Search

The pha1 gene cluster (pha 1A'-G) of Sinorhizobium meliloti has previously been characterized as a necessary component for proper invasion into plant root tissue. It has been suggested to encode a multi-subunit [K.sup.+]/[H.sup.+] antiporter, since mutations in the pha1 region rendered S. meliloti cells sensitive to [K.sup.+] and alkali, and because there is high amino acid sequence similarity to previously characterized multi-subunit cation/[H.sup.+] antiporters (Mrp antiporters). However, the detailed transport properties of the Phal system are yet to be determined. Interestingly, most of the Mrp antiporters are highly selective for [Na.sup.+], unlike the Phal system. Here, we report the functional expression of the Phal system in Escherichia coil and the measurement of cation/[H.sup.+] antiport activity. We showed that the Phal system is indeed a [K.sup.+]/[H.sup.+] antiporter with a pH optimum under mildly alkaline conditions. Moreover, we found that the Phal system can transport [Na.sup.+]; this was unexpected based on previous phenotypic analyses of pha1 mutants. Furthermore, we demonstrated that the cation selectivity of the Phal system was altered when the pH was lowered from the optimum. The downregulation of [Na.sup.+]/[H.sup.+] and [K.sup.+]/[H.sup.+] antiport activities upon acidic shift appeared to occur via different processes, which might indicate the presence of distinct mechanisms for the regulation of the [K.sup.+]/[H.sup.+] and [Na.sup.+]/[H.sup.+] antiport activities of the Phal system.

Free-living Sinorhizobium meliloti lpxXL and acpXL mutants lack lipid A very-long-chain fatty acids (VLCFAs) and have reduced competitiveness in alfalfa. We demonstrate that LpxXL and AcpXL play important but distinct roles in bacteroid development and that LpxXL is essential for the modification of S. meliloti bacteroid lipid A with VLCFAs.

Stimulation of potassium uptake is the most rapid response to an osmotic upshock in bacteria. This cation accumulates by a number of different transport systems whose importance has not been previously addressed for rhizobia. In silico analyses reveal the presence of genes encoding four possible potassium uptake systems in the genome of Sinorhizobium meliloti 1021: Kup1, Kup2, Trk, and Kdp. The study of the relevance of these systems under a number of different growth conditions and in symbiosis showed that the integrity of Kup1 or Trk is essential for growth under laboratory conditions even in osmotically balanced media and the absence of both systems leads to a reduced infectivity and competitiveness of the bacteria in alfalfa roots. Trk is the main system involved in the accumulation of potassium after an osmotic upshift and the most important system for growth of S. meliloti under hyperosmotic conditions. The other three systems, especially Kup1, are also relevant during the osmotic adaptation of the cell, and the relative importance of the Kdp system increases at low potassium concentrations.

Cyclopropane fatty acyl synthases (CFA synthases) are enzymes that catalyse the addition of a methylene group across cis double bonds of monounsaturated fatty acyl chains in lipids. We have investigated the function of two putative genes, cfa1 and cfa2, proposed to code for CFA synthases in Sinorhizobium meliloti. Total fatty acid composition and fatty acid distributions within lipid classes for wild-type and cfa1 and cfa2 mutant strains grown under Pi starvation and in acidic culture conditions were obtained by GC/MS and by infusion ESI/MS/MS, respectively. For wild-type cells and the cfa1 mutant, total cyclopropane fatty acids (CFAs) increased by 10% and 15% under [P.sub.i] starvation and acidic conditions, respectively; whereas in the cfa2 mutant, CFAs were less than 0.1% of wild-type under both growth conditions. Reporter gene fusion experiments revealed that cfa1 and cfa2 were expressed at similar levels in free-living cells. Thus under the conditions we examined, cfa2 was required for the cyclopropanation of lipids in S. meliloti whereas the role of cfa1 remains to be determined. Analysis of intact lipids revealed that cyclopropanation occurred on cis-11-octadecenoic acid located in either the sn-1 or the sn-2 position in phospholipids and that cyclopropanation in the sn-2 position occurred to a greater extent in phosphatidylcholines and sulfoquinovosyldiacylglycerols under acidic conditions than under [P.sub.i] starvation. The cfa2 gene was also required for cyclopropanation of non-phosphorus-containing lipids. Principal components analysis revealed no differences in the cyclopropanation of four lipid classes. We concluded that cyclopropanation occurred independently of the polar head group. Neither cfa1 nor cfa2 was required for symbiotic nitrogen fixation.

The genes encoding malate synthase (glcB) and isocitrate lyase (aceA) and a 240-bp open reading frame (SMc00767) located downstream ofaceA were isolated and functionally characterized in Sinorhizobium meliloti. Independent and double interposon mutants of each gene were constructed, and the corresponding phenotypes were analyzed, aceA mutants failed to grow on acetate, and mutants deficient in SMc00767 were also affected in acetate utilization. In contrast, mutants deficient in glcB grew on acetate similar to wild-type strain Rm5000. Complementation experiments showed that aceA and SMc00767 gene constructs were able to restore the growth on acetate in the corresponding single mutants, aceA-glcB, aceA-SMc00767, and glcB-SMc00767 double knockouts were also unable to grow on acetate, but this ability was recovered when the wild-type aceA-glcB or aceA-SMc00767 loci were introduced into the double mutants. These data confirm the functional role of aceA and SMc00767 and show that glcB, in the absence of SMc00767, is required for acetate metabolism. Isocitrate lyase and malate synthase activities were measured in strain Rm5000, the mutant derivatives, and complemented strains, aceA and glcB were able to complement the enzymatic activity lacking in the corresponding single mutants. The enzymatic activities also showed that SMc00767 represses the activity of isocitrate lyase in cells grown on acetate. Gene fusions confirmed the repressor role of SMc00767, which regulates aceA expression at the transcriptional level. Comparison of the transcriptional profiles of the SMc00767 mutant and wild-type strain Rm5000 showed that SMc00767 represses the expression of a moderate number of open reading frames, including aceA; thus, we propose that SMc00767 is a novel repressor involved in acetate metabolism in S. meliloti. Genetic and functional analyses indicated that aceA and SMc00767 constitute a functional two-gene operon, which is conserved in other [alpha]-proteobacteria. Alfalfa plants infected with the aceA and glcB mutants were not impaired in nodulation or nitrogen fixation, and so the glyoxylate cycle is not required in the Rhizobium-legume symbiosis.

Unprecedented agricultural intensification and increased crop yield will be necessary to feed the burgeoning world population, whose global food demand is projected to double in the next 50 years. Although grain production has doubled in the past four decades, largely because of the widespread use of synthetic nitrogenous fertilizers, pesticides, and irrigation promoted by the 'Green Revolution,' this rate of increased agricultural output is unsustainable because of declining crop yields and environmental impacts of modern agricultural practices. The last 20 years have seen diminishing returns in crop yield in response to increased application of fertilizers, which cannot be completely explained by current ecological models. A common strategy to reduce dependence on nitrogenous fertilizers is the production of leguminous crops, which fix atmospheric nitrogen via symbiosis with nitrogen-fixing rhizobia bacteria, in rotation with nonleguminous crops. Here we show previously undescribed in vivo evidence that a subset of organochlorine pesticides, agrichemicals, and environmental contaminants induces a symbiotic phenotype of inhibited or delayed recruitment of rhizobia bacteria to host plant roots, fewer root nodules produced, lower rates of nitrogenase activity, and a reduction in overall plant yield at time of harvest. The environmental consequences of synthetic chemicals compromising symbiotic nitrogen fixation are increased dependence on synthetic nitrogenous fertilizer, reduced soil fertility, and unsustainable long-term crop yields. legume | nitrogen fixation | symbiosis | Sinorhizobium meliloti

Sinorhizobium meliloti hpdA, which encodes the herbicide target 4-hydroxyphenylpyruvate dioxygenase, is positively regulated by HpdR. Gel mobility shift and DNase I footprinting analyses revealed that HpdR binds to a region that spans two conserved direct-repeat sequences within the hpdR-hpdA intergenic space. HpdR-dependent hpdA transcription occurs in the presence of 4-hydroxyphenylpyruvate, tyrosine, and phenylalanine, as well as during starvation. doi:10.1128/JB.01662-06

Sinorhizobium meliloti participates in a nitrogen-fixing symbiosis with legume plant host species of the genera Medicago, Melilotus, and Trigonella. We recently identified an S. meliloti two-component sensory histidine kinase, CbrA, which is absolutely required to establish a successful symbiosis with Medicago sativa (K. E. Gibson, G. R. Campbell, J. Lloret, and G. C. Walker, J. Bacteriol. 188:4508-4521, 2006). In addition to having a symbiotic defect, the cbrA::Tn5 mutant also has free-living phenotypes that suggest a cell envelope perturbation. Because the bases for these phenotypes are not well understood, we undertook an identification of CbrA-regulated genes. We performed a microarray analysis and compared the transcriptome of the cbrA::Tn5 mutant to that of the wild type. Our global analysis of gene expression identified 162 genes that are differentially expressed in the cbrA::Tn5 mutant, including those encoding proteins involved in motility and chemotaxis, metabolism, and cell envelope function. With regard to those genes with a known role in symbiosis, we observed increased expression of nine genes with overlapping functions in bacterial invasion of its host, which suggests that the mutant could be competent for invasion. Since these CbrA-repressed genes are vital to the invasion process, it appears that down-regulation of CbrA activity is important at this stage of nodule development. In contrast, our previous work showed that CbrA is required for bacteria to establish themselves within the host as nitrogen-fixing symbionts. Therefore, we propose a model in which CbrA functions as a developmental switch during symbiosis. doi:10.1128/JB.01834-06

A Tn5 mutant strain of Sinorhizobium meliloti with an insertion in tpiA (systematic identifier SMc01023), a putative triose phosphate isomerase (TPI)-encoding gene, was isolated. The tpiA mutant grew more slowly than the wild type on rhamnose and did not grow with glycerol as a sole carbon source. The genome of S. meliloti wild-type Rml021 contains a second predicted TPI-encoding gene, tpiB (SMc01614). We have constructed mutations and confirmed that both genes encode functional TPI enzymes, tpiA appears to be constitutively expressed and provides the primary TPI activity for central metabolism, tpiB has been shown to be required for growth with erythritol. TpiB activity is induced by growth with erythritol; however, basal levels of TpiB activity present in tpiA mutants allow for growth with gluconeogenic carbon sources. Although tpiA mutants can be complemented by tpiB, tpiA cannot substitute for mutations in tpiB with respect to erythritol catabolism. Mutations in tpiA or tpiB alone do not cause symbiotic defects; however, mutations in both tpiA and tpiB caused reduced symbiotic nitrogen fixation. doi:10.1128/JB.01707-06

Sinorhizobium meliloti is a gram-negative soil bacterium found either in free-living form or as a nitrogen-fixing endosymbiont of leguminous plants such as Medicago sativa (alfalfa). S. meliloti synthesizes an unusual sulfate-modified form of lipopolysaccharide (LPS). A recent study reported the identification of a gene, lpsS, which encodes an LPS sulfotransferase activity in S. meliloti. Mutants bearing a disrupted version of lpsS exhibit an altered symbiosis, in that they elicit more nodules than wild type. However, under free-living conditions, the lpsS mutant displayed no change in LPS sulfation. These data suggest that the expression of lpsS is differentially regulated, such that it is transcriptionally repressed during free-living conditions but upregulated during symbiosis. Here, I show that the expression of lpsS is upregulated in strains that constitutively express the symbiotic regulator SyrA. SyrA is a small protein that lacks an apparent DNA binding domain and is predicted to be located in the cytoplasmic membrane yet is sufficient to upregulate lpsS transcription. Furthermore, SyrA can mediate the transcriptional upregulation of exo genes involved in the biosynthesis of the symbiotic exopolysaccharide succinoglycan. The SyrA-mediated transcriptional upregulation of lpsS and exo transcription is blocked in mutants harboring a mutation in chvI, which encodes the response regulator of a conserved two-component system. Thus, SyrA likely acts indirectly to promote transcriptional upregulation of lpsS and exo genes through a mechanism that requires the ExoS/ChvI two-component system.

The type IV secretion system (T4SS) of the plant intracellular symbiont Sinorhizobium meliloti 1021 is required for conjugal transfer of DNA. However, it is not required for host invasion and persistence, unlike the T4SSs of closely related mammalian intracellular pathogens. A comparison of the requirement for a bacterial T4SS in plant versus animal host invasion suggests an important difference in the intracellular niches occupied by these bacteria.

Nitrogen-fixing rhizobial bacteroids import dicarboxylates by using the DctA transporter. G114 of DctA is highly conserved. A G114D mutant is inactive, but DctA with a small amino acid (G114A) or a helix disrupter (G114P) retains significant activity. G114 probably interacts with other membrane helices in stabilizing a substrate-binding pocket.

During the initial stages of symbiosis with the host plant Medicago sativa, Sinorhizobium meliloti must overcome an oxidative burst produced by the plant in order for proper symbiotic development to continue. While identifying mutants defective in symbiosis and oxidative stress defense, we isolated a mutant with a transposon insertion mutation of sitA, which encodes the periplasmic binding protein of the putative iron/ manganese ABC transporter SitABCD. Disruption of sitA causes elevated sensitivity to the reactive oxygen species hydrogen peroxide and superoxide. Disruption of sitA leads to elevated catalase activity and a severe decrease in superoxide dismutase B (SodB) activity and protein level. The decrease in SodB level strongly correlates with the superoxide sensitivity of the sitA mutant. We demonstrate that all free-living phenotypes of the sitA mutant can be rescued by the addition of exogenous manganese but not iron, a result that strongly implies that SitABCD plays an important role in manganese uptake in S. meliloti.

Employing a novel two-part screen, we identified Sinorhizobium meliloti mutants that were both sensitive to hydrogen peroxide and symbiotically defective on the host plant Medicago sativa. The mutations affect a wide variety of cellular processes and represent both novel and previously identified genes important in symbiosis.

Many bacterial species contain multiple copies of the genes that encode the chaperone GroEL and its cochaperone, GroES, including all of the fully sequenced root-nodulating bacteria that interact symbiotically with legumes to generate fixed nitrogen. In particular, in Sinorhizobium meliloti there are four groESL operons and one groEL gene. To uncover functional redundancies of these genes during growth and symbiosis, we attempted to construct strains containing all combinations of groEL mutations. Although a double groEL1 groEL2 mutant cannot be constructed, we demonstrate that the quadruple groEL1 groESL3 groEL4 groESL5 and groEL2 groESL3 groEL4 groESL5 mutants are viable. Therefore, like E. coli and other species, S. meliloti requires only one groEL gene for viability, and either groELl or groEL2 will suffice. The groEL1 groESL5 double mutant is more severely affected for growth at both 30[degrees]C and 40[degrees]C than the single mutants, suggesting overlapping functions in stress response. During symbiosis the quadruple groEL2 groESL3 groEL4 groESL5 mutant acts like the wild type, but the quadruple groEL1 groESL3 groEL4 groESL5 mutant acts like the groEL1 single mutant, which cannot fully induce nod gene expression and forms ineffective nodules. Therefore, the only groEL gene required for symbiosis is groEL1. However, we show that the other groE genes are expressed in the nodule at lower levels, suggesting minor roles during symbiosis. Combining our data with other data, we conclude that groESL1 encodes the housekeeping GroEL/GroES chaperone and that groESL5 is specialized for stress response.

The genome of the symbiotic soil bacterium Sinorhizobium meliloti contains eight genes coding for methylaccepting chemotaxis proteins (MCPs) McpS to McpZ and one gene coding for a transducer-like protein, IcpA. Seven of the MCPs are localized in the cytoplasmic membrane via two membrane-spanning regions, whereas McpY and IcpA lack such hydrophobic regions. The periplasmic regions of McpU, McpV, and McpX contain the small-ligand-binding domain Cache. In addition, McpU possesses the ligand-binding domain TarH. By probing gene expression with lacZ fusions, we have identified mcpU and mcpX as being highly expressed. Deletion of any one of the receptor genes caused impairments in the chemotactic response toward most organic acids, amino acids, and sugars in a swarm plate assay. The data imply that chemoreceptor proteins in S. meliloti can sense more than one class of carbon source and suggest that many or all receptors work as an ensemble. Tactic responses were virtually eliminated for a strain lacking all nine receptor genes. Capillary essays revealed three important sensors for the strong attractant proline: McpU, McpX, and McpY. Receptor deletions variously affected free-swimming speed and attractant-induced chemokinesis. Noticeably, cells lacking mcpU were swimming 9% slower than the wild-type control. We infer that McpU inhibits the kinase activity of CheA in the absence of an attractant. Cells lacking one of the two soluble receptors were impaired in chemokinetic proficiency by more than 50%. We propose that the internal sensors, IcpA and the PAS domain containing McpY, monitor the metabolic state of S. meliloti.

The NA[D.sup.+]-dependent malic enzyme (DME) and the NAD[P.sup.+]-dependent malic enzyme (TME) of Sinorhizobium meliloti are representatives of a distinct class of malic enzymes that contain a 440-amino-acid N-terminal region homologous to other malic enzymes and a 330-amino-acid C-terminal region with similarity to phos-photransacetylase enzymes (PTA). We have shown previously that dme mutants of S. meliloti fail to fix [N.sub.2] ([Fix.sup.-]) in alfalfa root nodules, whereas tme mutants are unimpaired in their [N.sub.2]-fixing ability ([Fix.sup.+]). Here we report that the amount of DME protein in bacteroids is 10 times greater than that of TME. We therefore investigated whether increased TME activity in nodules would allow TME to function in place of DME. The tme gene was placed under the control of the dme promoter, and despite elevated levels of TME within bacteroids, no symbiotic nitrogen fixation occurred in dme mutant strains. Conversely, expression of dme from the tme promoter resulted in a large reduction in DME activity and symbiotic [N.sub.2] fixation. Hence, TME cannot replace the symbiotic requirement for DME. In further experiments we investigated the DME PTA-like domain and showed that it is not required for [N.sub.2] fixation. Thus, expression of a DME C-terminal deletion derivative or the Escherichia coli NA[D.sup.+]-dependent malic enzyme (sfcA), both of which lack the PTA-like region, restored wild-type [N.sub.2] fixation to a dme mutant. Our results have defined the symbiotic requirements for malic enzyme and raise the possibility that a constant high ratio of NADPH + [H.sup.+] to NADP in nitrogen-fixing bacteroids prevents TME from functioning in [N.sub.2]-fixing bacteroids.

Sinorhizobium meliloti exists either in a free-living state in the soil or in symbiosis within legume nodules, where the bacteria differentiate into nitrogen-fixing bacteroids. Expression of genes involved in nitrogen fixation and associated respiration is governed by two intermediate regulators, NifA and FixK, respectively, which are controlled by a two-component regulatory system FixLJ in response to low-oxygen conditions. In order to identify the FixLJ regulon, gene expression profiles were determined in microaerobic free-living cells as well as during the symbiotic life of the bacterium for the wild type and a fixJ null-mutant strain. We identified 122 genes activated by FixJ in either state, including 87 novel targets. FixJ controls 74% of the genes induced in microaerobiosis (2% oxygen) and the majority of genes expressed in mature bacteroids. Ninety-seven percent of Fix J-activated genes are located on the symbiotic plasmid pSymA. Transcriptome profiles of a nifA and a fixK mutant showed that NifA activates a limited number of genes, all specific to the symbiotic state, whereas FixK controls more than 90 genes, involved in free-living and/or symbiotic life. This study also revealed that FixJ has no other direct targets besides those already known. FixJ is involved in the regulation of functions such as denitrification or amino acid/polyamine metabolism and transport. Mutations in selected novel FixJ targets did not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa roots. From these results, we propose an updated model of the FixJ region.

Sinorhizobium meliloti produces an exopolysaccharide called succinoglycan that plays a critical role in promoting symbiosis with its host legume, alfalfa (Medicago sativa). We performed a transposon mutagenesis and screened for mutants with altered succinoglycan production and a defect in symbiosis. In this way, we identified a putative two-component histidine kinase associated with a PAS sensory domain, now designated CbrA (calcofluor-bright regulator A). The cbrA::Tn5 mutation causes overproduction of succinoglycan and results in increased accumulation of low-molecular-weight forms of this exopolysaccharide. Our results suggest the cbrA::Tn5 allele leads to this succinoglycan phenotype through increased expression of exo genes required for succinoglycan biosynthesis and modification. Interestingly, CbrA-dependent regulation of exo and exs genes is observed almost exclusively during stationary-phase growth. The cbrA::Tn5 mutant also has an apparent cell envelope defect, based on increased sensitivity to a number of toxic compounds, including the bile salt deoxycholate and the hydrophobic dye crystal violet. Growth of the cbrA mutant is also slowed under oxidative-stress conditions. The CbrA-regulated genes exsA and exsE encode putative inner membrane ABC transporters with a high degree of similarity to lipid exporters. ExsA is homologous to the Escherichia coli MsbA protein, which is required for lipopolysacharide transport, while ExsE is a member of the eukaryotic family of ABCD/hALD peroxisomal membrane proteins involved in transport of very long-chain fatty acids, which are a unique component of the lipopolysaccharides of alphaproteobacteria. Thus, CbrA could play a role in regulating the lipopolysaccharide or lipoprotein components of the cell envelope.

Sinorhizobium meliloti is a gram-negative soil bacterium, capable of establishing a nitrogen-fixing symbiosis with its legume host, alfalfa (Medicago sativa). Quorum sensing plays a crucial role in this symbiosis, where it influences the nodulation process and the synthesis of the symbiotically important exopolysaccharide II (EPS II). S. meliloti has three quorum-sensing systems (Sin, Tra, and Mel) that use N-acyl homoserine lactones as their quorum-sensing signal molecule. Increasing evidence indicates that certain eukaryotic hosts involved in symbiotic or pathogenic relationships with gram-negative bacteria produce quorum-sensing-interfering (QSI) compounds that can cross-communicate with the bacterial quorum-sensing system. Our studies of alfalfa seed exudates suggested the presence of multiple signal molecules capable of interfering with quorum-sensing-regulated gene expression in different bacterial strains. In this work, we choose one of these QSI molecules (SWI) for further characterization. SWI inhibited violacein production, a phenotype that is regulated by quorum sensing in Chromobacterium violaceum. In addition, this signal molecule also inhibits the expression of the S. meliloti exp genes, responsible for the production of EPS II, a quorum-sensing-regulated phenotype. We identified this molecule as L-canavanine, an arginine analog, produced in large quantities by alfalfa and other legumes.

Cohesive ends of 16-3, a temperate phage of Rhizobium meliloti 41, have been identified as 10-base-long, 3'-protruding complementary G/C-rich sequences, terS and terL encode the two subunits of 16-3 terminase. Significant homologies were detected among the terminase subunits of phage 16-3 and other phages from various ecosystems.

To understand the mechanisms of ectoine-induced osmoprotection in Sinorhizobium meKloti, a proteomic examination of S. meliloti cells grown in minimal medium supplemented with ectoine was undertaken. This revealed the induction of 10 proteins. The protein products of eight genes were identified by using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Five of these genes, with four other genes whose products were not detected on two-dimensional gels, belong to the same gene cluster, which is localized on the pSymB megaplasmid. Four of the nine genes encode the characteristic components of an ATP-binding cassette transporter that was named ehu, for eetoine/hydroxyectoine uptake. This transporter was encoded by four genes (ehuA, chuB, ehuC, and ehuD) that formed an operon with another gene cluster that contains five genes, named eutABCDE for ectoine utilization. On the basis of sequence homologies, eutABCDE encode enzymes with putative and hypothetical functions in ectoine catabolism. Analysis of the properties of ehuA and eutA mutants suggests that S. meliloti possesses at least one additional eetoine catabolic pathway as well as a lower-affinity transport system for ectoine and hydroxyectoine. The expression of chuB, as determined by measurements of UidA activity, was shown to be induced by ectoine and hydroxyectoine but not by glycine betaine or by high osmolality.

The exopolysaccharide galactoglucan promotes the establishment of symbiosis between the nitrogen-fixing Gram-negative soil bacterium Sinorhizobium meliloti 2011 and its host plant alfalfa. The transcriptional regulator ExpG activates expression of galactoglucan biosynthesis genes by direct binding to the expA1, expGlexpD1 and expE1 promoter regions. ExpG is a member of the MarR family of regulatory proteins. Analysis of target sequences of an ExpG[(His).sub.6] fusion protein in the exp promoter regions resulted in the identification of a binding site composed of a conserved palindromic region and two associated sequence motifs. Association and dissociation kinetics of the specific binding of ExpG[(His).sub.6] to this binding site were characterized by standard biochemical methods and by single-molecule spectroscopy based on the atomic force microscope (AFM). Dynamic force spectroscopy indicated a distinct difference in the kinetics between the wild-type binding sequence and two mutated binding sites, leading to a closer understanding of the ExpG-DNA interaction. Abbreviations: AFM, atomic force microscopy; EMSA, electrophoretic mobility shift assay; EPS, exopolysaccharide; EPS I, succinoglycan; EPS II, galactoglucan; HTH, helix-turn-helix; [k.sub.on], on-rate; [k.sub.off], off-rate; [K.sub.d], dissociation constant.

The over expression and characterization of the three distinct heme-containing hydroperoxidases, katA, katB and katC from Sinohizobium meliloti is described. The results suggests that the combination of regulatory processes and of the kinetic parameters of the three hydroperoxidases appears to lead to an optimization of the S.meliloti capaity of H2O2 scavenging.

In Sinorhizobium meliloti, choline is the direct precursor of phosphatidylcholine, a major lipid membrane component in the Rhizobiaceae family, and glycine betaine, an important osmoprotectant. Moreover, choline is an efficient energy source which supports growth. Using a PCR strategy, we identified three chromosomal genes (choXWV) which encode components of an ABC transporter: ChoX (binding protein), ChoW (permease), and ChoV (ATPase). Whereas the best homology scores were obtained with components of betaine ProU-like systems, Cho is not involved in betaine transport. Site-directed mutagenesis of choX strongly reduced (60 to 75%) the choline uptake activity, and purification of ChoX, together with analysis of the ligand-binding specificity, showed that ChoX binds choline with a high affinity ([K.sub.D], 2.7 [micro]M) and acetylcholine with a low affinity ([K.sub.D], 2.7 [micro]M) but binds none of the betaines. Uptake competition experiments also revealed that ectoine, various betaines, and choline derivatives were not effective competitors for Cho-mediated choline transport. Thus, Cho is a highly specific high-affinity choline transporter. Choline transport activity and ChoX expression were induced by choline but not by salt stress. Western blotting experiments with antibodies raised against ChoX demonstrated the presence of ChoX in bacteroids isolated from nitrogen-fixing nodules obtained from Medicago sativa roots. The choX mutation did not have an effect on growth under standard conditions, and neither Nod nor Fix phenotypes were impaired in the mutant, suggesting that the remaining choline uptake system(s) still present in the mutant strain can compensate for the lack of Cho transporter.

Results show that two DNA segments from Sinorhizobium meliloti exhibit sequence similarity to rpoH genes, encoding the sigma(sup)32 RpoH heat -shock transcription factor. Of the two genes, rpoH1 and rpoH2, the former upon mutation impairs growth and affect nitrogen fixation during symbiosis with alfalfa.

Research has been conducted on Rhizobium meliloti flagellar, chemotaxis and motility genes. The probing of the gene expression on Western blots and with lacZ fusions has been carried out.

Exopolysaccharide galactoglucan (EPS II) production in the wild-type strain of the soil bacterium Sinorhizobium meliloti is induced by phosphate-limiting conditions or by extra copies of the exp gene cluster. Based on similarities to the transcriptional regulators of the MarR family, an additional putative regulatory gene, expG, was determined in the exp gene cluster. Employing exp-lacZ transcriptional fusions, a stimulating effect of extra copies of this expG on the transcription of all exp complementation groups was determined.

The capability of Rhizobium meliloti Rm1021 to synthesize succinoglycan is needed for it to invade the nodules. The symbiotic deficiency of exo mutants that are defected or modified in succinoglycan production was studied by utilizing R. meliloti cells that express green fluorescent protein. Findings suggested that succinoglycan is needed for both the initiation and elongation of infection threads during nodule entry.

A study was conducted on the influence of the acetyl and succinyl modifications of succinoglycan on the susceptibility of succinoglycan to cleavage by the ExoK and ExsH glycanases. Findings indicated that nonacetylated succinoglycan had a high degree of susceptibility to cleavage by the two glycanases, while nonsuccinylated succinoglycan has a low degree of susceptibility. These suggested that the molecular weight distribution of each of the modified forms of succinoglycan can be manipulated with a high degree of control by culturing various Rhizobium meliloti strains.

The dependence of autoxidation rate on oxygen concentration for Rhizobium meliloti FixL and Aplysia kurodai myoglobin with pentacoordinate met forms was determined. Results showed that the autoxidation mechanism used by the two proteins involved the reaction between a noncoordinated oxygen molecule and an intermediate with water coordinated to the ferrous heme iron. However, the absence of any link between the coordination of water and oxygen protection effect may also involved other mechanisms such as the transfer of electrons from oxyheme to deoxyheme.

The Rhizobium meliloti ExoK and ExsH glycanases have been proposed to contribute to production of low molecular weight (LMW) succinoglycan by depolymerizing high molecular weight succinoglycan chains in R. meliloti cultures. We expressed and purified ExoK and ExsH and determined that neither enzyme can extensively cleave succinoglycan prepared from R. meliloti cultures, although neutral/heat treatment and acid/heat treatment convert succinoglycan to forms that can be cleaved efficiently by both enzymes. These results were somewhat surprising, given that the exo[K.sup.+] and exs[H.sup.+] genes play a crucial role in production of LMW succinoglycan in R. meliloti cultures. We demonstrated by Western blot analyses that R. meliloti expresses ExoK and ExsH, that both proteins can be detected extracellularly, and that ExsH secretion depends on the prs[D.sup.+]/prs[E.sup.+] genes, consistent with previous predictions based on mutant analyses. Furthermore, we determined that the depolymerization activities associated with purified ExoK and ExsH are comparable with exo[K.sup.+] and exs[H.sup.+]-dependent depolymerization activities expressed in R. meliloti cultures. We resolved the apparent contradiction between the results of our previous genetic analyses and depolymerization assays by determining that ExoK and ExsH can cleave high molecular weight succinoglycan that is being produced actively by R. meliloti, but not succinoglycan that has accumulated in cultures, to yield LMW succinoglycan. We propose that ExoK and ExsH dynamically regulate the molecular weight distribution of succinoglycan by cleaving nascent succinoglycan only during a limited period after its synthesis, perhaps before it undergoes a time-dependent change in its conformation or aggregation state.

Research was conducted to investigate the effects of ionic and osmotic strength on the glucosyltransferase of Rhizobium meliloti responsible for cyclic beta-(1,2)-glucan biosynthesis. The regulation of the biosynthesis of these glucans were examine in vitro with membrane preparations from R meliloti. Results indicate that NdvA and the C terminus of NdvB are not essential for osmotic regulation of cyclic beta-(1,2)-glucan biosynthesis.

The phosphotransfer between the purified CheA, CheY1 and CheY2 proteins and the spontaneous dephosphorylation rates of CheY1-P and CheY2-P were examined in vitro to describe the mechanism behind the reactions. The results support a four-step phosphotransfer including ATP-dependent autophosphorylation of CheA. CheY1 was found to act as a 'phosphatase' of CheY2-P by becoming a sink for phosphate in the presence of unphosphorylated CheA. It was also found to regulate the active, phosphorylated state of CheY2-P, the main response regulator.

A research was conducted to study the responses of Rhizobium meliloti to phosphate stress and to determine the phosphate level at which R. meliloti induces its P stress response. Transposon Tn5 was conjugated from Escherichia coli S17-1 into R. meliloti via the suicide vector pSUP2021. Alfalfa seedlings in sterile growth box units were utilized to determine the symbiosis phenotype of the phoB mutant. Results indicated that the loss of a functional PhoB did not disturb the symbiotic function of R. meliloti regardless of the available phosphate.

The CcrM DNA methyltransferase was examined to determine its significant functions in Rhizobium meliloti and Caulobacter crescentus. Results of the analysis revealed that CcrM DNA methyltransferase is significant for viability, but does not take part in the DNA restriction-modification processes. Furthermore, C. crescentus and R. meliloti CcrM genes are interchangeable, making CcrM genes important elements of the cell cycle.

The effects of the alterations within the activation domain of the sigma54-dependent activator DctD on the transcriptional activation was analyzed. Mutant forms of DctD that failed to activate transcription were isolated and characterized. Results of the study revealed that selected purified mutant proteins have normal DNA-binding activity and interacted with sigma54 and core RNA polymerase.

Diphosphopyridine-nucleotide-dependent malic enzyme (DME) of Rhizobium meliloti is needed for symbiotic nitrogen fixation since it forms a pathway in which malate is converted to acetyl-CoA in bacteroids. This enzyme shows maximal activity with NAD+ as cofactor but has reduced activity when NADP+ is used. Acetyl-CoA strongly inhibited DME activity but not TME activity. The dme gene was expressed in free-living cells and in nitrogen-fixing bacteroids while tme gene was repressed in bacteroids.

The nature of carbon and nitrogen sources effects the coproduction of exopolysaccharide (EPS) and poly-beta-hydroxybutyrate (PHB) by the M5N1 and Su47 strains of Rhizobium melilot. Bacterial growth in nitrogen-deprived medium is limited, and facilitates EPS production rather than PHB production. Cells grown in glucose show a lag phase in EPS production, which is absent in EPS production from fructose and in PHB synthesis. The lag phase is related to the extracellular conversion of glucose to gluconic or 2-ketogluconic acids.

A gene encoding a variant of green fluorescent protein (GFP) from Aequorea victoria was placed under the control of a promoter which is constitutive in Rhizobium meliloti. The heterologous GFP gene was expressed at high levels during all stages of symbiosis. This enabled the visualization of R. meliloti cells as they grew in the rhizosphere, on the root surface and inside infection threads. Also, nodules that were infected with bacteria which were synthesizing GFP fluoresced when exposed to blue light. GFP-tagged bacteria could be observed inside infection threads, allowing the measurement of the growth rate.

Effective invasion of alfalfa by Rhizobium meliloti Rm1021 normally requires the presence of succinoglycan, an exopolysaccharide (EPS) produced by the bacterium. However, Rm1021 has the ability to produce a second EPS (EPS II) that can suppress the symbiotic defects of succinoglycan-deficient strains. EPS II is a polymer of modified glucose-([Beta]-1,3)-galactose subunits and is produced by Rm1021 derivatives carrying either an expR101 or mucR mutation. If the ability to synthesize succinoglycan is blocked genetically, expR101 derivatives of Rm1021 are nodulation-proficient, whereas mucR derivatives of Rm1021 are not. The difference in nodulation proficiency between these two classes of EPS II-producing strains is due to the specific production of a low molecular weight form of EPS II by expR101 strains. A low molecular weight EPS II fraction consisting of 15-20 EPS II disaccharide subunits efficiently allows nodule invasion by noninfective strains when present in amounts as low as 7 pmol per plant, suggesting that low molecular weight EPS II may act as a symbiotic signal during infection.

Ammonia inhibits plasmid conjugation between Rhizobium meliloti strains. A study of the effect of nutritional factors on two nonsymbiotic plasmids pRmeGR4a and pRmeGR4b of R. meliloti strains indicates that glutamate promotes transconjugation. The conjugation is reduced by a factor of 100 in media with glutamate and ammonium as nitrogen sources, in comparison with media containing only glutamate. The inhibition of conjugal transfer between R. meliloti strains by ammonia is however, unobservable in heterologous plasmids.

The 'thy' autoselective system can efficiently maintain the stability of the plasmid vector in Rhizobium (R.) meliloti under field and greenhouse conditions, and can make thymidylate synthase-deficient (Thy-) rhizobia nodulate the host. But the expression of the thy gene needs to be regulated to avert its negative effects on symbiosis. The thy system is based on the thy gene that encodes thymidylate synthase. The system comprises a plasmid vector from Lactococcus lactis and a Thy- spontaneous mutant of R. meliloti. The plasmid is free-replicating and has a broad range of hosts.

The C4-dicarboxylic acid transport protein D (DCDT) of Rhizobium meliloti crosslinks with sigma-54 of Salmonella typhimurium and the beta subunit of RNA polymerase of Escherichia coli. No crosslinking is seen between DCDT and proteins not involved in transcription initiation. Thus DCDT initiates transcription by directly interacting with sigma-54-RNA polymerase holoenzyme (E-sigma-54). The interaction of both a truncated form and intact DCDT with E-sigma-54 is similar. Transcription initiation can be controlled by both an active and inactive form of DCDT.

The ability of Rhizobium meliloti to tolerate high concentrations of salt in marshes may be related to changes in its lipopolysaccharide (LPS) structure in response to salt stress. The EFB1 strain of the bacteria can grow in concentrations of NaCl as high as 500mM. Electrophoresis and antibody assays indicated that the LPS of this strain changed in response to ionic stress and osmotic stress.

The cloning and sequencing of the poly-beta-hydroxybutyrate (PHB) biosynthetic genes phaA, phaB and ORF1, in Rhizobium meliloti 41, can be done using heterologus hybridization on a cosmid library. Another gene, phaC, clone is obtained from a R. meliloti PHB-Tn5 mutant. Escherichia coli transducted with these clones expresses phaA and phaB and synthesize PHB. The defect in PHB-mutant of R. meliloti can be removed by the phaC clone. Details of a study conducted on the genes and an analysis of the results are given.

LS1 The Rhizobium meliloti AK631 strain, with a mutation in the exoB gene, does not produce extracellular polysaccharide (EPS) but is able to symbiotically infect alfalfa. However, a lpsZ mutation in AK631 prevents the fix, suggesting that EPS' function is performed by a Escherichia coli group II K polysaccharide analog cell surface factor. The inability to fix is restored in both the lpsZ mutant and a lpsZ- wild-type when lpsZ is introduced and K polysaccharide expression is modified.

The Rhizobium (R.) meliloti strain 042B, from alfalfa grown in the Xinjiang Autonomous Region of China can nodulate and fix nitrogen with soybean plants indicating that the symbiotic capabilities of R. meliloti are not restricted to alfalfa and other legumes. R. strains generally form nodules only on a selected range of host plants though the host specificity varies in different strains. The R. meliloti strain can fix more nitrogen per hectare than other rhizobia including soybean rhizobia.

Analysis of rhizopine scylloinosamine (SI) synthesis gene (mos) shows that the Rhizobium meliloti strain RM220-3 producing rhizopine SI in nodules contains three ORFs namely, ORF1, mosB and mosC arranged in a 3.9 kb operon unit. The L5-30 mos operon contains four ORFs and a construct developed from the L5-30 mos locus having truncated mosA gene produces SI instead of 3-O-MSI.

A study of NodH-dependent in vitro sulfotransferase activity in Rhizobium meliloti using protein extracts and specifically purified recombinant NodH protein reveals that tetrameric Nod factors are that the most important and high affinity of NodH for suitable tetrameric substrate ensures its alteration, contributing to the fidelity of host-specific activity.