-like Rhizobium leguminosarum gene belonging to the glutathione S-transferase (GST) gene family were analyzed in Escherchia coli. Analysis of Rhizobium leguminasarum gene overexpressed in Escherichia coli indicated the presence of a 21990 Da polypeptide with a 203 amino acid sequence that was encoded by the GST gene. Furthermore, the GST gene exhibited an antigenic domain that was different from mammalian GST genes.

The specific genes involved in nodulation and nitrogen fixation in Rhizobium species are found in a single large symbiotic plasmid (pSym). Hybridization of pSym of Rhizobium etli against 32P-labelled single-stranded complementary DNAs showed that the transcriptional activity of pSym is governed by environmental stimuli. Under aerobic conditions, transcription of discrete regions of pSym are determined by carbon or nitrogen concentration. Increased gene expression was observed under conditions of poor carbon or nitrogen sources and during microaerobiosis, indicating that oxygen was a regulator of transcription. Oxygen was also suggested as a determinant of the location of transcription in pSym.

Most strains of Rhizobium leguminosarum have gene repC-like sequences that may serve as suitable PCR primer sites. Thirty nine out of forty one strains tested, and several Sinorhizobium strains amplify repC fragment sequences of 750bp. There is a possibility of repC groups corresponding to Rhizobium plasmid incompatibility groups. RepC sequences from R. leguminosarum show great genetic diversity.

The chromosome and symbiosis-related genotypes of rhizopine-producing strains of Rhizobium meliloti and R. leguminosarum cannot be identified on the basis of chromosomal characteristics. Rhizopine synthesis is related to certain symbiotic plasmid genotypes but is independent of the genome of the host plant. Rhizopine synthesis (mos) genes show more variations than rhizopine catabolism (moc) genes. Less divergence in the R. leguminosarum mos and moc genes shows that they have evolved after R. meliloti.

Insertion of a nodC-phoA reporter plasmid with nodD into a symbiotic plasmid-absent Rhizobium leguminosarum strain that is chemically mutagenized helps delineate the host genes that affect nod gene expression in R. leguminosarum. The mutant resulting from insertion of the symbiotic plasmid pRL1Jl after curing the nodC-phoA plasmid exhibits normal nodule formation with 80% symbiotic nitrogen fixation. Introduction of the nodC-phoA allele on PRL1Jl leads to low level nod gene activation. Genetic analysis reveals the presence of IS 50 in the mutant regulatory gene dctB, leading to reduced nod gene expression is not affected by other mutations in dctB, indicating that the effect is allele-specific.

Cloning and sequencing of the gene associated with the uptake of 4-hydroxybenzoate in Rhizobium leguminosarum reveals that 4-hydroxybenzoate hydroxylase (PobA) is essential for the uptake of 4-hydroxybenzoate. Mutants of R. leguminosarum lacking PobA fail to accumulate 4-hydroxybenzoate, and addition of a 2.0kb EcoRI-PstI DNA fragment, having DNA sequence similarity with the structural gene for PobA, restores the uptake of 4-hydroxybenzoate.

A Tn5 mutant (RU301) of R. leguminosarum that lacks the O-antigen, and which has rough colony morphology and is unable to swarm in TY agar, produces only lipopolysaccharide layer (LPS II). RU301 fails to grow on organic acids but high concentration of calcium or magnesium restores the growth. Southern blotting shows the location at transposon insertion in RU301 in a 3 kb EcoRI fragment. There is a separation of 10 kb between dctD and the region mutated in strain RU301. Additional calcium is not necessary for all LPS rough mutants, and gene mutation in RU301 is essential for nitrogen fixation.

Genetic and enzymatic characterization of the exoB mutant of Rhizobium sp reveals that, like other exoB mutants, Cal10 cannot produce UDP-glucose 4'-epimerase. The residual exopolysaccharide (EPS), obtained from Cal10 mutant, is a neutral beta glucan and the galactose forms a part of acidic EPS fraction. ExoB mutant induces effective nodules on sulla, a legume that develops indeterminate types of nodule. EPS is not required for successful nodulation in this symbiotic relationship.

psi operon of Rhizobium leguminosarum biovar phaseoli encodes three open reading frames, PsiA, PsiB and ORF-P. PsiA gene inhibits exopolysaccharide synthesis, causing R. leguminosarum strains to color with dye calcofluor. phoA defective gene insertions at PsiA and PsiB are identified and sequenced. PsiA-phoA and PsiB-phoA protein fusions and alkaline phosphatase activity reveal PsiA and PsiB located in the inner bacterial membrane.

Two Cpn60 homologues were discovered during the hybridization of an E. coli Cpn60 probe to total DNA from Rhizobium leguminosarum. One of the homologues which did not complement a Cpn60 mutation either for bacteriophagic growth or growth at high temperature, exhibited close homology to Cpn60 sequences from other prokaryotes. Cpn60 enables proteins to fold correctly into their final active conformation.

An investigation was conducted to identify the genes conferring competitive behavior in the competititive strain, Rhizobium leguminosarum biovar phaseoli strain KIM5s. Cosmid libraries of strain KIM5s were constructed and used to transform an inferior competitive strain, CE3. The experiments identifed nine highly competitive derivatives. However, plasmid curing experiments showed that the competitive phenotype of the transformed strains were not conferred by the introduced DNA. Therefore, the altered competitiveness is due to spontaneous genetic changes. The nine competitive strains will be useful for further genetic analysis of nodulation competitiveness.

Research has been conducted on Rhizobium leguminosarum fur mutants. The authors report that these mutants have been unaffected in iron-dependent operon regulation, and discuss differences and similarities in the Fur superfamily in rhizobia and other Gram-negative bacteria.

Research has been conducted on plasmid-borne Rhizobium etli katG gene encoding catalase-peroxidase KatG. The authors describe the cloning of this gene, and characterize its effect on oxygen stress resistance.

Research has been conducted on mimosine, a toxin present in tree-legume leucaena. Results demonstrate that leucaena-nodulating Rhizobium sp. strain TAL1145 degrades mimosine and that the mid genes from the TAL1145 fragment are required for mimosine degradation to HP.

Research has been conducted on Rhizobium leguminosarum gene mutations. Results demonstrate that these mutations reduce growth rates in Fe-replete and -depleted medium but do not affect symbiotic N (sub)2 fixation.

Research has been conducted on norCBQD genes encoding Bradyrhizobium japonicum USDA110 nitric oxide reductase. The isolation and characterization of these genes have been carried out, and the coding properties, genetic organization of norCBQD genes and the phenotypic analysis of their mutants are described.

The authors comment on the publications dealing with bacterial genome sequence. The topics of interest include potentials and limitations in genome sequencing, and the impostance of lateral gene transfers in bacterial adaptation.

A study was conducted to analyze alternative minitransposons that may be utilized to complement Tn5 derivatives. The number of restriction sites in the mTn5s was significantly minimize to increase the range of endonucleases which can be used to cut mutated target DNA in preparation for cloning. Results indicated novel constructs that provide useful tools for insertional mutagenesis, transcriptional signal localization and gene regulation studies.