Asunto(s)
Halobacterium/genética , Halobacterium/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Teoría de Sistemas , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Galactosa/metabolismo , Genes Arqueales , Genes Fúngicos , Genes Reguladores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
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.
Asunto(s)
Archaea/genética , Regulón , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Archaea , Genes Arqueales , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de AminoácidoRESUMEN
Halobacterium species display a variety of responses to light, including phototrophic growth, phototactic behavior, and photoprotective mechanisms. The complete genome sequence of Halobacterium species NRC-1 (Proc Natl Acad Sci USA 97: 12176-12181, 2000), coupled with the availability of a battery of methods for its analysis makes this an ideal model system for studying photobiology among the archaea. Here, we review: (1) the structure of the 2.57 Mbp Halobacterium NRC-1 genome, including a large chromosome, two minichromosomes, and 91 transposable IS elements; (2) the purple membrane regulon, which programs the accumulation of large quantities of the light-driven proton pump, bacteriorhodopsin, and allows for a period of phototrophic growth; (3) components of the sophisticated pathways for color-sensitive phototaxis; (4) the gas vesicle gene cluster, which codes for cell buoyancy organelles; (5) pathways for the production of carotenoid pigments and retinal, (6) processes for the repair of DNA damage; and (7) putative homologs of circadian rhythm regulators. We conclude with a discussion of the power of systems biology for comprehensive understanding of Halobacterium NRC-1 photobiology.
RESUMEN
We report the complete sequence of an extreme halophile, Halobacterium sp. NRC-1, harboring a dynamic 2,571,010-bp genome containing 91 insertion sequences representing 12 families and organized into a large chromosome and 2 related minichromosomes. The Halobacterium NRC-1 genome codes for 2,630 predicted proteins, 36% of which are unrelated to any previously reported. Analysis of the genome sequence shows the presence of pathways for uptake and utilization of amino acids, active sodium-proton antiporter and potassium uptake systems, sophisticated photosensory and signal transduction pathways, and DNA replication, transcription, and translation systems resembling more complex eukaryotic organisms. Whole proteome comparisons show the definite archaeal nature of this halophile with additional similarities to the Gram-positive Bacillus subtilis and other bacteria. The ease of culturing Halobacterium and the availability of methods for its genetic manipulation in the laboratory, including construction of gene knockouts and replacements, indicate this halophile can serve as an excellent model system among the archaea.
Asunto(s)
Genoma Bacteriano , Halobacterium/genética , Evolución Biológica , Membrana Celular/metabolismo , Reparación del ADN , Replicación del ADN , Metabolismo Energético , Halobacterium/metabolismo , Membrana Dobles de Lípidos , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Recombinación Genética , Transducción de Señal , Transcripción GenéticaRESUMEN
Transcription from the bop promoter in the haloarchaeon Halobacterium NRC-1, is highly induced under oxygen-limiting conditions. A DNA gyrase inhibitor, novobiocin, was previously shown to block bop gene induction and suggested that DNA supercoiling mediates transcriptional induction. A region of non-B structure was found 3' to the TATA box within an 11 bp alternating purine-pyrimidine sequence (RY box), which correlated to both increased DNA supercoiling and transcriptional induction. Here, saturation mutagenesis of the RY box region has been used to show that single-base substitutions of A(r)G either 23 or 19 bp 5' to the transcription start site temper the effect of DNA supercoiling based on novobiocin insensitivity of transcription. Mutagenesis of the region 5' to the TATA box showed its involvement in DNA supercoiling modulation of transcription, defined the 3' end of the upstream activator sequence (UAS) regulatory element, and ruled out the requirement for a TFB (TFIIB) Recognition Element. Spacing between the TATA box and UAS was found to be critical for promoter activity because insertion of partial or whole helical turns between the two elements completely inhibited transcription indicating that the UAS element does not function as a transcriptional enhancer. The results are discussed in the context of DNA melting and flexibility around the TATA box region and the involvement of multiple regulatory and transcription factors in bop promoter activity.
Asunto(s)
Proteínas Arqueales , ADN de Archaea , ADN Superhelicoidal , Elementos de Facilitación Genéticos , Genes Arqueales , Halobacterium/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción TFIIB , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , TATA BoxAsunto(s)
Proteínas Arqueales/metabolismo , Regulación Bacteriana de la Expresión Génica , Halobacterium/genética , Proteínas Nucleares/metabolismo , Factor de Transcripción TFIIB , Factores de Transcripción TFII/metabolismo , Factores de Transcripción/metabolismo , Dedos de Zinc , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Homología de Secuencia de Aminoácido , Factor de Transcripción TFIID , Factores de Transcripción/genética , Factores de Transcripción TFII/genéticaRESUMEN
Degenerate oligonucleotides were used to randomize 21 bp of the 53-bp minimal bop promoter in three 7-bp segments, including the putative TATA box and the upstream activator sequence (UAS). The mutagenized bop promoter and the wild-type structural gene and transcriptional terminator were inserted into a shuttle plasmid capable of replication in the halophilic archaeon Halobacterium sp. strain S9. Active promoters were isolated by screening transformants of an orange (Pum- bop) Halobacterium mutant for purple (Pum+ bop+) colonies on agar plates and analyzed for bop mRNA and/or bacteriorhodopsin content. Sequence analysis yielded the consensus sequence 5'-tyT(T/a)Ta-3', corresponding to the promoter TATA box element 30 to 25 bp 5' of the transcription start site. A putative UAS, 5'-ACCcnactagTTnG-3', located 52 to 39 bp 5' of the transcription start site was found to be conserved in active promoters. This study provides direct evidence for the requirement of the TATA box and UAS for bop promoter activity.