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Genetic and nutritional analyses of mutants of the extremely halophilic archaeon Halobacterium sp. strain NRC-1 showed that open reading frame (ORF) Vng1581C encodes a protein with nucleoside triphosphate:adenosylcobinamide-phosphate nucleotidyltransferase enzyme activity. This activity was previously associated with the cobY gene of the methanogenic archaeon Methanobacterium thermoautotrophicum strain DeltaH, but no evidence was obtained to demonstrate the direct involvement of this protein in cobamide biosynthesis in archaea. Computer analysis of the Halobacterium sp. strain NRC-1 ORF Vng1581C gene and the cobY gene of M. thermoautotrophicum strain DeltaH showed the primary amino acid sequence of the proteins encoded by these two genes to be 35% identical and 48% similar. A strain of Halobacterium sp. strain NRC-1 carrying a null allele of the cobY gene was auxotrophic for cobinamide-GDP, a known intermediate of the late steps of cobamide biosynthesis. The auxotrophic requirement for cobinamide-GDP was corrected when a wild-type allele of cobY was introduced into the mutant strain, demonstrating that the lack of cobY function was solely responsible for the observed block in cobamide biosynthesis in this archaeon. The data also show that Halobacterium sp. strain NRC-1 possesses a high-affinity transport system for corrinoids and that this archaeon can synthesize cobamides de novo under aerobic growth conditions. To the best of our knowledge this is the first genetic and nutritional analysis of cobalamin biosynthetic mutants in archaea.

Biogenesis of the light-driven proton pump bacteriorhodopsin in the archaeon Halobacterium salinarum requires coordinate synthesis of the bacterioopsin apoprotein and carotenoid precursors of retinal, which serves as a covalently bound cofactor. As a step towards elucidating the mechanism and regulation of carotenoid metabolism during bacteriorhodopsin biogenesis, we have identified an H. salinarum gene required for conversion of lycopene to beta-carotene, a retinal precursor. The gene, designated crtY, is predicted to encode an integral membrane protein homologous to lycopene beta-cyclases identified in bacteria and fungi. To test crtY function, we constructed H. salinarum strains with in-frame deletions in the gene. In the deletion strains, bacteriorhodopsin, retinal, and beta-carotene were undetectable, whereas lycopene accumulated to high levels ( approximately 1.3 nmol/mg of total cell protein). Heterologous expression of H. salinarum crtY in a lycopene-producing Escherichia coli strain resulted in beta-carotene production. These results indicate that H. salinarum crtY encodes a functional lycopene beta-cyclase required for bacteriorhodopsin biogenesis. Comparative sequence analysis yields a topological model of the protein and provides a plausible evolutionary connection between heterodimeric lycopene cyclases in bacteria and bifunctional lycopene cyclase-phytoene synthases in fungi.

Tn5 is a composite transposon consisting of two IS50 sequences in inverted orientation with respect to a unique, central region encoding several antibiotic resistances. The IS50R element encodes two proteins in the same reading frame which regulate the transposition reaction: the transposase (Tnp), which is required for transposition, and an inhibitor of transposition (Inh). The inhibitor is a naturally occurring deletion variant of Tnp which lacks the N-terminal 55 amino acids. In this report, we present the purification of both the Tnp and Inh proteins and an analysis of their DNA binding properties. Purified Tnp, but not Inh, was found to bind specifically to the outside end of Tn5. Inh, however, stimulated the binding activity of Tnp to outside-end DNA and was shown to be present with Tnp in these bound complexes. Inh was also found to exist as a dimer in solution. These results indicate that the N-terminal 55 amino acids of Tnp are required for sequence-specific binding. They also suggest that Inh inhibits transposition by forming mixed oligomers with Tnp which still bind to the ends of the transposon but are defective for later stages of the transposition reaction.

Site-specific mutagenesis has identified amino acids involved in bR proton transport. Biophysical studies of the mutants have elucidated the roles of two membrane-embedded residues: Asp-85 serves as the acceptor for the proton from the isomerized retinylidene Schiff base, and Asp-96 participates in reprotonation of this group. The functions of Arg-82, Leu-93, Asp-212, Tyr-185, and other residues that affect bR properties when substituted are not as well understood. Structural characterization of the mutant proteins will clarify the effects of substitutions at these positions. Current efforts in the field remain directed at understanding how retinal isomerization is coupled to proton transport. In particular, there has been more emphasis on determining the structures of bR and its photointermediates. Since well-ordered crystals of bR have not been obtained, continued electron diffraction studies of purple membrane offer the best opportunity for structure refinement. Other informative techniques include solid-state nuclear magnetic resonance of isotopically labeled bR (56) and electron paramagnetic resonance of bR tagged with nitroxide spin labels (2, 3, 13, 15). Site-directed mutagenesis will be essential in these studies to introduce specific sites for derivatization with structural probes and to slow the decay of intermediates. Thus, combining molecular biology and biophysics will continue to provide solutions to fundamental problems in bR.