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A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria.

Quiles-Puchalt N, Tormo-Más MÁ, Campoy S, Toledo-Arana A, Monedero V, Lasa I, Novick RP, Christie GE, Penadés JR - Nucleic Acids Res. (2013)

Bottom Line: These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages.To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription.This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biomedicina de Valencia (IBV-CSIC), 46010 Valencia, Spain, Centro de Investigación y Tecnología Animal, Instituto Valenciano de Investigaciones Agrarias (CITA-IVIA), Apdo. 187, 12.400 Segorbe, Castellón, Spain, Departamento de Ciencias Biomédicas, Universidad CEU Cardenal Herrera, 46113 Moncada, Valencia, Spain, Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain, Instituto de Agrobiotecnología, CSIC-Universidad Pública de Navarra, 31006 Pamplona, Navarra, Spain, Laboratorio de Bacterias Lacticas y Probioticos, Instituto de Agroquimica y Tecnologia de Alimentos-CSIC, 46980 Paterna, Valencia, Spain, Skirball Institute Program in Molecular Pathogenesis and Departments of Microbiology and Medicine, New York University Medical Center, 540 First Avenue, New York, NY 10016, USA, Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298-0678, USA and Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, G12 8TA Glasgow, UK.

ABSTRACT
The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria.

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DNA requirements for Ltr binding. (A) Derivatives of strains RN10359 (lysogenic for 80α), JP7904 (lysogenic for ϕ29) and JP2348 (lysogenic for ϕ55), containing each of the indicated plasmids, were SOS induced and assayed for β-lactamase activity under standard conditions. (B) Scheme of the chimeric structures generated to analyse the role of the different regions contained in the terS promoter regions form three different phages (80α, ϕ29 and ϕ55). (C) Derivatives of strains RN10359 (lysogenic for 80α) and JP2348 (lysogenic for ϕ55) containing each of the indicated plasmids were SOS induced and assayed for β-lactamase activity under standard conditions.
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gkt508-F7: DNA requirements for Ltr binding. (A) Derivatives of strains RN10359 (lysogenic for 80α), JP7904 (lysogenic for ϕ29) and JP2348 (lysogenic for ϕ55), containing each of the indicated plasmids, were SOS induced and assayed for β-lactamase activity under standard conditions. (B) Scheme of the chimeric structures generated to analyse the role of the different regions contained in the terS promoter regions form three different phages (80α, ϕ29 and ϕ55). (C) Derivatives of strains RN10359 (lysogenic for 80α) and JP2348 (lysogenic for ϕ55) containing each of the indicated plasmids were SOS induced and assayed for β-lactamase activity under standard conditions.

Mentions: The results described earlier in the text do not determine whether the upstream DNA repeats are sufficient to support Ltr binding. To address this question, we initially compared activation of gene expression using plasmids pJP1207, pJP1214 and pJP1221. These pCN41 derivative plasmids contain the terS promoter region (including the repeats) and the 5′ coding region of the terS gene from phages 80α, ϕ29 and ϕ55, respectively. Phages 80α and ϕ29 encode a RinA protein, and as shown in Supplementary Figure S3, the putative binding regions for the RinA proteins (which we have defined as a block of 59 nt just distal to rinA, including the direct repeats) are basically identical in these two phages. In contrast, the DNA just downstream of this block of nucleotides, including the intergenic region and the terS gene, is completely different. Phage ϕ55 encodes LtrC, and as shown in Supplementary Figure S3, has a repeat-containing region different from that present in phages 80α and ϕ29. However, as indicated previously, the cloned intergenic region from phage ϕ55 is basically identical to that present in ϕ29 and completely different from that of phage 80α (Supplementary Figure S3). Plasmids pJP1207 (80α), pJP1214 (ϕ29) and pJP1221 (ϕ55) were introduced into lysogenic strains RN10359 (80α), JP7904 (ϕ29) and JP2348 (ϕ55), the resident prophages were SOS induced and the β-lactamase reporter was measured. As shown in Figure 7A, the β-lactamase reporter was uniquely expressed in those strains carrying cognate pair phage–plasmid with the DNA repeat region corresponding to the resident prophage, confirming the specificity of the Ltr proteins for their corresponding repeats. Although the RinA-expressing phages 80α and ϕ29 cross-reacted, expression of the reporter gene was higher in those strains containing the specific phage and its cognate cloned DNA sequence. An explanation for this is given later in the text.Figure 7.


A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria.

Quiles-Puchalt N, Tormo-Más MÁ, Campoy S, Toledo-Arana A, Monedero V, Lasa I, Novick RP, Christie GE, Penadés JR - Nucleic Acids Res. (2013)

DNA requirements for Ltr binding. (A) Derivatives of strains RN10359 (lysogenic for 80α), JP7904 (lysogenic for ϕ29) and JP2348 (lysogenic for ϕ55), containing each of the indicated plasmids, were SOS induced and assayed for β-lactamase activity under standard conditions. (B) Scheme of the chimeric structures generated to analyse the role of the different regions contained in the terS promoter regions form three different phages (80α, ϕ29 and ϕ55). (C) Derivatives of strains RN10359 (lysogenic for 80α) and JP2348 (lysogenic for ϕ55) containing each of the indicated plasmids were SOS induced and assayed for β-lactamase activity under standard conditions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753634&req=5

gkt508-F7: DNA requirements for Ltr binding. (A) Derivatives of strains RN10359 (lysogenic for 80α), JP7904 (lysogenic for ϕ29) and JP2348 (lysogenic for ϕ55), containing each of the indicated plasmids, were SOS induced and assayed for β-lactamase activity under standard conditions. (B) Scheme of the chimeric structures generated to analyse the role of the different regions contained in the terS promoter regions form three different phages (80α, ϕ29 and ϕ55). (C) Derivatives of strains RN10359 (lysogenic for 80α) and JP2348 (lysogenic for ϕ55) containing each of the indicated plasmids were SOS induced and assayed for β-lactamase activity under standard conditions.
Mentions: The results described earlier in the text do not determine whether the upstream DNA repeats are sufficient to support Ltr binding. To address this question, we initially compared activation of gene expression using plasmids pJP1207, pJP1214 and pJP1221. These pCN41 derivative plasmids contain the terS promoter region (including the repeats) and the 5′ coding region of the terS gene from phages 80α, ϕ29 and ϕ55, respectively. Phages 80α and ϕ29 encode a RinA protein, and as shown in Supplementary Figure S3, the putative binding regions for the RinA proteins (which we have defined as a block of 59 nt just distal to rinA, including the direct repeats) are basically identical in these two phages. In contrast, the DNA just downstream of this block of nucleotides, including the intergenic region and the terS gene, is completely different. Phage ϕ55 encodes LtrC, and as shown in Supplementary Figure S3, has a repeat-containing region different from that present in phages 80α and ϕ29. However, as indicated previously, the cloned intergenic region from phage ϕ55 is basically identical to that present in ϕ29 and completely different from that of phage 80α (Supplementary Figure S3). Plasmids pJP1207 (80α), pJP1214 (ϕ29) and pJP1221 (ϕ55) were introduced into lysogenic strains RN10359 (80α), JP7904 (ϕ29) and JP2348 (ϕ55), the resident prophages were SOS induced and the β-lactamase reporter was measured. As shown in Figure 7A, the β-lactamase reporter was uniquely expressed in those strains carrying cognate pair phage–plasmid with the DNA repeat region corresponding to the resident prophage, confirming the specificity of the Ltr proteins for their corresponding repeats. Although the RinA-expressing phages 80α and ϕ29 cross-reacted, expression of the reporter gene was higher in those strains containing the specific phage and its cognate cloned DNA sequence. An explanation for this is given later in the text.Figure 7.

Bottom Line: These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages.To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription.This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biomedicina de Valencia (IBV-CSIC), 46010 Valencia, Spain, Centro de Investigación y Tecnología Animal, Instituto Valenciano de Investigaciones Agrarias (CITA-IVIA), Apdo. 187, 12.400 Segorbe, Castellón, Spain, Departamento de Ciencias Biomédicas, Universidad CEU Cardenal Herrera, 46113 Moncada, Valencia, Spain, Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain, Instituto de Agrobiotecnología, CSIC-Universidad Pública de Navarra, 31006 Pamplona, Navarra, Spain, Laboratorio de Bacterias Lacticas y Probioticos, Instituto de Agroquimica y Tecnologia de Alimentos-CSIC, 46980 Paterna, Valencia, Spain, Skirball Institute Program in Molecular Pathogenesis and Departments of Microbiology and Medicine, New York University Medical Center, 540 First Avenue, New York, NY 10016, USA, Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298-0678, USA and Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, G12 8TA Glasgow, UK.

ABSTRACT
The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria.

Show MeSH
Related in: MedlinePlus