Halobacterium halobium strains lysogenic for phage phi H contain a protein resembling coliphage repressors

Abstract

DNA-binding proteins such as bacteriophage repressors belong to the helix-turn-helix family. Ionic interactions drive DNA binding, which means that repressors bind DNA most tightly at low salt concentrations. This raises the question of how gene expression might be regulated in obligate halophiles, which maintain internal salt concentrations of about 5 M. As a model system we have investigated the phage OH, which infects the archaebacterium Halobacterium halobium. Previous genetic data and transcriptional mapping had suggested a region of the phage genome where a repressor might bind. A modified electrophoretic mobility shift assay was used to identify an activity, present only in lysogens, that specifically binds this region. Methylation interference and DNA sequencing were used to identify four similar binding sites, which are arranged so that two copies of a dimer might bind on one face of the DNA helix. Binding of a protein at these sites could block RNA polymerase from initiating a transcript found only during lytic growth. A nearby divergent promoter produces a lysogen-specific transcript, T6, which encodes a member of the helix-turn-helix family of DNA-binding proteins. By expressing the gene in Escherichia coli, we confirmed that T6 specifies the DNA binding activity detected biochemically. The data show that the basic DNA-binding motif of repressors can be adapted even for the unfavorable conditions of high salt concentration.Selective gene expression is primarily dependent on the activity of sequence-specific DNA-binding proteins. The identification, purification, and characterization of such proteins are a major focus of contemporary research in molecular biology. DNA-binding proteins are typically prepared by extracting cells or nuclei by buffers of high salt concentration. This procedure is effective because high salt concentrations dissociate protein-DNA complexes. The thermodynamic origin of this dissociation is in the large electrostatic component of the binding energy (16, 19). Protein-DNA interaction can be considered to be an ion-exchange reaction in which protein binding accompanies counterion displacement. In this situation, increasing the free cation concentration shifts the equilibrium in favor of dissociation. This raises the question of how protein-DNA interaction is driven in organisms with high internal salt concentrations. We have chosen to study the halophilic archaebacterium Halobacterium halobium, which maintains an internal salt concentration matching that of the external environment, typically about 5 M (5).Many DNA-binding proteins, such as bacteriophage repressors, belong to the helix-turn-helix family (14). Extensive genetic (17), biochemical (19), and structural (1, 30) data exist on their interaction with DNA. To take advantage of the previous work on phage repressors

Transcriptional regulation in Archaea: In vivo demonstration of a repressor binding site in a methanogen

“…By comparison, the palindromic sequence in the M. maripaludis nifH operator starts just two nucleotides from the transcription start, and repressor binding there could also interfere with some step in transcriptional initiation. We are aware of one other example of transcriptional regulation by repression in Archaea; a repressor present in phage H lysogens of Halobacterium halobium apparently functions by binding to palindromic sites immediately upstream of the TATA box for a lytic gene (27,28). However, despite similarities between bacterial repression and the observations made to date in Archaea, the picture is still incomplete and it would be premature to suggest that repression in Archaea occurs by a bacterial mechanism.…”

“…29-31), and evidently in H. halobium H as well (27), nearby copies of similar palindromes allow higher order cooperative interactions to occur due to repressor tetramers. In the M. maripaludis nifH promoter region, however, although a second palindrome exists, no evidence for any role was obtained.…”

Genomic and genetic dissection of an archaeal regulon

“…and S.D., unpublished observations). Previously, one halophage HTH protein was shown to bind DNA in band-shift assays (26). Finally, the redox-sensing PAS͞PAC region was also present in Bat (3,11).…”

The particle SSV1 from the extremely thermophilic archaeon Sulfolobus is a virus: demonstration of infectivity and of transfection with viral DNA.

“…In one of the two kingdoms, the Euryarchaeota (1), two phage/host systems have been studied in some molecular detail: the temperate phage DH of Halobacterium halobium (4)(5)(6) and the transducing phage TM1 of Methanobacterium thermoautotrophicum (7,8). In the other kingdom of the archaeal domain, the Crenarchaeota (1), comprising extreme thermophiles, a family of viruses of the anaerobic host Thermoproteus tenax, TTV1 through TTV4 (2,9,10), is of limited usefulness, because no plaque assay is available.…”