Genetic and topological analyses of the bop promoter of Halobacterium halobium: stimulation by DNA supercoiling and non-B-DNA structure

Abstract

The bop gene of wild-type Halobacterium halobium NRC-1 is transcriptionally induced more than 20-fold under microaerobic conditions. bop transcription is inhibited by novobiocin, a DNA gyrase inhibitor, at concentrations subinhibitory for growth. The exposure of NRC-1 cultures to novobiocin concentrations inhibiting bop transcription was found to partially relax plasmid DNA supercoiling, indicating the requirement of high DNA supercoiling for bop transcription. Next, the bop promoter region was cloned on an H. halobium plasmid vector and introduced into NRC-1 and S9, a bop overproducer strain. The cloned promoter was active in both H. halobium strains, but at a higher level in the overproducer than in the wild type. Transcription from the bop promoter on the plasmid was found to be inhibited by novobiocin to a similar extent as was transcription from the chromosome. When the cloned promoter was introduced into S9 mutant strains with insertions in either of two putative regulatory genes, brp and bat, no transcription was detectable, indicating that these genes serve to activate transcription from the bop promoter in trans. Deletion analysis of the cloned bop promoter from a site ϳ480 bp upstream of bop showed that a 53-bp region 5 to the transcription start site is sufficient for transcription, but a 28-bp region is not. An 11-bp alternating purine-pyrimidine sequence within the functional promoter region, centered 23 bp 5 to the transcription start point, was found to display DNA supercoiling-dependent sensitivity to S1 nuclease and OsO 4 , which is consistent with a non-B-DNA conformation similar to that of left-handed Z-DNA and suggests the involvement of unusual DNA structure in supercoiling-stimulated bop gene transcription.The bop gene, encoding bacterio-opsin, the purple membrane protein of Halobacterium halobium, was one of the first archaeal genes to be cloned and sequenced (11). After cloning, several studies focused on the transcription of the bop gene and its regulation by oxygen and light. The start site for transcription was shown to map just 2 nucleotides upstream of the coding region by purification of the message, capping of its 5Ј end, and sequencing (6). Although this established bop mRNA as a primary transcript, no easily identifiable promoter could be found. Only a weak similarity to the TATA-like (box A) element was present (17). Interestingly, an 11-bp-long alternating purine-pyrimidine sequence centered 23 bp 5Ј to the transcription start site was observed and hypothesized to influence transcription by adopting left-handed Z-DNA conformation, which would be stabilized by high salt concentration and DNA supercoiling in H. halobium.The bop gene was shown to be transcriptionally induced at least 20-fold under microaerobic conditions in wild-type H. halobium strains (36, 43). Interestingly, bop gene transcription was inhibited by novobiocin, suggesting the involvement of DNA supercoiling in the modulation of promoter activity (42). Moreover, the involvement of two upstream genes in bop gene tr...

brp and blh Are Required for Synthesis of the Retinal Cofactor of Bacteriorhodopsin in Halobacterium salinarum

“…Insertions in brp greatly decrease BR and bop mRNA levels (15)(16)(17)(18). This result can be interpreted to imply that Brp modulates bop expression (18,21). However, it has also been recognized that the effect of brp insertions may be attributable to an indirect effect on bat expression (18,21).…”

“…1) that includes bop, which encodes BO; bat, which encodes a transacting factor (Bat) that contains a region homologous to the PAS domain of the oxygen sensor NifL (19,20) and activates bop expression under microaerobic conditions (21,22); and blp, a gene of unknown function (23). Insertions in brp greatly decrease BR and bop mRNA levels (15)(16)(17)(18). This result can be interpreted to imply that Brp modulates bop expression (18,21).…”

“…However, cellular factors may be required to prevent the photooxidation or photoisomerization of the cofactor during its transport or binding to BO. These functions may be mediated by a retinal-specific chaperone or by a multifunctional enzyme that converts ␤-carotene to retinal and transports or binds retinal to BO.As a first step to identify cellular factors that mediate these processes, we chose to study the brp gene, which encodes a putative membrane protein (Brp) implicated in BR biogenesis (15)(16)(17)(18). The brp gene is part of a gene cluster (Fig.…”

“…As a first step to identify cellular factors that mediate these processes, we chose to study the brp gene, which encodes a putative membrane protein (Brp) implicated in BR biogenesis (15)(16)(17)(18). The brp gene is part of a gene cluster (Fig.…”

In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a β-Carotene 15,15′-Dioxygenase

“…The Blh protein is a putative membrane protein closely related to bacteriorhodopsin (40). Bacteriorhodopsin is a membrane protein containing seven transmembrane ␣-helices and a covalently bound molecule of retinal.…”