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Abstract A glucose dehydrogenase (GDH) that is induced 5-fold by phosphate starvation, has been characterized from a bacterium isolated from alkaline Indian vertisol soils. The bacterium was identified as Enterobacter asburiae based on 16S rRNA analysis. Concomitant with GDH induction, glucose was oxidized and secreted as gluconate (50 mM). Excretion of this acid caused a reduction in soil pH and the release of phosphate and iron. Mutants deficient in GDH activity failed to release phosphate from alkaline soils indicating that GDH activity is required to solubilize phosphate.

One of the largest contributions to biologically available nitrogen comes from the reduction of N₂ to ammonia by rhizobia in symbiosis with legumes. Plants supply dicarboxylic acids as a carbon source to bacteroids, and in return they receive ammonia. However, metabolic exchange must be more complex, because effective N₂ fixation by Rhizobium leguminosarum bv viciae bacteroids requires either one of two broad-specificity amino acid ABC transporters (Aap and Bra). It was proposed that amino acids cycle between plant and bacteroids, but the model was unconstrained because of the broad solute specificity of Aap and Bra. Here, we constrain the specificity of Bra and ectopically express heterologous transporters to demonstrate that branched-chain amino acid (LIV) transport is essential for effective N₂ fixation. This dependence of bacteroids on the plant for LIV is not due to their known down-regulation of glutamate synthesis, because ectopic expression of glutamate dehydrogenase did not rescue effective N₂ fixation. Instead, the effect is specific to LIV and is accompanied by a major reduction in transcription and activity of LIV biosynthetic enzymes. Bacteroids become symbiotic auxotrophs for LIV and depend on the plant for their supply. Bacteroids with aap bra null mutations are reduced in number, smaller, and have a lower DNA content than wild type. Plants control LIV supply to bacteroids, regulating their development and persistence. This makes it a critical control point for regulation of symbiosis.

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 approximately equal 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.

Rhizobium leguminosarum synthesizes polyhydroxybutyrate and glycogen as its main carbon storage compounds. To examine the role of these compounds in bacteroid development and in symbiotic efficiency, single and double mutants of R. leguminosarum bv. viciae were made which lack poly-hydroxybutyrate synthase (phaC), glycogen synthase (glgA), or both. For comparison, a single phaC mutant also was isolated in a bean-nodulating strain of R. leguminosarum bv. phaseoli. In one large glasshouse trial, the growth of pea plants inoculated with the R. leguminosarum bv. viciae phaC mutant were significantly reduced compared with wild-type-inoculated plants. However, in subsequent glasshouse and growth-room studies, the growth of pea plants inoculated with the mutant were similar to wild-type-inoculated plants. Bean plants were unaffected by the loss of polyhydroxybutyrate biosynthesis in bacteroids. Pea plants nodulated by a glycogen synthase mutant, or the glgA/phaC double mutant, grew as well as the wild type in growth-room experiments. Light and electron micrographs revealed that pea nodules infected with the glgA mutant accumulated large amounts of starch in the II/III interzone. This suggests that glycogen may be the dominant carbon storage compound in pea bacteroids. Polyhydroxybutyrate was present in bacteria in the infection thread of pea plants but was broken down during bacteroid formation. In nodules infected with a phaC mutant of R. leguminosarum bv. viciae, there was a drop in the amount of starch in the II/III interzone, where bacteroids form. Therefore, we propose a carbon burst hypothesis for bacteroid formation, where polyhydroxybutyrate accumulated by bacteria is degraded to fuel bacteroid differentiation.

A gene, cpaA, with similarity to calcium proton antiporters has been identified adjacent to lpcAB in Rhizobium leguminosarum. LpcA is a galactosyl transfererase while LpcB is a 2-keto-3-deoxyoctonate transfererase, both of which are required to form the lipopolysaccharide (LPS) core in R. leguminosarum. Mutations in lpcAB result in a rough LPS phenotype with a requirement for elevated calcium concentrations to allow growth, suggesting that truncation of the LPS core exposes a highly negatively charged molecule. This is consistent with the LPS core being one of the main sites for binding calcium in the Gram-negative outer membrane. Strain RU1109 (cpa::Tn5-lacZ) has a normal LPS layer, as measured by silver staining and Western blotting. This indicates that cpaA mutants are not grossly affected in their LPS layer. LacZ fusion analysis indicates that cpaA is constitutively expressed and is not directly regulated by the calcium concentration. Over-expression of cpaA increased the concentration of calcium required for growth, consistent with CpaA mediating calcium export from the cytosol. The location of lpcA, lpcB and cpaA as well as the phenotype of lpcB mutants suggests that CpaA might provide a specific export pathway for calcium to the LPS core.

A glucose dehydrogenase (GDH) that is induced 5-fold by phosphate starvation, has been characterized from a bacterium isolated from alkaline Indian vertisol soils. The bacterium was identified as Enterobacter asburiae based on 16S rRNA analysis. Concomitant with GDH induction, glucose was oxidized and secreted as gluconate (50 mM). Excretion of this acid caused a reduction in soil pH and the release of phosphate and iron. Mutants deficient in GDH activity failed to release phosphate from alkaline soils indicating that GDH activity is required to solubilize phosphate.

Optical traps exploit the radiation forces of laser light to manipulate microscopic particles. The ability to manipulate biological material and quantify the force required has been exploited in the biosciences; from the isolation of single cells to kinetic measurements of single motor molecules. This review describes the theory of optical trapping and using recent publications gives examples of how it has been employed across a broad spectrum of biological research.[PUBLICATION ABSTRACT]

A gene, cpaA, with similarity to calcium proton antiporters has been identified adjacent to lpcAB in Rhizobium leguminosarum. LpcA is a galactosyl transferase while LpcB is a 2-keto-3-deoxyoctonate transferase, both of which are required to form the lipopolysaccharide (LPS) core in R. leguminosarum. Mutations in lpcAB result in a rough LPS phenotype with a requirement for elevated calcium concentrations to allow growth, suggesting that truncation of the LPS core exposes a highly negatively charged molecule. This is consistent with the LPS core being one of the main sites for binding calcium in the Gram-negative outer membrane. Strain RU1109 ( cpaA::Tn 5-lacZ) has a normal LPS layer, as measured by silver staining and Western blotting. This indicates that cpaA mutants are not grossly affected in their LPS layer. LacZ fusion analysis indicates that cpaA is constitutively expressed and is not directly regulated by the calcium concentration. Over-expression of cpaA increased the concentration of calcium required for growth, consistent with CpaA mediating calcium export from the cytosol. The location of lpcA, lpcB and cpaA as well as the phenotype of lpcB mutants suggests that CpaA might provide a specific export pathway for calcium to the LPS core.