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Four genes essential for bacteriochlorophyll biosynthesis were known to be encoded within a 45 kb region of the Rhodobacter sphaeroides genome, the boundaries of which are defined by puh and puf genes for reaction-centre and light-harvesting LH1 complexes. The cluster is represented by eight overlapping inserts cloned in the mobilizable vector pSUP202. We have used localized transposon Tn5 mutagenesis to characterize this cluster further; a total of 87 independent insertions were generated which identify nine genes for bacteriochlorophyll biosynthesis, six for carotenoid biosynthesis, and puhA encoding the reaction-centre H subunit. This work provides an essential framework for a detailed study of the structure and expression of genes for photosynthesis in this bacterium.

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The expression site-associated gene ESAG 6 was previously implicated in transferrin binding in the protozoan parasite Trypanosoma brucei. ESAG 6 and the closely related ESAG 7 of T. brucei strain AnTatl.3 have now been expressed in insect cells using the baculovirus expression system. Expression of ESAG 6 alone in insect cells gives rise to a glycosylated protein of =52 kDa, which is cell surfaceassociated through a glycosylphosphatidylnoltol anchor at its C terminus. The ESAG 7 product of about 42 kDa is also glycosylated, but lacks the glycosylphosphatidylinositol modification, and is located intracellularly. No terrm-binding activity is observed when either ESAG is expressed independently. However, their coexpression results in a cell surface complex of ESAG 6 and 7 products that specifically binds trusferrin. This shows that both ESAG 6 and 7 products are necessary and s ent for binding to transferrin.Trypanosoma brucei is widespread in Africa and causes a wasting disease-Nagana-in cattle and sleeping sickness in humans. In the mammalian host the parasite exists extracellularly in the blood and tissues, where it evades the immune response by sequentially expressing a repertoire of antigenically distinct surface proteins, encoded by the variant surface glycoprotein (VSG) genes. Each transcriptionally active VSG gene is located in a so-called expression site, which contains at least eight other cotranscribed expression siteassociated genes (ESAGs) (1). Although highly immunogenic, the VSGs are unsuitable for immunization against the parasitic infection. Potential targets for protective immunization may be surface proteins that deliver essential nutrients to the parasite and that are invariant.Living extracellularly, the parasite has access to the host's serum components. It has been demonstrated that T. brucei requires the major serum iron-transport protein, transferrin (TF), for growth (2). In T. brucei, a specific TF-binding protein (TFBP) that could function as a receptor in transferrin uptake was sought using TF-Sepharose as an affinity matrix (3). N-terminal sequencing of a purified TFBP isolated in extremely small ajnounts from T. brucei strain MITat 1.4 (clone 117a) revealed it to be identical to the products of two highly similar ESAGs, ESAGs 6 and 7 (3, 4). The putative products of these genes share about 85% sequence identity. Both contain a potential signal peptide, suggesting they are secreted or located on the surface. The ESAG 6 but not ESAG 7 product has a potential recognition site for attachment of a glycosylphosphatidylinositol (GPI) anchor at the C-terminal end. ESAG 6 was originally reported as encoding the TFBP (3). Subsequent work, however, has suggested that the data were misinterpreted (M. Ligtenberg and P. Borst, personal communication; this work; refs. 5 and 6) and that ESAG 6 and 7 products are involved in TF binding (this work;refs. 6 and 7).In this paper we report the expression of both proteins individually and together in insect cells and unequivocally demonstrate that in...

The puc operon of Rhodobacter sphaeroides encoding polypeptides of the major light-harvesting complex, LH2, has been found to be linked to hemF, a gene encoding a putative anaerobic coproporphyrinogen III oxidase. The puc-hemF region of the R. sphaeroides genome has been investigated by insertional mutagenesis, complementation analysis of these insertional mutants and DNA sequencing. A third gene, designated pucC, has been found immediately downstream of pucA and has been shown to be essential for LH2 expression. pucC is cotranscribed with pucB and pucA; however, hemF and the pucBAC operon were found not to be transcriptionally linked. Ultrastructural studies indicated that the morphology of the intracytoplasmic membrane may depend upon expression of pucC as well as pucBA.