Identification of a phage-coded DNA-binding protein that regulates transcription from late promoters in bacteriophage P4.
The genetic element P4 can propagate as a temperate phage or as a multicopy plasmid in its host Escherichia coli. Late in the lytic cycle and in the plasmid condition, transcription of the P4 essential genes depends on the activation of the late promoters P(LL) and P(sid), which control the transcription of the left and right operons, respectively. Both P4 late promoters are positively regulated by the product of the P4 delta gene, which is transcribed from P(sid). We have identified a new P4 gene, vis, that appears to play a relevant role in P4 late transcription control. vis is the first gene downstream of P(LL) and codes for a basic 88 amino acid protein with a potential helix-turn-helix motif. Expression of the cloned vis gene suppresses all the phenotypic traits exhibited by P4 vir1, a mutant that carries a promoter-up mutation in the late promoter P(LL). By Northern hybridization analysis we showed that vis negatively regulates transcription from P(LL) and enhances transcription from P(sid). Thus, vis auto-regulates its expression by repressing its own promoter and enhancing transcription of delta, which is required for P(LL) activation. The vis gene was fused with the glutathione S-transferase gene and the GST-Vis fusion protein was partially purified. By gel retardation assays and DNA footprinting we demonstrated that GST-Vis binds to a 32 bp long region immediately downstream of P(LL). We also showed, by gel retardation, that GST-Vis binds to the P sid region. A sequence present in both P(LL) and P(sid) regions may represent the Vis binding consensus sequence. The dual role of Vis on the control of P4 late transcription may be required for a regulated expression of the replication functions when P4 propagates in the plasmid state.
Polo
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Dipartimento di Genetica e di Biologia dei microrganismi Università di Milano, Italy.
Sturniolo
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Dehó
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Ghisotti
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000235:genetics; D036022:Capsid Proteins; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000187:drug effects / Q000821:virology; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification; D018257:Helix-Loop-Helix Motifs; D008969:Molecular Sequence Data; D009876:Operon; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification; D014774:Virulence
D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C095129:Sid protein, Bacteriophage P4; D014764:Viral Proteins; C098826:Vis protein, Bacteriophage P4; D005982:Glutathione Transferase