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A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

Donovan C, Heyer A, Pfeifer E, Polen T, Wittmann A, Krämer R, Frunzke J, Bramkamp M - Nucleic Acids Res. (2015)

Bottom Line: Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction.Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication.Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology I, Ludwig-Maximilians-University Munich, Großhaderner Str. 2-4, 82152 Planegg-Martinsried, Germany Institute for Biochemistry, University of Cologne, Zülpicherstr. 47, 50674 Cologne, Germany.

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AlpC assembles into filaments in C. glutamicum. (A) eCFP-AlpC (CDC020) assembles into short, straight filaments when expressed at physiological concentration. Cells were grown in CGXII containing 4% glucose. Excision of the CGP3 prophage was induced with 0.6 μg/ml mitomycin C for 3 h prior to microscopic analysis. (B) AlpC-CFP (CDC021) assembles into long, curved filaments when overproduced in C. glutamicum, red arrowhead. Cells were grown in BHI and induced by addition of 0.5 mM IPTG 1 h prior to microscopic analysis. Mutation of the conserved aspartic acid of the phosphate 2 motif of AlpC reduces filament assembly (AlpCD301A-CFP, CDC022) (lower panel). (C) Z-stack analysis of the subcellular distribution of AlpC-CFP (CDC021) filaments. Cells overproducing AlpC-CFP contain numerous filaments of various orientation, curvature and length (Movie S1). (D) AlpC-CFP filaments are dynamic. Images were acquired at 5 s intervals (top left) for 1 min (Movie S2). The red line denotes the position in the cell used to generate the kymograph (right). (E) AlpCD301A-CFP filaments are static. Images were acquired at 5 s intervals (top left) for 100 s (Movie S3). As in (C), a kymograph is shown on the right. Scale bar, 2 μm
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Figure 3: AlpC assembles into filaments in C. glutamicum. (A) eCFP-AlpC (CDC020) assembles into short, straight filaments when expressed at physiological concentration. Cells were grown in CGXII containing 4% glucose. Excision of the CGP3 prophage was induced with 0.6 μg/ml mitomycin C for 3 h prior to microscopic analysis. (B) AlpC-CFP (CDC021) assembles into long, curved filaments when overproduced in C. glutamicum, red arrowhead. Cells were grown in BHI and induced by addition of 0.5 mM IPTG 1 h prior to microscopic analysis. Mutation of the conserved aspartic acid of the phosphate 2 motif of AlpC reduces filament assembly (AlpCD301A-CFP, CDC022) (lower panel). (C) Z-stack analysis of the subcellular distribution of AlpC-CFP (CDC021) filaments. Cells overproducing AlpC-CFP contain numerous filaments of various orientation, curvature and length (Movie S1). (D) AlpC-CFP filaments are dynamic. Images were acquired at 5 s intervals (top left) for 1 min (Movie S2). The red line denotes the position in the cell used to generate the kymograph (right). (E) AlpCD301A-CFP filaments are static. Images were acquired at 5 s intervals (top left) for 100 s (Movie S3). As in (C), a kymograph is shown on the right. Scale bar, 2 μm

Mentions: In order to study the behavior of AlpC at physiological concentration, the native alpC locus was replaced with an ecfp-alpC allele (CDC020, Supplementary Table S1). Under conditions of prophage induction using mitomycin C, eCFP-AlpC (CDC020) readily assembled into filaments (Figure 3A). AlpC filaments or foci were observed in the vast majority of cells. The filaments were varying in length, straight and mostly found pointing to the cell membrane at different angles. These results suggest that AlpC expression and filament formation occurs in response to induction of the CGP3 prophage.


A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

Donovan C, Heyer A, Pfeifer E, Polen T, Wittmann A, Krämer R, Frunzke J, Bramkamp M - Nucleic Acids Res. (2015)

AlpC assembles into filaments in C. glutamicum. (A) eCFP-AlpC (CDC020) assembles into short, straight filaments when expressed at physiological concentration. Cells were grown in CGXII containing 4% glucose. Excision of the CGP3 prophage was induced with 0.6 μg/ml mitomycin C for 3 h prior to microscopic analysis. (B) AlpC-CFP (CDC021) assembles into long, curved filaments when overproduced in C. glutamicum, red arrowhead. Cells were grown in BHI and induced by addition of 0.5 mM IPTG 1 h prior to microscopic analysis. Mutation of the conserved aspartic acid of the phosphate 2 motif of AlpC reduces filament assembly (AlpCD301A-CFP, CDC022) (lower panel). (C) Z-stack analysis of the subcellular distribution of AlpC-CFP (CDC021) filaments. Cells overproducing AlpC-CFP contain numerous filaments of various orientation, curvature and length (Movie S1). (D) AlpC-CFP filaments are dynamic. Images were acquired at 5 s intervals (top left) for 1 min (Movie S2). The red line denotes the position in the cell used to generate the kymograph (right). (E) AlpCD301A-CFP filaments are static. Images were acquired at 5 s intervals (top left) for 100 s (Movie S3). As in (C), a kymograph is shown on the right. Scale bar, 2 μm
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Figure 3: AlpC assembles into filaments in C. glutamicum. (A) eCFP-AlpC (CDC020) assembles into short, straight filaments when expressed at physiological concentration. Cells were grown in CGXII containing 4% glucose. Excision of the CGP3 prophage was induced with 0.6 μg/ml mitomycin C for 3 h prior to microscopic analysis. (B) AlpC-CFP (CDC021) assembles into long, curved filaments when overproduced in C. glutamicum, red arrowhead. Cells were grown in BHI and induced by addition of 0.5 mM IPTG 1 h prior to microscopic analysis. Mutation of the conserved aspartic acid of the phosphate 2 motif of AlpC reduces filament assembly (AlpCD301A-CFP, CDC022) (lower panel). (C) Z-stack analysis of the subcellular distribution of AlpC-CFP (CDC021) filaments. Cells overproducing AlpC-CFP contain numerous filaments of various orientation, curvature and length (Movie S1). (D) AlpC-CFP filaments are dynamic. Images were acquired at 5 s intervals (top left) for 1 min (Movie S2). The red line denotes the position in the cell used to generate the kymograph (right). (E) AlpCD301A-CFP filaments are static. Images were acquired at 5 s intervals (top left) for 100 s (Movie S3). As in (C), a kymograph is shown on the right. Scale bar, 2 μm
Mentions: In order to study the behavior of AlpC at physiological concentration, the native alpC locus was replaced with an ecfp-alpC allele (CDC020, Supplementary Table S1). Under conditions of prophage induction using mitomycin C, eCFP-AlpC (CDC020) readily assembled into filaments (Figure 3A). AlpC filaments or foci were observed in the vast majority of cells. The filaments were varying in length, straight and mostly found pointing to the cell membrane at different angles. These results suggest that AlpC expression and filament formation occurs in response to induction of the CGP3 prophage.

Bottom Line: Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction.Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication.Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology I, Ludwig-Maximilians-University Munich, Großhaderner Str. 2-4, 82152 Planegg-Martinsried, Germany Institute for Biochemistry, University of Cologne, Zülpicherstr. 47, 50674 Cologne, Germany.

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Related in: MedlinePlus