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Identification and molecular characterization of bacteriophage phiAxp-2 of Achromobacter xylosoxidans

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ABSTRACT

A novel Achromobacter xylosoxidans bacteriophage, phiAxp-2, was isolated from hospital sewage in China. The phage was morphologically and microbiologically characterized, and its one-step growth curve, host range, genomic sequence, and receptor were determined. Its morphology showed that phiAxp-2 belongs to the family Siphoviridae. Microbiological characterization demonstrated that pH 7 is most suitable for phage phiAxp-2; its titer decreased when the temperature exceeded 50 °C; phiAxp-2 is sensitive to ethanol and isopropanol; and the presence of calcium and magnesium ions is necessary to accelerate cell lysis and improve the formation of phiAxp-2 plaques. Genomic sequencing and a bioinformatic analysis showed that phage phiAxp-2 is a novel bacteriophage, consisting of a circular, double-stranded 62,220-bp DNA molecule with a GC content of 60.11% that encodes 86 putative open reading frames (ORFs). The lipopolysaccharide of A. xylosoxidans is involved in the adsorption of phiAxp-2.

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Effects of different treatments applied to the host bacterium on phiAxp-2 adsorption, which is shown as residual pfu percentages.(a) Effect of proteinase K treatment on the adsorption of phiAxp-2 to A. xylosoxidans strain A22732. (b) Effect of periodate treatment on the adsorption of phiAxp-2 to A. xylosoxidans strain A22732. The controls (LB and “A22732+ acetate”), untreated (A22732), and treated groups (“A22732+ ProtK”, treated with proteinase K; “A22732+ IO4−”, treated with periodate) were tested for adsorption, as indicated on the x axes. Error bars denote statistical variations. Significance was determined with one-sample Student’s t test when the treated and untreated groups were compared. *P < 0.05. (c) Inactivation of phiAxp-2 by LPS derived from A. xylosoxidans A22732. Percentage infectivity was determined after incubation for 1 h at 37 °C. Error bars denote statistical variations.
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f7: Effects of different treatments applied to the host bacterium on phiAxp-2 adsorption, which is shown as residual pfu percentages.(a) Effect of proteinase K treatment on the adsorption of phiAxp-2 to A. xylosoxidans strain A22732. (b) Effect of periodate treatment on the adsorption of phiAxp-2 to A. xylosoxidans strain A22732. The controls (LB and “A22732+ acetate”), untreated (A22732), and treated groups (“A22732+ ProtK”, treated with proteinase K; “A22732+ IO4−”, treated with periodate) were tested for adsorption, as indicated on the x axes. Error bars denote statistical variations. Significance was determined with one-sample Student’s t test when the treated and untreated groups were compared. *P < 0.05. (c) Inactivation of phiAxp-2 by LPS derived from A. xylosoxidans A22732. Percentage infectivity was determined after incubation for 1 h at 37 °C. Error bars denote statistical variations.

Mentions: The adsorption of the phage to the bacterial surface is the first and most important step in the phage infection process. Both the lipopolysaccharide (LPS) and outer membrane proteins located on the surfaces of Gram-negative bacteria can be used as phage receptors. In the present study, protease K and periodate were used to destroy the A. xylosoxidans outer membrane proteins and LPS, respectively, to determine the attachment site for phage phiAxp-2 on the cell surface of A. xylosoxidans (Fig. 7a,b). Phage adsorption to LPS-deficient A. xylosoxidans cells was inhibited, indicating that phage-specific adhesion is mediated by LPS (Fig. 7b). These results were confirmed with a phage inactivation assay performed with pure LPS isolated from strain A22732. These experiments showed a direct correlation between the LPS concentration and the inhibition of viral particle infectivity (Fig. 7c), and approximately 12.5 μg/ml LPS inhibited the activity of 50% of 2.8 × 103 pfu phiAxp-2. LPS of E. coli 0111:B4 was used as the negative control and showed no phage-inactivating capacity compared with A. xylosoxidans LPS, indicating that A. xylosoxidans LPS is the specific receptor for phage phiAxp-2.


Identification and molecular characterization of bacteriophage phiAxp-2 of Achromobacter xylosoxidans
Effects of different treatments applied to the host bacterium on phiAxp-2 adsorption, which is shown as residual pfu percentages.(a) Effect of proteinase K treatment on the adsorption of phiAxp-2 to A. xylosoxidans strain A22732. (b) Effect of periodate treatment on the adsorption of phiAxp-2 to A. xylosoxidans strain A22732. The controls (LB and “A22732+ acetate”), untreated (A22732), and treated groups (“A22732+ ProtK”, treated with proteinase K; “A22732+ IO4−”, treated with periodate) were tested for adsorption, as indicated on the x axes. Error bars denote statistical variations. Significance was determined with one-sample Student’s t test when the treated and untreated groups were compared. *P < 0.05. (c) Inactivation of phiAxp-2 by LPS derived from A. xylosoxidans A22732. Percentage infectivity was determined after incubation for 1 h at 37 °C. Error bars denote statistical variations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f7: Effects of different treatments applied to the host bacterium on phiAxp-2 adsorption, which is shown as residual pfu percentages.(a) Effect of proteinase K treatment on the adsorption of phiAxp-2 to A. xylosoxidans strain A22732. (b) Effect of periodate treatment on the adsorption of phiAxp-2 to A. xylosoxidans strain A22732. The controls (LB and “A22732+ acetate”), untreated (A22732), and treated groups (“A22732+ ProtK”, treated with proteinase K; “A22732+ IO4−”, treated with periodate) were tested for adsorption, as indicated on the x axes. Error bars denote statistical variations. Significance was determined with one-sample Student’s t test when the treated and untreated groups were compared. *P < 0.05. (c) Inactivation of phiAxp-2 by LPS derived from A. xylosoxidans A22732. Percentage infectivity was determined after incubation for 1 h at 37 °C. Error bars denote statistical variations.
Mentions: The adsorption of the phage to the bacterial surface is the first and most important step in the phage infection process. Both the lipopolysaccharide (LPS) and outer membrane proteins located on the surfaces of Gram-negative bacteria can be used as phage receptors. In the present study, protease K and periodate were used to destroy the A. xylosoxidans outer membrane proteins and LPS, respectively, to determine the attachment site for phage phiAxp-2 on the cell surface of A. xylosoxidans (Fig. 7a,b). Phage adsorption to LPS-deficient A. xylosoxidans cells was inhibited, indicating that phage-specific adhesion is mediated by LPS (Fig. 7b). These results were confirmed with a phage inactivation assay performed with pure LPS isolated from strain A22732. These experiments showed a direct correlation between the LPS concentration and the inhibition of viral particle infectivity (Fig. 7c), and approximately 12.5 μg/ml LPS inhibited the activity of 50% of 2.8 × 103 pfu phiAxp-2. LPS of E. coli 0111:B4 was used as the negative control and showed no phage-inactivating capacity compared with A. xylosoxidans LPS, indicating that A. xylosoxidans LPS is the specific receptor for phage phiAxp-2.

View Article: PubMed Central - PubMed

ABSTRACT

A novel Achromobacter xylosoxidans bacteriophage, phiAxp-2, was isolated from hospital sewage in China. The phage was morphologically and microbiologically characterized, and its one-step growth curve, host range, genomic sequence, and receptor were determined. Its morphology showed that phiAxp-2 belongs to the family Siphoviridae. Microbiological characterization demonstrated that pH 7 is most suitable for phage phiAxp-2; its titer decreased when the temperature exceeded 50&thinsp;&deg;C; phiAxp-2 is sensitive to ethanol and isopropanol; and the presence of calcium and magnesium ions is necessary to accelerate cell lysis and improve the formation of phiAxp-2 plaques. Genomic sequencing and a bioinformatic analysis showed that phage phiAxp-2 is a novel bacteriophage, consisting of a circular, double-stranded 62,220-bp DNA molecule with a GC content of 60.11% that encodes 86 putative open reading frames (ORFs). The lipopolysaccharide of A. xylosoxidans is involved in the adsorption of phiAxp-2.

No MeSH data available.


Related in: MedlinePlus