An epoxide hydrolase was purified to homogeneity from the epichlorohydrin-utilizing bacterium Pseudomonas sp. strain AD1. The enzyme was found to be a monomeric protein with a molecular mass of 35 kDa. With epichlorohydrin as the substrate, the enzyme followed Michaelis-Menten kinetics with a K , value of 0.3 mM and a Vmax of 34 pmol . min-' . mg protein-'. The epoxide hydrolase catalyzed the hydrolysis of several epoxides, including epichlorohydrin, epibromohydrin, epoxyoctane and styrene epoxide. With all chiral compounds tested, both stereoisomers were converted. Amino acid sequencing of cyanogen bromide-generated peptides did not yield sequences with similarities to other known proteins.

Growth of a glutamate transport-deficient mutant of Rhodobacter sphaeroides on glutamate as sole carbon and nitrogen source can be restored by the addition of millimolar amounts of Na ؉ . Uptake of glutamate (K t of 0.2 M) by the mutant strictly requires Na ؉ (K m of 25 mM) and is inhibited by ionophores that collapse the proton motive force (pmf). The activity is osmotic-shock-sensitive and can be restored in spheroplasts by the addition of osmotic shock f luid. Transport of glutamate is also observed in membrane vesicles when Na ؉ , a proton motive force, and purified glutamate binding protein are present. Both transport and binding is highly specific for glutamate. The Na ؉ -dependent glutamate transporter of Rb. sphaeroides is an example of a secondary transport system that requires a periplasmic binding protein and may define a new family of bacterial transport proteins.Rhodobacter sphaeroides is a phototrophic, Gram-negative, mesophilic bacterium that can grow on a wide variety of compounds aerobically in the dark and anaerobically in the light. For the uptake of nutrients such as amino acids, this organism mainly relies on binding protein-dependent transport systems rather than secondary transport systems (1). Binding protein dependent transport systems were first identified by Heppel et al. (2), who showed that these systems are osmotic shock-sensitive due to the release of a substratebinding protein from the periplasmic space. These transport systems belong to the family of ATP binding cassette (ABC) proteins (3-5) and have been studied in great detail in cells, membrane vesicles, and in purified and reconstituted form. Typically, they are multisubunit systems that consist of a soluble periplasmic substrate binding protein that interacts with a membrane protein complex composed of two identical or homologous integral membrane proteins and two identical or homologous ATP-binding proteins. Transport requires the hydrolysis of ATP (4) and is blocked by vanadate, an inhibitor of P-type ATPases (6). Binding protein-dependent systems often exhibit a much higher affinity for the substrate as compared with secondary transport systems, and do not require the proton motive force (pmf) as a driving force for transport (3,4,5,7).Uptake of the anionic amino acids glutamate and aspartate and their respective amides glutamine and asparagine by Rb. sphaeroides occurs via a single ABC transport system (8) that utilizes two distinct binding proteins with specificity for glutamate͞glutamine and aspartate͞asparagine, respectively. We have previously isolated a mutant, strain MJ7, that is defective in this ABC-glutamate transporter and is unable to grow on glutamate as carbon (C-) and nitrogen (N-) source (9). Both growth and transport of glutamate can be restored by expressing the Escherichia coli GltP, a secondary H ϩ :glutamate transport system, in Rb. sphaeroides MJ7 (9).We now show that growth of Rb. sphaeroides strain MJ7 on glutamate can also be restored by the inclusion of millimolar amounts of Na ϩ in the m...

The mechanism of L-glutamate uptake was studied in Rhodobacter sphaeroides. Uptake of L-glutamate is mediated by a high-affinity (K t of 1.2 M), shock-sensitive transport system that is inhibited by vanadate and dependent on the internal pH.

Rhodobacter sphaeroides is chemotactic to glutamate and most other amino acids. In Escherichia coli, chemotaxis involves a membrane-bound sensor that either binds the amino acid directly or interacts with the binding protein loaded with the amino acid. In R. sphaeroides, chemotaxis is thought to require both the uptake and the metabolism of the amino acid. Glutamate is accumulated by the cells via a binding protein-dependent system. To determine the role of the binding protein and transport in glutamate taxis, mutants were created by Tn5 insertion mutagenesis and selected for growth in the presence of the toxic glutamine analogue gamma-glutamyl-hydrazide. One of the mutants, R. sphaeroides MJ7, was defective in glutamate uptake but showed wild-type levels of binding protein. The mutant showed no chemotactic response to glutamate. Both glutamate uptake and chemotaxis were recovered when the gltP gene, coding for the H(+)-linked glutamate carrier of E. coli, was expressed in R. sphaeroides MJ7. It is concluded that the chemotactic response to glutamate strictly requires uptake of glutamate, supporting the view that intracellular metabolism is needed for chemotaxis in R. sphaeroides.

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