CONTROL OF LYTIC DEVELOPMENT IN THE STREPTOMYCES TEMPERATE PHAGE PHI-C31

S E WILSON, C J INGHAM, Irene Hunter, Margaret Caroline MacHin Smith

Research output: Contribution to journalArticle

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Abstract

The repressor gene, c, is required for maintenance of lysogeny in the Streptomyces phage phi C31. The c gene expresses three in-frame N-terminally different protein isoforms at least one of which is thought to bind to a 17 bp highly (c) under bar onserved (I) under bar nverted (r) under bar epeat (CIR) sequence found at 18(or more) loci throughout the phi C31 genome. Here we present evidence that one of these loci, CIR6, and its interaction with the products of the repressor gene are critical in the control of the lytic pathway in phi C31. To the right of CIR6, according to the standard map of phi C31, an 'immediate-early' promoter, ap1, was discovered after insertion of a fragment containing CIR6 upstream of a promoterless kanamycin-resistance gene, aphII, to form pCIA2. pCIA2 conferred kanamycin resistance upon Streptomyces coelicolor A3(2) but not upon a phi C31 lysogen of S. coelicolor. Operator-constitutive (OC) mutants of pCIA2 were isolated and the mutations lay in CIR6, i.e. CIR6:G14T and CIR6:C2A. Primer extension analysis of RNA prepared from an induced, temperature-sensitive lysogen of S. coelicolor localized a mRNA 5' endpoint 21 bp to the right of CIR6. The importance of the ap1/CIR6 region in the regulation of lytic growth was demonstrated by the analysis of a virulent mutant, phi C31 vir1, capable of forming plaques on an S. coelicolor phi C31 lysogen. phi C31 vir1 contained a DNA inversion with the breakpoints lying within the integrase gene (which lies approximately 7 kbp to the right of CIR6) and in the essential early region between CIR6 and the -10 sequence for ap1. The separation of ap1 from its operator was thought to be the basis for the virulent phenotype in phi C31 vir1. Band-shift assays and DNase I footprinting experiments using purified 42 kDa repressor isoform confirmed that CIRs 5 and 6 were indeed the targets for binding of this protein. The 42 kDa repressor bound to CIR6 with higher affinity than to CIR5 in spite of their identical core sequences. Repressor bound at CIR6 facilitated binding at CIR5. The high-affinity binding to CIR6 was abolished with the OC mutant, CIR6:G14T. Hydroxyl radical footprinting and dimethyl sulphate methylation protection of the 42 kDa repressor-CIR6 interaction suggested that the protein bound in the major groove and to one face of the DNA.

Original languageEnglish
Pages (from-to)131-143
Number of pages13
JournalMolecular Microbiology
Volume16
Issue number1
Publication statusPublished - Apr 1995

Keywords

  • GEL-ELECTROPHORESIS
  • CLONING VECTORS
  • GENE-EXPRESSION
  • EARLY REGION
  • DNA
  • TRANSCRIPTION
  • REPRESSOR
  • BINDING
  • BACTERIOPHAGE-PHI-C31
  • PHAGE-PHI-C31

Cite this

WILSON, S. E., INGHAM, C. J., Hunter, I., & Smith, M. C. M. (1995). CONTROL OF LYTIC DEVELOPMENT IN THE STREPTOMYCES TEMPERATE PHAGE PHI-C31. Molecular Microbiology, 16(1), 131-143.

CONTROL OF LYTIC DEVELOPMENT IN THE STREPTOMYCES TEMPERATE PHAGE PHI-C31. / WILSON, S E ; INGHAM, C J ; Hunter, Irene; Smith, Margaret Caroline MacHin.

In: Molecular Microbiology, Vol. 16, No. 1, 04.1995, p. 131-143.

Research output: Contribution to journalArticle

WILSON, SE, INGHAM, CJ, Hunter, I & Smith, MCM 1995, 'CONTROL OF LYTIC DEVELOPMENT IN THE STREPTOMYCES TEMPERATE PHAGE PHI-C31', Molecular Microbiology, vol. 16, no. 1, pp. 131-143.
WILSON SE, INGHAM CJ, Hunter I, Smith MCM. CONTROL OF LYTIC DEVELOPMENT IN THE STREPTOMYCES TEMPERATE PHAGE PHI-C31. Molecular Microbiology. 1995 Apr;16(1):131-143.
WILSON, S E ; INGHAM, C J ; Hunter, Irene ; Smith, Margaret Caroline MacHin. / CONTROL OF LYTIC DEVELOPMENT IN THE STREPTOMYCES TEMPERATE PHAGE PHI-C31. In: Molecular Microbiology. 1995 ; Vol. 16, No. 1. pp. 131-143.
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abstract = "The repressor gene, c, is required for maintenance of lysogeny in the Streptomyces phage phi C31. The c gene expresses three in-frame N-terminally different protein isoforms at least one of which is thought to bind to a 17 bp highly (c) under bar onserved (I) under bar nverted (r) under bar epeat (CIR) sequence found at 18(or more) loci throughout the phi C31 genome. Here we present evidence that one of these loci, CIR6, and its interaction with the products of the repressor gene are critical in the control of the lytic pathway in phi C31. To the right of CIR6, according to the standard map of phi C31, an 'immediate-early' promoter, ap1, was discovered after insertion of a fragment containing CIR6 upstream of a promoterless kanamycin-resistance gene, aphII, to form pCIA2. pCIA2 conferred kanamycin resistance upon Streptomyces coelicolor A3(2) but not upon a phi C31 lysogen of S. coelicolor. Operator-constitutive (OC) mutants of pCIA2 were isolated and the mutations lay in CIR6, i.e. CIR6:G14T and CIR6:C2A. Primer extension analysis of RNA prepared from an induced, temperature-sensitive lysogen of S. coelicolor localized a mRNA 5' endpoint 21 bp to the right of CIR6. The importance of the ap1/CIR6 region in the regulation of lytic growth was demonstrated by the analysis of a virulent mutant, phi C31 vir1, capable of forming plaques on an S. coelicolor phi C31 lysogen. phi C31 vir1 contained a DNA inversion with the breakpoints lying within the integrase gene (which lies approximately 7 kbp to the right of CIR6) and in the essential early region between CIR6 and the -10 sequence for ap1. The separation of ap1 from its operator was thought to be the basis for the virulent phenotype in phi C31 vir1. Band-shift assays and DNase I footprinting experiments using purified 42 kDa repressor isoform confirmed that CIRs 5 and 6 were indeed the targets for binding of this protein. The 42 kDa repressor bound to CIR6 with higher affinity than to CIR5 in spite of their identical core sequences. Repressor bound at CIR6 facilitated binding at CIR5. The high-affinity binding to CIR6 was abolished with the OC mutant, CIR6:G14T. Hydroxyl radical footprinting and dimethyl sulphate methylation protection of the 42 kDa repressor-CIR6 interaction suggested that the protein bound in the major groove and to one face of the DNA.",
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T1 - CONTROL OF LYTIC DEVELOPMENT IN THE STREPTOMYCES TEMPERATE PHAGE PHI-C31

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AU - Hunter, Irene

AU - Smith, Margaret Caroline MacHin

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N2 - The repressor gene, c, is required for maintenance of lysogeny in the Streptomyces phage phi C31. The c gene expresses three in-frame N-terminally different protein isoforms at least one of which is thought to bind to a 17 bp highly (c) under bar onserved (I) under bar nverted (r) under bar epeat (CIR) sequence found at 18(or more) loci throughout the phi C31 genome. Here we present evidence that one of these loci, CIR6, and its interaction with the products of the repressor gene are critical in the control of the lytic pathway in phi C31. To the right of CIR6, according to the standard map of phi C31, an 'immediate-early' promoter, ap1, was discovered after insertion of a fragment containing CIR6 upstream of a promoterless kanamycin-resistance gene, aphII, to form pCIA2. pCIA2 conferred kanamycin resistance upon Streptomyces coelicolor A3(2) but not upon a phi C31 lysogen of S. coelicolor. Operator-constitutive (OC) mutants of pCIA2 were isolated and the mutations lay in CIR6, i.e. CIR6:G14T and CIR6:C2A. Primer extension analysis of RNA prepared from an induced, temperature-sensitive lysogen of S. coelicolor localized a mRNA 5' endpoint 21 bp to the right of CIR6. The importance of the ap1/CIR6 region in the regulation of lytic growth was demonstrated by the analysis of a virulent mutant, phi C31 vir1, capable of forming plaques on an S. coelicolor phi C31 lysogen. phi C31 vir1 contained a DNA inversion with the breakpoints lying within the integrase gene (which lies approximately 7 kbp to the right of CIR6) and in the essential early region between CIR6 and the -10 sequence for ap1. The separation of ap1 from its operator was thought to be the basis for the virulent phenotype in phi C31 vir1. Band-shift assays and DNase I footprinting experiments using purified 42 kDa repressor isoform confirmed that CIRs 5 and 6 were indeed the targets for binding of this protein. The 42 kDa repressor bound to CIR6 with higher affinity than to CIR5 in spite of their identical core sequences. Repressor bound at CIR6 facilitated binding at CIR5. The high-affinity binding to CIR6 was abolished with the OC mutant, CIR6:G14T. Hydroxyl radical footprinting and dimethyl sulphate methylation protection of the 42 kDa repressor-CIR6 interaction suggested that the protein bound in the major groove and to one face of the DNA.

AB - The repressor gene, c, is required for maintenance of lysogeny in the Streptomyces phage phi C31. The c gene expresses three in-frame N-terminally different protein isoforms at least one of which is thought to bind to a 17 bp highly (c) under bar onserved (I) under bar nverted (r) under bar epeat (CIR) sequence found at 18(or more) loci throughout the phi C31 genome. Here we present evidence that one of these loci, CIR6, and its interaction with the products of the repressor gene are critical in the control of the lytic pathway in phi C31. To the right of CIR6, according to the standard map of phi C31, an 'immediate-early' promoter, ap1, was discovered after insertion of a fragment containing CIR6 upstream of a promoterless kanamycin-resistance gene, aphII, to form pCIA2. pCIA2 conferred kanamycin resistance upon Streptomyces coelicolor A3(2) but not upon a phi C31 lysogen of S. coelicolor. Operator-constitutive (OC) mutants of pCIA2 were isolated and the mutations lay in CIR6, i.e. CIR6:G14T and CIR6:C2A. Primer extension analysis of RNA prepared from an induced, temperature-sensitive lysogen of S. coelicolor localized a mRNA 5' endpoint 21 bp to the right of CIR6. The importance of the ap1/CIR6 region in the regulation of lytic growth was demonstrated by the analysis of a virulent mutant, phi C31 vir1, capable of forming plaques on an S. coelicolor phi C31 lysogen. phi C31 vir1 contained a DNA inversion with the breakpoints lying within the integrase gene (which lies approximately 7 kbp to the right of CIR6) and in the essential early region between CIR6 and the -10 sequence for ap1. The separation of ap1 from its operator was thought to be the basis for the virulent phenotype in phi C31 vir1. Band-shift assays and DNase I footprinting experiments using purified 42 kDa repressor isoform confirmed that CIRs 5 and 6 were indeed the targets for binding of this protein. The 42 kDa repressor bound to CIR6 with higher affinity than to CIR5 in spite of their identical core sequences. Repressor bound at CIR6 facilitated binding at CIR5. The high-affinity binding to CIR6 was abolished with the OC mutant, CIR6:G14T. Hydroxyl radical footprinting and dimethyl sulphate methylation protection of the 42 kDa repressor-CIR6 interaction suggested that the protein bound in the major groove and to one face of the DNA.

KW - GEL-ELECTROPHORESIS

KW - CLONING VECTORS

KW - GENE-EXPRESSION

KW - EARLY REGION

KW - DNA

KW - TRANSCRIPTION

KW - REPRESSOR

KW - BINDING

KW - BACTERIOPHAGE-PHI-C31

KW - PHAGE-PHI-C31

M3 - Article

VL - 16

SP - 131

EP - 143

JO - Molecular Microbiology

JF - Molecular Microbiology

SN - 0950-382X

IS - 1

ER -