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 PAS/PAC domain of Bat in redox-sensing 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 gamma-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.

Figure 1

Map of the purple membrane regulon and carotenoid biosynthesis pathway. (a) A map for the chromosomal locus coding for bop, brp, bat, blp, and crtB1 genes. The site for ISH1 insertion in bat in strain SD20 is indicated. The four mapped promoters, P_bop, P_brp, P_blp, and P_crtB1, are indicated with bent arrows either above or below the map. (b) The pathway for carotenoid biosynthesis. Steps 1 and 2 in bacteria are catalyzed by the crtB1 gene product: phytoene synthase; steps 3 and 4 in plants are catalyzed by phytoene desaturase (Pds); and step 5 in Halobacterium sp. is catalyzed by Brp.

Figure 2

Alignments for Halobacterium NRC-1 promoters (blp, bop, brp, and crtB1) with the bop promoter consensus derived by saturation mutagenesis (a) (13, 14), the consensus representing nucleotides conserved within the four promoters (b), the tomato pds gene, and the γ-proteobacterium pop gene promoters. Nucleotides conserved in at least three Halobacterium NRC-1 promoters are shaded in yellow, other identities to a are shaded in gray, and gaps are indicated with a dot. The transcription start site (+1) is shaded in blue. The bop promoter RY box is underlined; the sixth and tenth nucleotides within this element were identified to be the sites for supercoiling sensitivity (14).

Figure 3

Expression profiles for bop, blp, brp, and crtB1. Message levels for the four bop regulon genes in stationary phase cultures of the bat∷ISH1 strain, SD20 (a); early (1), mid- to late-exponential (2), and stationary phase (3) cultures of the wild-type strain NRC-1 (b); and early (1), mid- to late-exponential (2), and stationary phase (3) cultures of the purple membrane overproducing strain, S9 (c). (d) Sensitivity of transcription to changes in DNA supercoiling was tested by quantifying message levels from cultures grown with (1) and without (2) novobiocin.

Figure 4

Bat, the transcription regulator of the purple membrane regulon. (a) Positions and amino acids within the redox-responsive PAS (shaded orange)/PAC (shaded blue) domain, the photoresponsive GAF domain (purple font), and the HTH DNA binding motif (green font) are shown. The amino acids altered within the PAC domain in S9 Bat are indicated with a red font. (b) The double-frameshift mutation in the S9 bat gene. Deletion of a “G” (red font) and an insertion of an “A” (blue font) four codons apart has resulted in an altered amino acid sequence (shown below and above the aligned DNA sequences). The amino acid corresponding to the N85C mutation in the E. coli Aer protein is shaded in black (27).

Figure 5

GAF and other domains in predicted archaeal proteins. (a) Multiple sequence alignment of archaeal GAF domains. The amino acids with identities to the signature are indicated in a red font. Alignments for both GAF domains identified in Archaeoglobus fulgidus Afu0448 and Halobacterium CHP2334 (Hal_2334) are included in the alignment. (b) Domain organization in the archaeal putative cytoplasmic signal-transducing proteins. Regions containing GAF domains are shown as red boxes, PAS domains as green boxes, PAC domains as blue boxes, HTH domains as pink boxes, and histidine kinase domains as yellow boxes.