Genomic and genetic dissection of an archaeal regulon

Abstract

The extremely halophilic archaeon Halobacterium sp. NRC-1 can grow phototrophically by means of light-driven proton pumping by bacteriorhodopsin in the purple membrane. Here, we show by genetic analysis of the wild type, and insertion and double-frame shift mutants of Bat that this transcriptional regulator coordinates synthesis of a structural protein and a chromophore for purple membrane biogenesis in response to both light and oxygen. Analysis of the complete Halobacterium sp. NRC-1 genome sequence showed that the regulatory site, upstream activator sequence (UAS), the putative binding site for Bat upstream of the bacterio-opsin gene (bop), is also present upstream to the other Bat-regulated genes. The transcription regulator Bat contains a photoresponsive cGMP-binding (GAF) domain, and a bacterial AraC type helix-turn-helix DNA binding motif. We also provide evidence for involvement of the PAS͞PAC domain of Bat in redoxsensing activity by genetic analysis of a purple membrane overproducer. Five additional Bat-like putative regulatory genes were found, which together are likely to be responsible for orchestrating the complex response of this archaeon to light and oxygen. Similarities of the bop-like UAS and transcription factors in diverse organisms, including a plant and a ␥-proteobacterium, suggest an ancient origin for this regulon capable of coordinating light and oxygen responses in the three major branches of the evolutionary tree of life. Finally, sensitivity of four of five regulon genes to DNA supercoiling is demonstrated and correlated to presence of alternating purine-pyrimidine sequences (RY boxes) near the regulated promoters.H alobacterium species produce a specialized region in the cell membrane, named purple membrane, which consists of a two-dimensional crystalline lattice of a single chromoprotein, bacteriorhodopsin (BR). BR contains a 1:1 complex between a protein component bacterio-opsin and a chromophore retinal, and carries out light-driven proton pumping across the membrane (1, 2). BR is highly induced in late exponential to stationary phase of growth in response to high light intensity and low oxygen tension (3, 4). Proton pumping by BR is used to drive ATP synthesis and can sustain a period of phototrophic growth (1, 2). The mechanism of proton translocation across the membrane by BR has been the subject of extensive study by biophysical and mutational approaches (5, 6). Recently, retinal proteins similar to BR have been discovered in some fungi and uncultivated marine planktonic bacteria, indicating a much wider distribution in nature than originally appreciated (7,8).In Halobacterium sp. NRC-1, the proteins for biogenesis of the purple membrane are coded by the bop gene, specifying bacterioopsin, and several nearby genes, e.g., brp, bat, blp, and crtB1, thought to be involved in synthesis of the chromophore, genetic regulation, or biogenesis of the membrane (Fig. 1a) (9, 10). Early studies showed that insertions into bacterio-opsin gene activator (bat) or bacterio-opsin related prote...

Parallel evolution of transcriptome architecture during genome reorganization

“…While the blh-crtY and atp operons are both involved in ATP synthesis, it is not clear how this benefits the halophiles, given that these genes are regulated in an exactly opposing manner under changing oxygen conditions ). Consistent with this and prior studies (Baliga et al 2001(Baliga et al , 2002Bonneau et al 2007), growth-associated regulation of crtY-blh transcript was also anti-correlated with atpIKECFAB (r ; À0.8) and highly correlated with other TUs of phototrophy genes including blp, bat-brp, and bop (r ; 0.9). Interestingly, our data reveal that the relatively new promoter of atpD is gaining similar regulatory control as that of the parent operon.…”

Genomic Analysis of Anaerobic Respiration in the ArchaeonHalobacteriumsp. Strain NRC-1: Dimethyl Sulfoxide and TrimethylamineN-Oxide as Terminal Electron Acceptors

“…The bacterio-opsin gene bop displayed the highest up-regulation (15-fold) of all ORFs. The strong differential expression of the bop gene is consistent with its regulation by the oxygenor redox-sensing activator Bat (2,3,40,41). Genes involved in aerobic respiration in NRC-1 were not down-regulated in the absence of oxygen.…”

“…strain NRC-1 under anaerobic conditions. Previous work utilized a combination of genetic and genomic studies to establish the purple membrane regulon, which leads to coordinate synthesis of bacterio-opsin and its chromophore, retinal, to allow for light-driven proton pumping (2,8,40,41). The previous work led to the identification of a novel positive regulator, Bat, which controls the expression of the bacterio-opsin (bop) gene, as well as the first and last steps in the retinal biosynthetic pathway.…”

Understanding the Adaptation of Halobacterium Species NRC-1 to Its Extreme Environment through Computational Analysis of Its Genome Sequence

“…Recent success in solving the structure of the large ribosomal subunit from a related halophile, Haloarcula marismortui (Ban et al 2000), genomic and genetic dissection of the archaeal regulon for purple membrane synthesis in Halobacterium NRC-1 (Baliga et al 2001), and reverse genetic analysis of carotenoid and retinal biosynthesis (Peck et al 2001) further solidify halophiles as viable and indispensable model systems in both prokaryotic and eukaryotic fields. Undoubtedly, on-going work using genetic arrays, gene knockouts, and further computational analysis will yield insights into life in extreme environments generally and adaptation to hypersaline environments specifically.…”