Turlough M. Finan

The complete sequence of the 1,683-kb pSymB megaplasmid from the N2-fixing endosymbiont Sinorhizobium meliloti

Analysis of the 1,683,333-nt sequence of the pSymB megaplasmid from the symbiotic N2-fixing bacterium Sinorhizobium meliloti revealed that the replicon has a high gene density with a total of 1,570 protein-coding regions, with few insertion elements and regions duplicated elsewhere in the genome. The only copies of an essential arg-tRNA gene and the minCDE genes are located on pSymB. Almost 20% of the pSymB sequence carries genes encoding solute uptake systems, most of which were of the ATP-binding cassette family. Many previously unsuspected genes involved in polysaccharide biosynthesis were identified and these, together with the two known distinct exopolysaccharide synthesis gene clusters, show that 14% of the pSymB sequence is dedicated to polysaccharide synthesis. Other recognizable gene clusters include many involved in catabolic activities such as protocatechuate utilization and phosphonate degradation. The functions of these genes are consistent with the notion that pSymB plays a major role in the saprophytic competence of the bacteria in the soil environment.

The regulator gene phoB mediates phosphate stress-controlled synthesis of the membrane lipid diacylglyceryl-N,N,N-trimethylhomoserine in Rhizobium (Sinorhizobium) meliloti

Bacteria react to phosphate starvation by activating genes involved in the transport and assimilation of phosphate as well as other phosphorous compounds. Some soil bacteria have evolved an additional mechanism for saving phosphorus. Under phosphate‐limiting conditions, they replace their membrane phospholipids by lipids not containing phosphorus. Here, we show that the membrane lipid pattern of the free‐living microsymbiotic bacterium Rhizobium (Sinorhizobium) meliloti is altered at low phosphate concentrations. When phosphate is growth limiting, an increase in sulpholipids, ornithine lipids and the de novo synthesis of diacylglyceryl trimethylhomoserine (DGTS) lipids is observed. Rhizobium meliloti phoCDET mutants, deficient in phosphate uptake, synthesize DGTS constitutively at low or high medium phosphate concentrations, suggesting that reduced transport of phosphorus sources to the cytoplasm causes induction of DGTS biosynthesis. Rhizobium meliloti phoU or phoB mutants are unable to form DGTS at low or high phosphate concentrations. However, the functional complementation of phoU or phoB mutants with the phoB gene demonstrates that, of the two genes, only intact phoB is required for the biosynthesis of the membrane lipid DGTS.

Novel Pathway for Arsenic Detoxification in the Legume Symbiont Sinorhizobium meliloti

We report a novel pathway for arsenic detoxification in the legume symbiont Sinorhizobium meliloti. Although a majority of ars operons consist of three genes, arsR (transcriptional regulator), arsB [As(OH)3/H+ antiporter], and arsC (arsenate reductase), the S. meliloti ars operon includes an aquaglyceroporin (aqpS) in place of arsB. The presence of AqpS in an arsenic resistance operon is interesting, since aquaglyceroporin channels have previously been shown to adventitiously facilitate uptake of arsenite into cells, rendering them sensitive to arsenite. To understand the role of aqpS in arsenic resistance, S. meliloti aqpS and arsC were disrupted individually. Disruption of aqpS resulted in increased tolerance to arsenite but not arsenate, while cells with an arsC disruption showed selective sensitivity to arsenate. The results of transport experiments in intact cells suggest that AqpS is the only protein of the S. meliloti ars operon that facilitates transport of arsenite. Coexpression of S. meliloti aqpS and arsC in a strain of E. coli lacking the ars operon complemented arsenate but not arsenite sensitivity. These results imply that, when S. meliloti is exposed to environmental arsenate, arsenate enters the cell through phosphate transport systems and is reduced to arsenite by ArsC. Internally generated arsenite flows out of the cell by downhill movement through AqpS. Thus, AqpS confers arsenate resistance together with ArsC-catalyzed reduction. This is the first report of an aquaglyceroporin with a physiological function in arsenic resistance.

Mapping the Sinorhizobium meliloti 1021 solute-binding protein-dependent transportome

The number of solute-binding protein-dependent transporters in rhizobia is dramatically increased compared with the majority of other bacteria so far sequenced. This increase may be due to the high affinity of solute-binding proteins for solutes, permitting the acquisition of a broad range of growth-limiting nutrients from soil and the rhizosphere. The transcriptional induction of these transporters was studied by creating a suite of plasmid and integrated fusions to nearly all ATP-binding cassette (ABC) and tripartite ATP-independent periplasmic (TRAP) transporters of Sinorhizobium meliloti. In total, specific inducers were identified for 76 transport systems, amounting to ≈47% of the ABC uptake systems and 53% of the TRAP transporters in S. meliloti. Of these transport systems, 64 are previously uncharacterized in Rhizobia and 24 were induced by solutes not known to be transported by ABC- or TRAP-uptake systems in any organism. This study provides a global expression map of one of the largest transporter families (transportome) and an invaluable tool to both understand their solute specificity and the relationships between members of large paralogous families.

Analysis of C4-dicarboxylate transport genes in Rhizobium meliloti

A 5.1 kbp DNA fragment was isolated which complemented C4‐dicarboxylate transport mutants (dct) of Rhizobium meliloti. Characterization of this fragment by subcloning, transposon mutagenesis, and complementation analysis revealed three loci, designated dctA, dctB, and dctD. TnphoA‐generated alkaline phosphatase fusions to dctA suggested that this gene encodes the structural transport protein and allowed the determination of its direction of transcription. Analysis of the fusions in various mutant backgrounds demonstrated that dctB, dctD, and ntrA products are required for dctA expression. The dctA fusion was constitutively expressed in a dctA mutant background, but was not expressed in dctA dctB or dctA dctD double mutants. This suggests that the constitutive expression in a dctA mutant background is mediated through dctB and dctD. Three independent second‐site Dct+ revertant mutations in ntrA mutant strains mapped to the dct locus. Succinate transport in these revertant strains was constitutive, whereas in the wild type, succinate transport was inducible. These results are consistent with the direct requirement of the ntrA gene product for dctA expression. Alfalfa plants inoculated with the dctB and dctD mutants showed reduced nitrogen‐fixing activity. Nodules induced by dctA mutants failed to fix nitrogen. These symbiotic phenotypes are consistent with previous suggestions that dctA expression in bacteroids can occur independently of dctB and dctD.

Genomes of the Symbiotic Nitrogen-Fixing Bacteria of Legumes

Over the last several decades, there have been a large number of studies done on the genetics, biochemistry, physiology, ecology, and agronomics of the bacteria forming nitrogen-fixing symbioses with legumes. These bacteria, collectively referred to as the rhizobia, are taxonomically and

Genome prediction of PhoB regulated promoters in Sinorhizobium meliloti and twelve proteobacteria.

In proteobacteria, genes whose expression is modulated in response to the external concentration of inorganic phosphate are often regulated by the PhoB protein which binds to a conserved motif (Pho box) within their promoter regions. Using a position weight matrix algorithm derived from known Pho box sequences, we identified 96 putative Pho regulon members whose promoter regions contained one or more Pho boxs in the Sinorhizobium meliloti genome. Expression of these genes was examined through assays of reporter gene fusions and through comparison with published microarray data. Of 96 genes, 31 were induced and 3 were repressed by Pi starvation in a PhoB dependent manner. Novel Pho regulon members included several genes of unknown function. Comparative analysis across 12 proteobacterial genomes revealed highly conserved Pho regulon members including genes involved in Pi metabolism (pstS, phnC and ppdK). Genes with no obvious association with Pi metabolism were predicted to be Pho regulon members in S.meliloti and multiple organisms. These included smc01605 and smc04317 which are annotated as substrate binding proteins of iron transporters and katA encoding catalase. This data suggests that the Pho regulon overlaps and interacts with several other control circuits, such as the oxidative stress response and iron homeostasis.

NAD+ ‐dependent malic enzyme of Rhizobium meliloti is required for symbiotic nitrogen fixation

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 NADP+ 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.

Regulation and Properties of PstSCAB, a High-Affinity, High-Velocity Phosphate Transport System of Sinorhizobium meliloti

The properties and regulation of the pstSCAB-encoded Pi uptake system from the alfalfa symbiont Sinorhizobium meliloti are reported. We present evidence that the pstSCAB genes and the regulatory phoUB genes are transcribed from a single promoter that contains two PhoB binding sites and that transcription requires PhoB. S. meliloti strain 1021 (Rm1021) and its derivatives were found to carry a C deletion frameshift mutation in the pstC gene (designated pstC1021) that severely impairs activity of the PstSCAB Pi transport system. This mutation is absent in RCR2011, the parent of Rm1021. Correction of the pstC1021 mutation in Rm1021 by site-directed mutagenesis revealed that PstSCAB is a Pi-specific, high-affinity (Km, 0.2 microM), high-velocity (Vmax, 70 nmol/min/mg protein) transport system. The pstC1021 allele was shown to generate a partial pho regulon constitutive phenotype, in which transcription is activated by PhoB even under Pi-excess conditions that render PhoB inactive in a wild-type background. The previously reported symbiotic Fix- phenotype of phoCDET mutants was found to be dependent on the pstC1021 mutation, as Rm1021 phoCDET mutants formed small white nodules on alfalfa that failed to reduce N2, whereas phoCDET mutant strains with a corrected pstC allele (RmP110) formed pink nodules on alfalfa that fixed N2 like the wild type. Alfalfa root nodules formed by the wild-type RCR2011 strain expressed the low-affinity orfA-pit-encoded Pi uptake system and neither the pstSCAB genes nor the phoCDET genes. Thus, metabolism of alfalfa nodule bacteroids is not Pi limited.

The expression of a novel antisense gene mediates incompatibility within the large repABC family of α-proteobacterial plasmids

Large extrachromosomal replicons in many members of the α‐proteobacteria encode genes that are required for plant or animal pathogenesis or symbiosis. Most of these replicons encode repABC genes that control their replication and faithful segregation during cell division. In addition to its chromosome, the plant endosymbiont Sinorhizobium meliloti also maintains the 1.4 Mb pSymA and 1.7 Mb pSymB symbiotic megaplasmids both of which are repABC‐type replicons. In all repABC loci that have been characterized, an apparently untranslated intergenic region between the repB and repC genes encodes a strong incompatibility determinant (referred to as incα). Here we report the isolation of mutations within the incα regions of pSymA and pSymB that eliminate incompatibility. These mutations map to and inactivate a promoter in the intergenic region that drives the expression of an approximately 56 nucleotide untranslated RNA molecule that mediates incompatibility. This gene, that we have named incA, is transcribed antisense to the repABC genes. Our analysis suggests that the incA gene is conserved in repABC loci from a diverse spectrum of bacteria.