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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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The phage-encoded cI repressor controls rinA expression. (A) Transcriptional tiling map showing expression of the early (including rinA) and late genes from phage 80α. Strain RN10359, lysogenic for 80α, was SOS induced, and samples were analysed at the indicated times. (B) Schematic representation of the different blaZ transcriptional fusions obtained to analyse the control of the rinA gene. Plasmids were introduced into strain RN4220 and assayed at mid-exponential phase for β-lactamase activity under standard conditions. In addition, expression of the β-lactamase reporter was also assayed after infection with phage 80α. ND; not determined.
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gkt508-F9: The phage-encoded cI repressor controls rinA expression. (A) Transcriptional tiling map showing expression of the early (including rinA) and late genes from phage 80α. Strain RN10359, lysogenic for 80α, was SOS induced, and samples were analysed at the indicated times. (B) Schematic representation of the different blaZ transcriptional fusions obtained to analyse the control of the rinA gene. Plasmids were introduced into strain RN4220 and assayed at mid-exponential phase for β-lactamase activity under standard conditions. In addition, expression of the β-lactamase reporter was also assayed after infection with phage 80α. ND; not determined.

Mentions: To investigate further the regulation of ltr gene expression, we carried out transcriptional profiling using phage 80α as a model. We isolated RNA before or 30 and 60 min after SOS induction of strain RN10359, which is lysogenic for phage 80α, and this RNA was analysed using tiling microarrays. The tiling microarrays were designed to analyse the genomic landscape of transcription in a strand-specific manner at 14 nt resolution (see ‘Materials and Methods’ section). We expected that the tiling array format would permit us to map the position of phage 80α transcripts in a complementary way to the conventional molecular analysis. Figure 9A shows the tiling-array hybridization signals of phage 80α, using RNA obtained at different times after induction of the phage cycle. The first observation from this experiment is that phage 80α showed basal transcription of most of its genes in absence of induction. This basal transcription can be explained by the spontaneous low level of prophage induction that occurs in lysogenic cells. Second, there is a clear temporal difference between transcription of rinA, which is seen at 30 min post-induction, and the late (morphogenetic) operon, which does not increase until the 60 min time point. This is consistent with an essential role for rinA in activating late transcription. Finally, 30 min after induction of the phage lytic cycle, we observed what appears to be fairly uniform transcription on the top strand of a region encompassing the cro to the rinA genes. This region includes two predicted open reading frames (ORF11 and ORF19) that lie in the opposite orientation and would be expressed from the other strand, which is also confirmed by the tiling array. Such a distribution of mapped reads in ‘overlapping operons’ has been described previously in genome-wide profiling of S. aureus transcription (21). In that study, these overlapping reads appear to correlate with regions in which short antisense RNAs also map; therefore, it is not clear whether the apparent continuous transcription across these divergent open reading frames reflects a polycistronic mRNA that spans this region or the synthesis of an additional short transcript from the same strand that is an antisense RNA to the divergent ORF. Thus, there are two alternative interpretations of the results we obtained with 80α. One possibility is that that all the genes from cro to rinA belong to a single early operon, which is controlled by the phage-encoded cI repressor. An alternative interpretation is that there are multiple promoters, all co-temporally regulated, and that there are overlapping short RNAs leading to the appearance of uniform transcription across this region.Figure 9.


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)

The phage-encoded cI repressor controls rinA expression. (A) Transcriptional tiling map showing expression of the early (including rinA) and late genes from phage 80α. Strain RN10359, lysogenic for 80α, was SOS induced, and samples were analysed at the indicated times. (B) Schematic representation of the different blaZ transcriptional fusions obtained to analyse the control of the rinA gene. Plasmids were introduced into strain RN4220 and assayed at mid-exponential phase for β-lactamase activity under standard conditions. In addition, expression of the β-lactamase reporter was also assayed after infection with phage 80α. ND; not determined.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt508-F9: The phage-encoded cI repressor controls rinA expression. (A) Transcriptional tiling map showing expression of the early (including rinA) and late genes from phage 80α. Strain RN10359, lysogenic for 80α, was SOS induced, and samples were analysed at the indicated times. (B) Schematic representation of the different blaZ transcriptional fusions obtained to analyse the control of the rinA gene. Plasmids were introduced into strain RN4220 and assayed at mid-exponential phase for β-lactamase activity under standard conditions. In addition, expression of the β-lactamase reporter was also assayed after infection with phage 80α. ND; not determined.
Mentions: To investigate further the regulation of ltr gene expression, we carried out transcriptional profiling using phage 80α as a model. We isolated RNA before or 30 and 60 min after SOS induction of strain RN10359, which is lysogenic for phage 80α, and this RNA was analysed using tiling microarrays. The tiling microarrays were designed to analyse the genomic landscape of transcription in a strand-specific manner at 14 nt resolution (see ‘Materials and Methods’ section). We expected that the tiling array format would permit us to map the position of phage 80α transcripts in a complementary way to the conventional molecular analysis. Figure 9A shows the tiling-array hybridization signals of phage 80α, using RNA obtained at different times after induction of the phage cycle. The first observation from this experiment is that phage 80α showed basal transcription of most of its genes in absence of induction. This basal transcription can be explained by the spontaneous low level of prophage induction that occurs in lysogenic cells. Second, there is a clear temporal difference between transcription of rinA, which is seen at 30 min post-induction, and the late (morphogenetic) operon, which does not increase until the 60 min time point. This is consistent with an essential role for rinA in activating late transcription. Finally, 30 min after induction of the phage lytic cycle, we observed what appears to be fairly uniform transcription on the top strand of a region encompassing the cro to the rinA genes. This region includes two predicted open reading frames (ORF11 and ORF19) that lie in the opposite orientation and would be expressed from the other strand, which is also confirmed by the tiling array. Such a distribution of mapped reads in ‘overlapping operons’ has been described previously in genome-wide profiling of S. aureus transcription (21). In that study, these overlapping reads appear to correlate with regions in which short antisense RNAs also map; therefore, it is not clear whether the apparent continuous transcription across these divergent open reading frames reflects a polycistronic mRNA that spans this region or the synthesis of an additional short transcript from the same strand that is an antisense RNA to the divergent ORF. Thus, there are two alternative interpretations of the results we obtained with 80α. One possibility is that that all the genes from cro to rinA belong to a single early operon, which is controlled by the phage-encoded cI repressor. An alternative interpretation is that there are multiple promoters, all co-temporally regulated, and that there are overlapping short RNAs leading to the appearance of uniform transcription across this region.Figure 9.

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