The <i>bop</i> gene regulon
Halobacterium sp. sustains a short Period of phototrophic growth by synthesizing ATP using a light-driven proton pump, bacteriorhodopsin. The expression of bop, which encodes the protein component of bacteriorhodopsin, bacterioopsin, is induced in mid- to late-log phase with maximal expression in the stationary phase. Three cis-acting elements within the minimal 53 bp bop promoter were predicted to be responsible in mediating expression and regulation of this gene, viz. UAS, TATA box, and RY box. Finally, computational analysis of the complete genome sequence was projected as a means of identifying putative transcription factors and regulators.
I have used a saturation mutagenesis approach to derive a consensus for 35 bp of the 53 bp minimal bop promoter. In the process, we identified two cis-acting elements required for transcription of bop (TATA box and UAS). Based on the presence of an alternate TATA box consensus sequence and lack of a TFB responsive element (BRE) we predicted presence of multiple TBPs and TFBs in Halobacterium NRC-1. Complete genome sequence analysis identified a total of six TBPs and seven TF13s. The UAS was highly immutable at its 3′ and 5 ′ extremities.
Three nucleotides (−27A, −23 A and −19A), within the RY box, were identified to be the major sites for DNA supercoiling mediated regulation of bop.
We identified the UAS upstream to three other genes, viz. brp, blp and crtB1. Expression profiles for these genes indicated that (a) they are coordinately regulated in Halobacterium NRC-1 and S9, (b) Bat, the putative bacterio-opsin activator, is required for expression of bop, blp and crtB1 and (c) like the bop promoter, the crtB1 and brp promoters contain alternating purine-pyrimidine sequences (10 and 11 bp long respectively), and are also regulated by changes in DNA supercoiling.
A detailed analysis of Bat revealed the presence of a redox-sensing PAS/PAC domain, a cGMP responsive GAF domain and a DNA-binding helix-turn-helix motif. These results lead us to conclude that carotenoid and bacterio-opsin biosyntheses are coordinately regulated by Bat in Halobacterium NRC-1. Also, Bat probably functions as a repressor and activator in high- and low-oxygen tension respectively; and the repressor state is transdominant.
A model has been proposed to explain bop gene regulation by DNA supercoiling, multiple transcription factors, and Bat. (Abstract shortened by UMI.)