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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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Restriction fragment length polymorphism analysis of phiAxp-2 DNA.Genomic DNA from phage phiAxp-2 was digested with the enzymes indicated (HindIII) and run on an agarose gel (0.7%). The length of fragments generated by digestion of the linear genome or the circular genome was showed on the right side of the electrophoresis map.
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f3: Restriction fragment length polymorphism analysis of phiAxp-2 DNA.Genomic DNA from phage phiAxp-2 was digested with the enzymes indicated (HindIII) and run on an agarose gel (0.7%). The length of fragments generated by digestion of the linear genome or the circular genome was showed on the right side of the electrophoresis map.

Mentions: For the future application of phage phiAxp-2 to protect humans from A. xylosoxidans infections, the phage must be characterized in detail, including its genomic sequence. The genomic DNA of phiAxp-2 was extracted and purified, and its genome was completely sequenced and analyzed. Analysis of the sequence found that the restriction endonuclease HindIII had nine cutting sites in the genomic DNA. Thus, it was expected that when HindIII was used to digest the DNA, ten fragments would be generated if the DNA comprised a linear genome, but if the genome was circular, nine fragments would be generated. The HindIII digestion experiment generated nine fragments in the agarose gel (Fig. 3), revealing that the phiAxp-2 genome is a circular molecule. The complete circular double-stranded DNA genome of phage phiAxp-2 is 62,220 bp in length with a G+C content of 60.11%. This percentage is lower than those of the complete Achromobacter genomes sequenced so far (65–66% G+C content), but is higher than those of the sequenced A. xylosoxidans-specific phages, JWAlpha (KF787095) and JWDelta (KF787094), which are 54.4% and 54.2%, respectively5. Analysis of the phage phiAxp-2 genome revealed 86 putative open reading frames (ORFs). The National Center for Biotechnology Information (NCBI) database was scanned for homologues of the proteins encoded by the predicted ORFs using BlastP. Because the genome of phiAxp-2 diverges from other available phage genomes, only a limited number (33%) of protein functions could be predicted with similarity searches, highlighting the novelty of this phage. Therefore, a more detailed investigation is required to fully understand the nature of this novel phage. Twenty-eight ORFs showed some similarity in the BlastP analysis. Putative functions were assigned to 22 ORFs based on these similarities. Despite the low number of annotated ORFs, different modules can be recognized in the phiAxp-2 genome, which encodes proteins for (i) DNA replication, regulation, and modification, (ii) DNA packaging, and (iii) head and tail morphogenesis (Fig. 4). All of these functional clusters are located in ORF1–ORF34, which constitutes approximately 60% of the whole genome length. The remaining ORFs showed less or no similarity to other proteins in the NCBI database.


Identification and molecular characterization of bacteriophage phiAxp-2 of Achromobacter xylosoxidans
Restriction fragment length polymorphism analysis of phiAxp-2 DNA.Genomic DNA from phage phiAxp-2 was digested with the enzymes indicated (HindIII) and run on an agarose gel (0.7%). The length of fragments generated by digestion of the linear genome or the circular genome was showed on the right side of the electrophoresis map.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037462&req=5

f3: Restriction fragment length polymorphism analysis of phiAxp-2 DNA.Genomic DNA from phage phiAxp-2 was digested with the enzymes indicated (HindIII) and run on an agarose gel (0.7%). The length of fragments generated by digestion of the linear genome or the circular genome was showed on the right side of the electrophoresis map.
Mentions: For the future application of phage phiAxp-2 to protect humans from A. xylosoxidans infections, the phage must be characterized in detail, including its genomic sequence. The genomic DNA of phiAxp-2 was extracted and purified, and its genome was completely sequenced and analyzed. Analysis of the sequence found that the restriction endonuclease HindIII had nine cutting sites in the genomic DNA. Thus, it was expected that when HindIII was used to digest the DNA, ten fragments would be generated if the DNA comprised a linear genome, but if the genome was circular, nine fragments would be generated. The HindIII digestion experiment generated nine fragments in the agarose gel (Fig. 3), revealing that the phiAxp-2 genome is a circular molecule. The complete circular double-stranded DNA genome of phage phiAxp-2 is 62,220 bp in length with a G+C content of 60.11%. This percentage is lower than those of the complete Achromobacter genomes sequenced so far (65–66% G+C content), but is higher than those of the sequenced A. xylosoxidans-specific phages, JWAlpha (KF787095) and JWDelta (KF787094), which are 54.4% and 54.2%, respectively5. Analysis of the phage phiAxp-2 genome revealed 86 putative open reading frames (ORFs). The National Center for Biotechnology Information (NCBI) database was scanned for homologues of the proteins encoded by the predicted ORFs using BlastP. Because the genome of phiAxp-2 diverges from other available phage genomes, only a limited number (33%) of protein functions could be predicted with similarity searches, highlighting the novelty of this phage. Therefore, a more detailed investigation is required to fully understand the nature of this novel phage. Twenty-eight ORFs showed some similarity in the BlastP analysis. Putative functions were assigned to 22 ORFs based on these similarities. Despite the low number of annotated ORFs, different modules can be recognized in the phiAxp-2 genome, which encodes proteins for (i) DNA replication, regulation, and modification, (ii) DNA packaging, and (iii) head and tail morphogenesis (Fig. 4). All of these functional clusters are located in ORF1–ORF34, which constitutes approximately 60% of the whole genome length. The remaining ORFs showed less or no similarity to other proteins in the NCBI database.

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 °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.