Chemical synthesis of scyllo-inosamine and catabolism studies in Sinorhizobium meliloti

Rhizopines such as scyllo-inosamine (SIA) and L-3-O-methyl-scyllo-inosamine (3-O-MSI) play an intricate role as nutritional mediators during the establishment of the symbiotic relationship between legumes and rhizobia. The mechanism of action is not well understood. One challenge is the availability of rhizopines, which occur in only minute amounts in plant nodules. We herewith report an efficient synthesis of scyllo-inosamine and its biochemical activity in specific bacteria. SIA was prepared in 7 steps and 32% overall yield from readily available myo-inositol. The chemically synthesized SIA was tested to determine whether it can serve as sole carbon and nitrogen source for Sinorhizobium meliloti wild-type strain L5-30 and for strains carrying mutations in the rhizopine degradation (moc) genes. The analysis of the phenotype of the mutant strains revealed that the moc genes previously shown to be essential for the breakdown of the rhizopines isolated from root nodules are also essential for the utilization of the chemically synthesized SIA.     

Fatty acid‐releasing activities in Sinorhizobium meliloti include unusual diacylglycerol lipase

Phospholipids are well known for their membrane‐forming properties and thereby delimit any cell from the exterior world. In addition, membrane phospholipids can act as precursors for signals and other biomolecules during their turnover. Little is known about phospholipid signalling, turnover and remodelling in bacteria. Recently, we showed that a FadD‐deficient mutant of Sinorhizobium meliloti, unable to convert free fatty acids to their coenzyme A derivatives, accumulates free fatty acids during the stationary phase of growth. Enzymatic activities responsible for the generation of these free fatty acids were unknown in rhizobia. Searching the genome of S. meliloti, we identified a potential lysophospholipase (SMc04041) and two predicted patatin‐like phospholipases A (SMc00930, SMc01003). Although SMc00930 as well as SMc01003 contribute to the release of free fatty acids in S. meliloti, neither one can use phospholipids as substrates. Here we show that SMc01003 converts diacylglycerol to monoacylglycerol and a fatty acid, and that monoacylglycerol can be further degraded by SMc01003 to another fatty acid and glycerol. A SMc01003‐deficient mutant of S. meliloti transiently accumulates diacylglycerol, suggesting that SMc01003 also acts as diacylglycerol lipase (DglA) in its native background. Expression of the DglA lipase in Escherichia coli causes lysis of cells in stationary phase of growth.     

Functional analysis of Sinorhizobium meliloti genes involved in biotin synthesis and transport

External biotin greatly stimulates bacterial growth and alfalfa root colonization by Sinorhizobium meliloti strain 1021. Several genes involved in responses to plant-derived biotin have been identified in this bacterium, but no genes required for biotin transport are known, and not all loci required for biotin synthesis have been assigned. Searches of the S. meliloti genome database in combination with complementation tests of Escherichia coli biotin auxotrophs indicate that biotin synthesis probably is limited in S. meliloti 1021 by the poor functioning or complete absence of several key genes. Although several open reading frames with significant similarities to genes required for synthesis of biotin in gram-positive and gram-negative bacteria were found, only bioB, bioF, and bioH were demonstrably functional in complementation tests with known E. coli mutants. No sequence or complementation evidence was found for bioA, bioC, bioD, or bioZ. In contrast to other microorganisms, the S. meliloti bioB and bioF genes are not localized in a biotin synthesis operon, but bioB is cotranscribed with two genes coding for ABC transporter-like proteins, designated here bioM and bioN. Mutations in bioM and bioN eliminated growth on alfalfa roots and reduced bacterial capacity to maintain normal intracellular levels of biotin. Taken together, these data suggest that S. meliloti normally grows on exogenous biotin using bioM and bioN to conserve biotin assimilated from external sources.     

Conservation of a pseudomonad-like hydrocarbon degradative ferredoxin oxygenase complex involved in rhizopine catabolism in Sinorhizobium meliloti and Rhizobium leguminosarum bv. viciae

In Sinorhizobium meliloti the mocCABR genes have previously been shown to be required for rhizopine (3-O-methyl-scyllo-inosamine, 3-O-MSI) catabolism. We show that the mocDE(F) gene cluster is also needed. MocDE(F), which is involved in the catabolism of 3-O-MSI to its demethylated form scyllo-inosamine (SI) has homology to components that would comprise a ferredoxin-oxygenase system. The mocCABRDE(F) suite of genes is required for 3-O-MSI catabolism in both S. meliloti and R. leguminosarum bv. viciae. However, SI catabolism in S. meliloti requires mocCABR, whereas only mocCA are required for its catabolism in R. leguminosarum suggesting the two species require different chromosomal genes which act in concert with moc genes for the catabolism of rhizopine.     

Sinorhizobium meliloti genes involved in tolerance to the antimicrobial peptide protamine

The innate resistance of plants and animals to microbial infection is mediated in part by small cationic peptides with antimicrobial activity. We assessed the susceptibility of the alfalfa symbiont Sinorhizobium meliloti to the model antimicrobial peptide protamine. Twenty-one Tn5-induced mutants showing increased sensitivity to protamine were isolated, and nine were further characterized in detail. These nine mutants carried distinct transposon insertions that affected a total of seven different genes. Three of these genes are involved in exopolysaccharide and beta-(1,2)-glucan biosynthesis (exoT, exoU and ndvB), three other genes are implicated in nitrogen metabolism, such as a putative dyhidropyrimidinase, hutU and ureF, and the last gene exhibited similarity to the ATP binding cassette family of membrane transporters. Symbiotic defects ranging from severe to moderate were displayed by some of the protamine-hypersensitive mutants suggesting that S. meliloti possess active mechanisms to counteract hypothetical cationic peptides that may be produced by its host plant.     

Functional analysis of nine putative chemoreceptor proteins in Sinorhizobium meliloti

The genome of the symbiotic soil bacterium Sinorhizobium meliloti contains eight genes coding for methyl-accepting 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 assays 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.     

An ATP-dependent L-carnitine transporter in Listeria monocytogenes Scott A is involved in osmoprotection.

Listeria monocytogenes is a gram-positive, psychotrophic, food-borne pathogen which is able to grow in osmotically stressful environments. Carnitine (beta-hydroxy-L-tau-N-trimethyl aminobutyrate) can contribute significantly to growth of L. monocytogenes at high osmolarity (R. R. Beumer, M. C. te Giffel, L. J. Cox, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 60:1359-1363, 1994). Transport of L-[N-methyl-14C]carnitine in L. monocytogenes was shown to be energy dependent. Analysis of cell extracts revealed that L-carnitine was not further metabolized, which supplies evidence for its role as an osmoprotectant in L. monocytogenes. Uptake of L-carnitine proceeds in the absence of a proton motive force and is strongly inhibited in the presence of the phosphate analogs vanadate and arsenate. The L-carnitine permease is therefore most likely driven by ATP. Kinetic analysis of L-carnitine transport in glucose-energized cells revealed the presence of a high-affinity uptake system with a Km of 10 microM and a maximum rate of transport (Vmax) of 48 nmol min-1 mg of protein-1. L-[14C]carnitine transport in L. monocytogenes is significantly inhibited by a 10-fold excess of unlabelled L-carnitine, acetylcarnitine, and tau-butyrobetaine, whereas L-proline and betaine display, even at a 100-fold excess, only a weak inhibitory effect. In conclusion, an ATP-dependent L-carnitine transport system in L. monocytogenes is described, and its possible roles in cold adaptation and intracellular growth in mammalian cells are discussed.     

Interrelations between glycine betaine catabolism and methionine biosynthesis in Sinorhizobium meliloti strain 102F34

Methionine is produced by methylation of homocysteine. Sinorhizobium meliloti 102F34 possesses only one methionine synthase, which catalyzes the transfer of a methyl group from methyl tetrahydrofolate to homocysteine. This vitamin B(12)-dependent enzyme is encoded by the metH gene. Glycine betaine can also serve as an alternative methyl donor for homocysteine. This reaction is catalyzed by betaine-homocysteine methyl transferase (BHMT), an enzyme that has been characterized in humans and rats. An S. meliloti gene whose product is related to the human BHMT enzyme has been identified and named bmt. This enzyme is closely related to mammalian BHMTs but has no homology with previously described bacterial betaine methyl transferases. Glycine betaine inhibits the growth of an S. meliloti bmt mutant in low- and high-osmotic strength media, an effect that correlates with a decrease in the catabolism of glycine betaine. This inhibition was not observed with other betaines, like homobetaine, dimethylsulfoniopropionate, and trigonelline. The addition of methionine to the growth medium allowed a bmt mutant to recover growth despite the presence of glycine betaine. Methionine also stimulated glycine betaine catabolism in a bmt strain, suggesting the existence of another catabolic pathway. Inactivation of metH or bmt did not affect the nodulation efficiency of the mutants in the 102F34 strain background. Nevertheless, a metH strain was severely defective in competing with the wild-type strain in a coinoculation experiment.     

Sinorhizobium meliloti ExoR and ExoS proteins regulate both succinoglycan and flagellum production

The production of the Sinorhizobium meliloti exopolysaccharide, succinoglycan, is required for the formation of infection threads inside root hairs, a critical step during the nodulation of alfalfa (Medicago sativa) by S. meliloti. Two bacterial mutations, exoR95::Tn5 and exoS96::Tn5, resulted in the overproduction of succinoglycan and a reduction in symbiosis. Systematic analyses of the symbiotic phenotypes of the two mutants demonstrated their reduced efficiency of root hair colonization. In addition, both the exoR95 and exoS96 mutations caused a marked reduction in the biosynthesis of flagella and consequent loss of ability of the cells to swarm and swim. Succinoglycan overproduction did not appear to be the cause of the suppression of flagellum biosynthesis. Further analysis indicated that both the exoR95 and exoS96 mutations affected the expression of the flagellum biosynthesis genes. These findings suggest that both the ExoR protein and the ExoS/ChvI two-component regulatory system are involved in the regulation of both succinoglycan and flagellum biosynthesis. These findings provide new avenues of understanding of the physiological changes S. meliloti cells go through during the early stages of symbiosis and of the signal transduction pathways that mediate such changes.     

Role of a conserved membrane glycine residue in a dicarboxylate transporter from Sinorhizobium meliloti

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.     

Crystal structure of the ligand-binding protein EhuB from Sinorhizobium meliloti reveals substrate recognition of the compatible solutes ectoine and hydroxyectoine

In microorganisms, members of the binding-protein-dependent ATP-binding cassette transporter superfamily constitute an important class of transport systems. Some of them are involved in osmoprotection under hyperosmotic stress by facilitating the uptake of “compatible solutes”. Currently, the molecular mechanisms used by these transport systems to recognize compatible solutes are limited to transporters specific for glycine betaine and proline betaine. Therefore, this study reports a detailed analysis of the molecular principles governing substrate recognition in the Ehu system from Sinorhizobium meliloti, which is responsible for the uptake of the compatible solutes ectoine and hydroxyectoine. To contribute to a broader understanding of the molecular interactions underlying substrate specificity, our study focused on the substrate-binding protein EhuB because this protein binds the ligand selectively, delivers it to the translocation machinery in the membrane and is thought to be responsible for substrate specificity. The crystal structures of EhuB, in complex with ectoine and hydroxyectoine, were determined at a resolution of 1.9 Å and 2.3 Å, respectively, and allowed us to assign the structural principles of substrate recognition and binding. Based on these results, site-directed mutagenesis of amino acids involved in ligand binding was employed to address their individual contribution to complex stability. A comparison with the crystal structures of other binding proteins specific for compatible solutes revealed common principles of substrate recognition, but also important differences that might be an adaptation to the nature of the ligand and to the demands on protein affinity imposed by the environment.     

glnD and mviN are genes of an essential operon in Sinorhizobium meliloti

To evaluate the role of uridylyl-transferase, the Sinorhizobium meliloti glnD gene was isolated by heterologous complementation in Azotobacter vinelandii. The glnD gene is cotranscribed with a gene homologous to Salmonella mviN. glnD1::Omega or mviN1::Omega mutants could not be isolated by a powerful sucrose counterselection procedure unless a complementing cosmid was provided, indicating that glnD and mviN are members of an indispensable operon in S. meliloti.