Temporal Regulation of Viral Transcription during Development of Thermus thermophilus Bacteriophage ϕYS40

Sevostyanova, Anastasiya; Djordjevic, M.; Kuznedelov, Konstantin; Naryshkina, Tatyana; Gelfand, Mikhail S.; Severinov, Konstantin; Minakhin, Leonid

doi:10.1016/j.jmb.2006.11.050

Cited by 32 publications

(44 citation statements)

References 31 publications

(4 reference statements)

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“…In vivo primer extension reactions were done essentially as described elsewhere (33). P. aeruginosa PAO1 cells were infected with KZ (MOI, 5) at an OD 600 of 0.3 and harvested at time points specified for the experiments performed.…”

Section: Methodsmentioning

confidence: 99%

Development of Giant Bacteriophage ϕKZ Is Independent of the Host Transcription Apparatus

Ceyssens

Minakhin

Bossche

et al. 2014

J Virol

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251

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Pseudomonas aeruginosa bacteriophage KZ is the type representative of the giant phage genus, which is characterized by unusually large virions and genomes. By unraveling the transcriptional map of the ϳ280-kb KZ genome to single-nucleotide resolution, we combine 369 KZ genes into 134 operons. Early transcription is initiated from highly conserved AT-rich promoters distributed across the KZ genome and located on the same strand of the genome. Early transcription does not require phage or host protein synthesis. Transcription of middle and late genes is dependent on protein synthesis and mediated by poorly conserved middle and late promoters. Unique to KZ is its ability to complete its infection in the absence of bacterial RNA polymerase (RNAP) enzyme activity. We propose that transcription of the KZ genome is performed by the consecutive action of two KZ-encoded, noncanonical multisubunit RNAPs, one of which is packed within the virion, another being the product of early genes. This unique, rifampin-resistant transcriptional machinery is conserved within the diverse giant phage genus. IMPORTANCEThe data presented in this paper offer, for the first time, insight into the complex transcriptional scheme of giant bacteriophages. We show that Pseudomonas aeruginosa giant phage KZ is able to infect and lyse its host cell and produce phage progeny in the absence of functional bacterial transcriptional machinery. This unique property can be attributed to two phage-encoded putative RNAP enzymes, which contain very distant homologues of bacterial ␤ and ␤=-like RNAP subunits. T ranscription is driven by DNA-dependent RNA polymerases (RNAPs), which synthesize RNA from DNA templates (1). RNAPs can be classified into two unrelated families: small singlesubunit enzymes (ssRNAPs), encoded by some bacteriophages and also found in mitochondria and chloroplasts, and large multisubunit cellular enzymes (msRNAPs), transcribing genes in bacterial, archaeal, and eukaryal genomes. The catalytic activity of enzymes from both families is accomplished through a common two-metal-ion mechanism. The canonical bacterial msRNAP is a 400-kDa complex consisting of five core subunits (␣ 2 ␤␤=) which are directed to specific promoter sequences by a variety of factors (2). The two largest RNAP subunits, ␤ and ␤=, contain conserved double-psi beta-barrel (DPBB) domains that together form the active center (3-5). Members of the ssRNAP family rely on different catalytic domains and amino acid motifs for catalysis of the RNA polymerization reaction and are related to DNA polymerases and reverse transcriptases (6, 7). Bacterial RNAPs are inactivated by the antibiotic rifampin (Rif), which acts by binding to the ␤-subunit pocket deep inside the active-site channel, preventing synthesis of RNA sequences longer than 3 to 4 nucleotides (nt) (8).Bacterial RNAPs play a key role during the infection of bacterial cells by bacteriophages. Most known phages do not encode their own RNAP but redirect the host transcription machinery to viral promoters by rel...

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Section: Methodsmentioning

confidence: 99%

Development of Giant Bacteriophage ϕKZ Is Independent of the Host Transcription Apparatus

Ceyssens

Minakhin

Bossche

et al. 2014

J Virol

Self Cite

150

251

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“…For the biochemical analyses, T. thermophilus RNAP core and holoenzymes with the C-terminally 10His-tagged b9 subunit were purified from the T. thermophilus HB8 rpoCT10H strain, as described (Sevostyanova et al 2007). E. coli core RNAP was purchased from Epicentre.…”

Section: Protein Preparationmentioning

confidence: 99%

Structural basis for promoter specificity switching of RNA polymerase by a phage factor

Tagami

Sekine

Minakhin³

et al. 2014

Genes Dev.

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Transcription of DNA to RNA by DNA-dependent RNA polymerase (RNAP) is the first step of gene expression and a major regulation point. Bacteriophages hijack their host's transcription machinery and direct it to serve their needs. The gp39 protein encoded by Thermus thermophilus phage P23-45 binds the host's RNAP and inhibits transcription initiation from its major ''-10/-35'' class promoters. Phage promoters belonging to the minor ''extended -10'' class are minimally inhibited. We report the crystal structure of the T. thermophilus RNAP holoenzyme complexed with gp39, which explains the mechanism for RNAP promoter specificity switching. gp39 simultaneously binds to the RNAP b-flap domain and the C-terminal domain of the s subunit (region 4 of the s subunit [s 4 ]), thus relocating the b-flap tip and s 4 . The~45 Å displacement of s 4 is incompatible with its binding to the -35 promoter consensus element, thus accounting for the inhibition of transcription from -10/-35 class promoters. In contrast, this conformational change is compatible with the recognition of extended -10 class promoters. These results provide the structural bases for the conformational modulation of the host's RNAP promoter specificity to switch gene expression toward supporting phage development for gp39 and, potentially, other phage proteins, such as T4 AsiA.

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“…Two types of factors have been reported for T. thermophilus HB8; one is the typical 70 -type factor, designated A , and the other is an extracytoplasmic function factor, designated E (24). The putative sip promoter sequence is in good agreement with the sequence of the T. thermophilus A -dependent promoter (25). Multiple-sequence alignment of seven promoters with identified transcription initiation sites revealed unambiguous sequence conservation of the Ϫ10 and Ϫ35 promoter elements (see Table S2 in the supplemental material).…”

Section: Resultsmentioning

confidence: 64%

Silica-Induced Protein (Sip) in Thermophilic Bacterium Thermus thermophilus Responds to Low Iron Availability

Fujino

Nagayoshi

Iwase

et al. 2016

Appl Environ Microbiol

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Thermus thermophilus HB8 expresses silica-induced protein (Sip) when cultured in medium containing supersaturated silicic acids. Using genomic information, Sip was identified as a Fe 3؉ -binding ABC transporter. Detection of a 1-kb hybridized band in Northern analysis revealed that sip transcription is monocistronic and that sip has its own terminator and promoter. The sequence of the sip promoter showed homology with that of the A -dependent promoter, which is known as a housekeeping promoter in HB8. Considering that sip is transcribed when supersaturated silicic acids are added, the existence of a repressor is presumed. DNA microarray analysis suggested that supersaturated silicic acids and iron deficiency affect Thermus cells similarly, and enhanced sip transcription was detected under both conditions. This suggested that sip transcription was initiated by iron deficiency and that the ferric uptake regulator (Fur) controlled the transcription. Three Fur gene homologues (TTHA0255, TTHA0344, and TTHA1292) have been annotated in the HB8 genome, and electrophoretic mobility shift assays revealed that the TTHA0344 product interacts with the sip promoter region. In medium containing supersaturated silicic acids, free Fe 3؉ levels were decreased due to Fe 3؉ immobilization on colloidal silica. This suggests that, because Fe 3؉ ions are captured by colloidal silica in geothermal water, Thermus cells are continuously exposed to the risk of iron deficiency. Considering that Sip is involved in iron acquisition, Sip production may be a strategy to survive under conditions of low iron availability in geothermal water. IMPORTANCEThe thermophilic bacterium Thermus thermophilus HB8 produces silica-induced protein (Sip) in the presence of supersaturated silicic acids. Sip has homology with iron-binding ABC transporter; however, the mechanism by which Sip expression is induced by silicic acids remains unexplained. We demonstrate that Sip captures iron and its transcription is regulated by the repressor ferric uptake regulator (Fur). This implies that Sip is expressed with iron deficiency. In addition, it is suggested that negatively charged colloidal silica in supersaturated solution absorbs Fe 3؉ ions and decreases iron availability. Considering that geothermal water contains ample silicic acids, it is suggested that thermophilic bacteria are always facing iron starvation. Sip production may be a strategy for surviving under conditions of low iron availability in geothermal water. S ilica (SiO 2 ) is one of the most abundant substances in the Earth's crust. In aqueous solution, silica dissolves as monosilicic acid, Si(OH) 4 , the solubility of which depends on conditions such as temperature, pH, and salt concentrations (1). In geothermal water at high temperature and pressure underground, silicic acids are in equilibrium with the quartz in rock layers, with concentrations reaching around 600 ppm at 300°C. When this geothermal water is discharged to the surface, where the solubility of amorphous silica is about 300 ppm...

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Temporal Regulation of Viral Transcription during Development of Thermus thermophilus Bacteriophage ϕYS40

Cited by 32 publications

References 31 publications

Development of Giant Bacteriophage ϕKZ Is Independent of the Host Transcription Apparatus

Development of Giant Bacteriophage ϕKZ Is Independent of the Host Transcription Apparatus

Structural basis for promoter specificity switching of RNA polymerase by a phage factor

Silica-Induced Protein (Sip) in Thermophilic Bacterium Thermus thermophilus Responds to Low Iron Availability

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