Characterization of the Rhizobium (Sinorhizobium) meliloti high- and low-affinity phosphate uptake systems

Genetic studies have suggested that Rhizobium (Sinorhizobium) meliloti contains two distinct phosphate (Pi) transport systems, encoded by the phoCDET genes and the orfA-pit genes, respectively. Here we present data which show that the ABC-type PhoCDET system has a high affinity for Pi (Km, 0.2 microM) and that Pi uptake by this system is severely inhibited by phosphonates. This high-affinity uptake system was induced under Pi-limiting conditions and was repressed in the presence of excess Pi. Uptake via the OrfA-Pit system was examined in (i) a phoC mutant which showed increased expression of the orfA-pit genes as a result of a promoter-up mutation and (ii) a phoB mutant (PhoB is required for phoCDET expression). Pi uptake in both strains exhibited saturation kinetics (Km, 1 to 2 microM) and was not inhibited by phosphonates. This uptake system was active in wild-type cells grown with excess Pi and appeared to be repressed when the cells were starved for Pi. Thus, our biochemical data show that the OrfA-Pit and PhoCDET uptake systems are differentially expressed depending on the state of the cell with respect to phosphate availability.     

Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concetrations. Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.     

Sucrose is a nonaccumulated osmoprotectant in Sinorhizobium meliloti

Intracellular accumulation of sucrose in response to lowered water activity seems to occur only in photosynthetic organisms. Here we demonstrate, for the first time, the potent ability of this common sugar, supplied exogenously, to reduce growth inhibition of Sinorhizobium meliloti cells in media of inhibitory osmolarity. Independently of the nature of the growth substrates and the osmotic agent, sucrose appears particularly efficient in promoting the recovery of cytoplasmic volume after plasmolysis. Surprisingly, sucrose is not accumulated by the bacteria at an osmotically efficient level. Instead, it strongly stimulates the accumulation of the main endogenous osmolytes glutamate and N-acetylglutaminylglutamine amide (NAGGN). Examining cell volume changes during the hyperosmotic treatment, we found a close correlation between the enhancement of the osmotically active solute pool and the increase in cell volume. Sucrose shares several features with ectoine, another nonaccumulated osmoprotectant for S. meliloti. Overall, osmoregulation in S. meliloti appears to be strongly divergent from that in most bacteria.     

Rhizobium meliloti mutants deficient in phospholipid N-methyltransferase still contain phosphatidylcholine

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes. In addition to this structural function, PC is thought to play a major role in lipid turnover and signalling in eukaryotic systems. In prokaryotes, only some groups of bacteria, among them the members of the family Rhizobiaceae, contain PC. To understand the role of PC in bacteria, we have studied Rhizobium meliloti 1021, which is able to form nitrogen-fixing nodules on its legume host plants and therefore has a very complex phenotype. R. meliloti was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, and potential mutants defective in phospholipid N-methyltransferase were screened by using a colony autoradiography procedure. Filters carrying lysed replicas of mutagenized colonies were incubated with S-adenosyl-L-[methyl-14C]methionine. Enzymatic transfer of methyl groups to phosphatidylethanolamine (PE) leads to the formation of PC and therefore to the incorporation of radiolabel into lipid material. Screening of 24,000 colonies for reduced incorporation of radiolabel into lipids led to the identification of seven mutants which have a much-reduced specific activity of phospholipid N-methyltransferase. In vivo labelling of mutant lipids with [14C]acetate showed that the methylated PC biosynthesis intermediates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine are no longer detectable. This loss is combined with a corresponding increase in the potential methyl acceptor PE. These results indicate that PC biosynthesis via the methylation pathway is indeed blocked in the mutants isolated. However, mass spectrometric analysis of the lipids shows that PC was still present when the mutants had been grown on complex medium and that it was present in the mutants in wild-type amounts. In vivo labelling with [methyl-14C]methionine shows that in phospholipid N-methyltransferase-deficient mutants, the choline moiety of PC is not formed by methylation. These findings suggest the existence of a second pathway for PC biosynthesis in Rhizobium.     

Different phenotypic classes of Sinorhizobium meliloti mutants defective in synthesis of K antigen

For Sinorhizobium meliloti (also known as Rhizobium meliloti) AK631 to establish effective symbiosis with alfalfa, it must be able to synthesize a symbiotically active form of its K antigen, a capsular polysaccharide containing a Kdo (3-deoxy-D-manno-octulosonic acid) derivative. Previously isolated mutants defective in the synthesis of K antigen are resistant to bacteriophage phi16-3. By screening ca. 100,000 Tn5-mutagenized R. meliloti bacteria for resistance to bacteriophage phi16-3, we isolated 119 mutants, 31 of which could not be complemented by genes previously identified as being required for K-antigen synthesis. Of these 31 new mutants, 13 were symbiotically defective and lacked the K antigen. Through genetic and phenotypic analyses, we have grouped these mutants into four distinct classes. Although all of these mutants lack the K antigen, many also have altered lipopolysaccharides (LPS), suggesting that the biochemical pathways for the synthesis of K antigen and LPS have common enzymatic steps. In addition, we have found that these and other classes of K-antigen-defective mutants of S. meliloti AK631 exhibit unique patterns of sensitivities to phage strains to which the parental strain was resistant. Our studies have identified new classes of genes required for both the synthesis of K antigen and the symbiotic proficiency of S. meliloti AK631. Some of these classes of genes also play a role in LPS synthesis.     

Mechanism of gluconate synthesis in Rhizobium meliloti by using in vivo NMR

The dehydrogenation of [1-(13)C]- and [2-(13)C]glucose into gluconate was monitored by NMR spectroscopy in living cell suspensions of two Rhizobium meliloti strains. The synthesis of gluconate was accompanied, in the cellular environment, by the formation of two gluconolactones, a gamma-lactone being detected in addition to the expected delta-lactone. These lactones--as well as the gluconate--could be further metabolized by the cells. The delta-lactone was utilized faster than the gamma-lactone. The presence--in significant amounts--and the relative stability of the lactones raise the question of their possible physiological significance.     

Molecular and genetic characterization of the rhizopine catabolism (mocABRC) genes of Rhizobium meliloti L5-30

Rhizopine (L-3-O-methyl-scyllo-inosamine, 3-O-MSI) is a symbiosis-specific compound, which is synthesized in nitrogen-fixing nodules of Medicago sativa induced by Rhizobium meliloti strain L5-30. 3-O-MSI is thought to function as an unusual growth substrate for R. meliloti L5-30, which carries a locus (mos) responsible for its synthesis closely linked to a locus (moc) responsible for its degradation. Here, the essential moc genes were delimited by Tn5 mutagenesis and shown to be organized into two regions, separated by 3 kb of DNA. The DNA sequence of a 9-kb fragment spanning the two moc regions was determined, and four genes were identified that play an essential role in rhizopine catabolism (mocABC and mocR). The analysis of the DNA sequence and the amino acid sequence of the deduced protein products revealed that MocA resembles NADH-dependent dehydrogenases. MocB exhibits characteristic features of periplasmic-binding proteins that are components of high-affinity transport systems. MocC does not share significant homology with any protein in the database. MocR shows homology with the GntR class of bacterial regulator proteins. These results suggest that the mocABC genes are involved in the uptake and subsequent degradation of rhizopine, whereas mocR is likely to play a regulatory role.     

The common nodABC genes of Rhizobium meliloti are host-range determinants

Symbiotic bacteria of the genus Rhizobium synthesize lipo-chitooligosaccharides, called Nod factors (NFs), which act as morphogenic signal molecules on legume hosts. The common nodABC genes, present in all Rhizobium species, are required for the synthesis of the core structure of NFs. NodC is an N-acetylglucosaminyltransferase, and NodB is a chitooligosaccharide deacetylase; NodA is involved in N-acylation of the aminosugar backbone. Specific nod genes are involved in diverse NF substitutions that confer plant specificity. We transferred to R. tropici, a broad host-range tropical symbiont, the ability to nodulate alfalfa, by introducing nod genes of R. meliloti. In addition to the specific nodL and nodFE genes, the common nodABC genes of R. meliloti were required for infection and nodulation of alfalfa. Purified NFs of the R. tropici hybrid strain, which contained chitin tetramers and were partly N-acylated with unsaturated C16 fatty acids, were able to elicit nodule formation on alfalfa. Inactivation of the R. meliloti nodABC genes suppressed the ability of the NFs to nodulate alfalfa. Studies of NFs from nodA, nodB, nodC, and nodI mutants indicate that (i) NodA of R. meliloti, in contrast to NodA of R. tropici, is able to transfer unsaturated C16 fatty acids onto the chitin backbone and (ii) NodC of R. meliloti specifies the synthesis of chitin tetramers. These results show that allelic variation of the common nodABC genes is a genetic mechanism that plays an important role in signaling variation and in the control of host range.     

Cloning and identification of conjugative transfer origins in the Rhizobium meliloti genome

A simple approach was used to identify Rhizobium meliloti DNA regions with the ability to convert a nontransmissible vector into a mobilizable plasmid, i.e., to contain origins of conjugative transfer (oriT, mob). RecA-defective R. meliloti merodiploid populations, where each individual contained a hybrid cosmid from an R. meliloti GR4 gene library, were used as donors en masse in conjugation with another R. meliloti recipient strain, selecting transconjugants for vector-encoded antibiotic resistance. Restriction analysis of cosmids isolated from individual transconjugants resulted in the identification of 11 nonoverlapping DNA regions containing potential oriTs. Individual hybrid cosmids were confirmed to be mobilized from the original recA donors at frequencies ranging from 10(-2) to 10(-5) per recipient cell. DNA hybridization experiments showed that seven mob DNA regions correspond to plasmid replicons: four on symbiotic megaplasmid 1 (pSym1), one on pSym2, and another two on each of the two cryptic plasmids harbored by R. meliloti GR4. Another three mob clones could not be located to any plasmid and were therefore preliminarily assigned to the chromosome. With this strategy, we were able to characterize the oriT of the conjugative plasmid pRmeGR4a, which confirmed the reliability of the approach to select for oriTs. Moreover, transfer of the 11 mob cosmids from R. meliloti into Escherichia coli occurred at frequencies as high as 10(-1), demonstrating the R. meliloti gene transfer capacity is not limited to the family Rhizobiaceae. Our results show that the R. meliloti genome contains multiple oriTs that allow efficient DNA mobilization to rhizobia as well as to phylogenetically distant gram-negative bacteria.     

Identification of glucuronan lyase from a mutant strain of Rhizobium meliloti

This review attempts to capture the history of research involved in the understanding of lipid metabolism via investigation of the sn-glycerol-3-phosphate acyltransferase (glycerol-P acyltransferase), the first step in the synthesis of lipids in E. coli. We will review the original identification of this enzymatic activity and its subsequent characterization. The biochemical and genetic regulation of this enzyme and gene are discussed, as well as the unique structural characterization of this integral membrane protein. Future perspectives regarding the regulatory and structural aspects of this key enzyme are discussed.     

Different roles of CheY1 and CheY2 in the chemotaxis of Rhizobium meliloti

Biliary lipid secretion is a complex process involving a multitude of metabolic pathways. It has always been assumed that bile salt secretion (BSec) fully controls this process. Recently we have demonstrated, that mdr2 P-glycoprotein (P-gp) is an important controlling step as well. In this study we have analysed the control structure of this pathway with Metabolic Control Analysis. METHODS: FVB mice homozygous (+/+) or heterozygous (+/-) for mdr2 P-glycoprotein were infused via the tail vein with tauroursodeoxycholate in stepwise increasing concentrations. Bile was collected and biliary lipids were determined by standard techniques. RESULTS: To simplify the pathway we have lumped all reactions involved in BSsec into bile in one step. Since this step is not controlled by the canalicular BS concentration, the FCC of BS secretion on phospholipid secretion (PLsec) could be calculated from a plot between BS and PL secretion. The FCC of BSsec varied from 80% at low flux to a value of 90% at maximal BS output. The FCC of mdr2 P-gp was determined by varying the gene dose of mdr2 P-gp. Since PLsec showed linear kinetics towards canalicular BS the FCC could be calculated via the Deviation index. The values for the FCC of mdr2 P-gp in (+/+) mice vary from 80% at low flux to 125% at maximal BS output. CONCLUSIONS: Both BS secretion and mdr2 P-gp strongly control biliary phospholipid secretion. The sum of the FCCs of both steps is always much higher than 100% implicating the presence of step(s) which exert negative control. We hypothesize that steps controlling biliary water transport account for the negative control.     

Isolation of Sinorhizobium meliloti Tn5 mutants with altered cytochrome terminal oxidase expression and improved symbiotic performance

OBJECTIVES: We sought to investigate whether, in humans, the timing and incidence of a relapse of atrial fibrillation (AF) during the first month after cardioversion indicates the presence of electrical remodeling and whether this could be influenced by prevention of intracellular calcium overload during AF. BACKGROUND: Animal experiments have shown that AF induces shortening of the atrial refractory period, resulting in an increased vulnerability for reinduction of AF. This electrical remodeling was completely reversible within 1 week after cardioversion of AF and was presumably related to intracellular calcium overload. METHODS: Using transtelephonic monitoring in 61 patients cardioverted for chronic AF, we evaluated the daily incidence of recurrence of AF and determined, by Cox regression analysis, the influence of patient characteristics and medication on relapse of AF. RESULTS: During 1 month of follow-up, 35 patients (57%) had a relapse of AF, with a peak incidence during the first 5 days after cardioversion. Furthermore, in patients with a recurrence of AF, there was a positive correlation between the duration of the shortest coupling interval of the premature atrial beats after cardioversion and the timing of the recurrence of AF (p = 0.0013). Multivariate analysis revealed that the use of intracellular calcium-lowering drugs during AF was the only significant variable related to maintenance of sinus rhythm after cardioversion (p = 0.03). CONCLUSIONS: The daily distribution of recurrences of AF suggests a temporary vulnerable electrophysiologic state of the atria. Use of intracellular calcium-lowering medications during AF appeared to reduce recurrences, possibly due to a reduction of electrical remodeling during AF.     

Presence of a gene encoding choline sulfatase in Sinorhizobium meliloti bet operon: choline-O-sulfate is metabolized into glycine betaine

Glycine betaine is a potent osmoprotectant accumulated by Sinorhizobium meliloti to cope with osmotic stress. The biosynthesis of glycine betaine from choline is encoded by an operon of four genes, betICBA, as determined by sequence and mutant analysis. The betI and betC genes are separated by an intergenic region containing a 130-bp mosaic element that also is present between the betB and betA genes. In addition to the genes encoding a presumed regulatory protein (betI), the betaine aldehyde dehydrogenase (betB), and the choline dehydrogenase (betA) enzymes also found in Escherichia coli, a new gene (betC) was identified as encoding a choline sulfatase catalyzing the conversion of choline-O-sulfate and, at a lower rate, phosphorylcholine, into choline. Choline sulfatase activity was absent from betC but not from betB mutants and was shown to be induced indifferently by choline or choline-O-sulfate as were the other enzymes of the pathway. Unlike what has been shown in other bacteria and plants, choline-O-sulfate is not used as an osmoprotectant per se in S. meliloti, but is metabolized into glycine betaine. S. meliloti also can use this compound as the sole carbon, nitrogen, and sulfur source for growth and that depends on a functional bet locus. In conclusion, choline-O-sulfate and phosphorylcholine, which are found in higher plants and fungi, appear to be substrates for glycine betaine biosynthesis in S. meliloti.     

Expression and study of recombinant ExoM, a beta1-4 glucosyltransferase involved in succinoglycan biosynthesis in Sinorhizobium meliloti

Here we report on the overexpression and in vitro characterization of a recombinant form of ExoM, a putative beta1-4 glucosyltransferase involved in the assembly of the octasaccharide repeating subunit of succinoglycan from Sinorhizobium meliloti. The open reading frame exoM was isolated by PCR and subcloned into the expression vector pET29b, allowing inducible expression under the control of the T7 promoter. Escherichia coli BL21(DE3)/pLysS containing exoM expressed a novel 38-kDa protein corresponding to ExoM in N-terminal fusion with the S-tag peptide. Cell fractionation studies showed that the protein is expressed in E. coli as a membrane-bound protein in agreement with the presence of a predicted C-terminal transmembrane region. E. coli membrane preparations containing ExoM were shown to be capable of transferring glucose from UDP-glucose to glycolipid extracts from an S. meliloti mutant strain which accumulates the ExoM substrate (Glcbeta1-4Glcbeta1-3Gal-pyrophosphate-polyprenol). Thin-layer chromatography of the glycosidic portion of the ExoM product showed that the oligosaccharide formed comigrates with an authentic standard. The oligosaccharide produced by the recombinant ExoM, but not the starting substrate, was sensitive to cleavage with a specific cellobiohydrolase, consistent with the formation of a beta1-4 glucosidic linkage. No evidence for the transfer of multiple glucose residues to the glycolipid substrate was observed. It was also found that ExoM does not transfer glucose to an acceptor substrate that has been hydrolyzed from the polyprenol anchor. Furthermore, neither glucose, cellobiose, nor the trisaccharide Glcbeta1-4Glcbeta1-3Glc inhibited the transferase activity, suggesting that some feature of the lipid anchor is necessary for activity.     

Regulation of sodium-dependent phosphate transport in osteoclasts

Osteoclasts are the primary cells responsible for bone resorption. They are exposed to high ambient concentrations of inorganic phosphate (Pi) during the process of bone resorption and they possess specific Pi-transport system(s) capable of taking up Pi released by bone resorption. By immunochemical studies and PCR, we confirmed previous studies suggesting the presence of an Na-dependent Pi transporter related to the renal tubular "NaPi" proteins in the osteoclast. Using polyclonal antibodies to NaPi-2 (the rat variant), an approximately 95-kD protein was detected, localized in discrete vesicles in unpolarized osteoclasts cultured on glass coverslips. However, in polarized osteoclasts cultured on bone, immunofluorescence studies demonstrated the protein to be localized exclusively on the basolateral membrane, where it colocalizes with an Na-H exchanger but opposite to localization of the vacuolar H-ATPase. An inhibitor of phosphatidylinositol 3-kinase, wortmannin, and an inhibitor of actin cytoskeletal organization, cytochalasin D, blocked the bone-stimulated increase in Pi uptake. Phosphonoformic acid (PFA), an inhibitor of the renal NaPi-cotransporter, reduced NaPi uptake in the osteoclast. PFA also elicited a dose-dependent inhibition of bone resorption. PFA limited ATP production in osteoclasts attached to bone particles. Our results suggest that Pi transport in the osteoclast is a process critical to the resorption of bone through provision of necessary energy substrates.     

Succinoglycan is required for initiation and elongation of infection threads during nodulation of alfalfa by Rhizobium meliloti

Rhizobium meliloti Rm1021 must be able to synthesize succinoglycan in order to invade successfully the nodules which it elicits on alfalfa and to establish an effective nitrogen-fixing symbiosis. Using R. meliloti cells that express green fluorescent protein (GFP), we have examined the nature of the symbiotic deficiency of exo mutants that are defective or altered in succinoglycan production. Our observations indicate that an exoY mutant, which does not produce succinoglycan, is symbiotically defective because it cannot initiate the formation of infection threads. An exoZ mutant, which produces succinoglycan without the acetyl modification, forms nitrogen-fixing nodules on plants, but it exhibits a reduced efficiency in the initiation and elongation of infection threads. An exoH mutant, which produces symbiotically nonfunctional high-molecular-weight succinoglycan that lacks the succinyl modification, cannot form extended infection threads. Infection threads initiate at a reduced rate and then abort before they reach the base of the root hairs. Overproduction of succinoglycan by the exoS96::Tn5 mutant does not reduce the efficiency of infection thread initiation and elongation, but it does significantly reduce the ability of this mutant to colonize the curled root hairs, which is the first step of the invasion process. The exoR95::Tn5 mutant, which overproduces succinoglycan to an even greater extent than the exoS96::Tn5 mutant, has completely lost its ability to colonize the curled root hairs. These new observations lead us to propose that succinoglycan is required for both the initiation and elongation of infection threads during nodule invasion and that excess production of succinoglycan interferes with the ability of the rhizobia to colonize curled root hairs.     

Characterization of a novel acyl carrier protein, RkpF, encoded by an operon involved in capsular polysaccharide biosynthesis in Sinorhizobium meliloti

Rhizobial capsular polysaccharides (RKPs) play an important role in the development of a nitrogen-fixing symbiosis with the plant host and in Sinorhizobium meliloti AK631 functional rkpABCDEF genes are required for the production of RKPs. After cloning the rkpF gene, we overexpressed and purified the derived protein product (RkpF) in Escherichia coli. Like acyl carrier protein (ACP), the RkpF protein can be labeled in vivo with radioactive beta-alanine added to the growth medium. If homogeneous RkpF protein is incubated with radiolabeled coenzyme A in the presence of purified holo-ACP synthase from E. coli, an in vitro transfer of 4'-phosphopantetheine to the RkpF protein can be observed. The conversion from apo-RkpF protein to holo-RkpF protein seems to go along with a major conformational change of the protein structure, because the holo-RkpF protein runs significantly faster on native polyacrylamide gel electrophoresis than the apo-RkpF protein. Electrospray mass spectrometric analysis reveals a mass of 9,585 for the apo-RkpF protein and a mass of 9,927 for the holo-RkpF protein. Our data show that RkpF is a novel ACP.