The Rhodobacter sphaeroides 2.4.1 tryptophan-rich sensory protein gene, tspO (formerly crtK, ORF160) encodes a 17-kDa protein which has an unusually high content of aromatic amino acids in general and of Ltryptophan in particular. The TspO protein was localized to the outer membrane of aerobically grown R. sphaeroides 2.4.1 by use of a polyclonal antibody against the purified protein. This protein is present in severalfold higher levels in photosynthetic as opposed to aerobic grown cells. Although tspO lies within the crt gene cluster, null mutations have an intact carotenoid biosynthetic pathway. In the TSPO1 mutant there was an increased production of carotenoids and bacteriochlorophyll relative to the wild type, particularly when cells were grown aerobically or semiaerobically. When present in trans the tspO gene restored "normal" pigment production to TSPO1. The effect of the tspO gene on pigment production was shown to take place at the level of gene expression. Because the tspO gene product of R. sphaeroides 2.4.1 shows significant sequence homology and similarity to the peripheral-type benzodoazepine receptor from mammalian sources, TspO-specific antibodies when probed against liver and kidney mitochondrial protein showed strong cross-reactivity. The role of TspO in R. sphaeroides 2.4.1 and its relation to photosynthesis gene expression are discussed.Rhodobacter sphaeroides is a member of the proteobacteria, and it is characterized by its metabolic versatility including growth either chemoheterotrophically or photoheterotrophically. A decrease in oxygen level results in the induction of the photosynthetic membrane system designated the intracytoplasmic membrane (ICM).1 The ICM contains all of the components necessary to convert light energy into chemical energy during phototrophic growth. The ICM is also gratuitously synthesized by R. sphaeroides during anaerobic growth in the dark in the presence of an alternative electron acceptor, such as dimethyl sulfoxide (Me 2 SO). While ICM synthesis and composition are known to be tightly regulated, the molecular mechanisms which govern the biosynthesis and assembly of the ICM are only beginning to yield to molecular genetic analysis (1).Carotenoids (Crt) in addition to bacteriochlorophyll (Bchl) are an important structural component of the ICM. They take part in the entrapment and utilization of light energy, and furthermore, have an important antioxidative function during aerobic growth. The crt gene cluster of Rhodobacter capsulatus has been well characterized by (2, 3), and the genes and likely enzymatic activities which they encode have been discussed. In R. sphaeroides, these genes have been recently shown to occupy a similar arrangement as in R. capsulatus (4).According to the phenotype of a number of Crt mutants in R. capsulatus (3) and in R. sphaeroides NCIB8253 (4) enzymatic activities have been assigned to seven of the eight genes constituting this cluster but not to crtK, herein designated tspO (tryptophan-rich sensory protein). Based on the amino acid...

The rat mitochondrial outer membranelocalized benzodiazepine receptor (MBR) was expressed in wild-type and TspO ؊ (tryptophan-rich sensory protein) strains of the facultative photoheterotroph, Rhodobacter sphaeroides 2.4.1, and was shown to retain its structure within the bacterial outer membrane as assayed by its binding properties with a variety of MBR ligands. Functionally, it was able to substitute for TspO by negatively regulating the expression of photosynthesis genes in response to oxygen. This effect was reversed pharmacologically with the MBR ligand PK11195. These results suggest a close evolutionary and functional relationship between the bacterial TspO and the MBR. This relationship provides further support for the origin of the mammalian mitochondrion from a ''photosynthetic'' precursor. Finally, these findings provide novel insights into the physiological role that has been obscure for the MBR in situ.

Human peripheral-type cannabinoid receptor (CB2) was expressed in Escherichia coli as a fusion with the maltose-binding protein, thioredoxin, and a deca-histidine tag. Functional activity and structural integrity of the receptor in bacterial protoplast membranes was confirmed by extensive binding studies with a variety of natural and synthetic cannabinoid ligands. E. coli membranes expressing CB2 also activated cognate G-proteins in an in vitro coupled assay. Detergent-solubilized receptor was purified to 80%-90% homogeneity by affinity chromatography followed by ion-exchange chromatography. By high-resolution NMR on the receptor in DPC micelles, it was determined that purified CB2 forms 1:1 complexes with the ligands CP-55,940 and anandamide. The receptor was successfully reconstituted into phosphatidylcholine bilayers and the membranes were deposited into a porous substrate as tubular lipid bilayers for structural studies by NMR and scattering techniques. The peripheral cannabinoid receptor, CB2, belongs to the class of G-protein-coupled receptors (GPCRs). These are integral membrane proteins involved in a large number of physiological processes including sensory transduction, cell-to-cell communication, and immune and hormonal response (Martin 1986; Munro et al. 1993). GPCRs have become the object of intense investigations, in part because of their role as therapeutically important targets of drugs. One of the major difficulties in rational design and synthesis of potential modulators of GPCR activity is the lack of information on GPCR structure. It is notoriously difficult to crystallize GPCRs, most likely because their structure and conformation are intricately linked to the surrounding lipid matrix. Furthermore, in GPCR crystals the loop regions that connect the seven transmembrane helices are structurally heterogeneous (Ohguro et al. 1996), which prevents obtaining information about this important site by crystallography. Another limitation is that GPCRs in crystals are not functional. Here we explored the feasibility of expressing, purifying , and reconstituting CB2 for structural studies at functional conditions by methods like nuclear magnetic resonance and scattering techniques. With the notable exception of rhodopsin, most of the GPCRs are not available in large quantities from natural sources. Therefore , their expression in a heterologous host constitutes a more practical alternative. Escherichia coli offers important advantages as a potential expression host, including robustness of cultivation, ease of constructing expression strains, and the absence of expensive media requirements.