Table 1.
Comparison of the putative open reading frames (ORFs) of ϕAB6 with ϕAB1.
Fig 1.
Sequence alignment of tail fibers of A. baumannii phages.
(A) Amino acid sequence alignment of the phage tail fibers encoded by ϕAB1 ORF41 and ϕAB6 ORF40, respectively. (B) Predicted domain structure of ϕAB6 ORF40. The numbers on the top indicate size in amino acid residues. The domains are identified as follow: T7 tail (aa 10–112), T7 phage tail domain; PLN03010 (aa 218–274), the PLN03010 domain of polygalacturonase; and PL 3 (aa 226–419) pectate lyase 3 superfamily.
Fig 2.
Immunogold-labeling electron microscopy of ϕAB6 phage particles.
The primary antibody targeting ϕAB6 ORF40 is described in Materials and Methods. The secondary antibody was conjugated to 6-nm diameter gold particles (black dots). Scale bar, 100 nm.
Fig 3.
Translucent halo formation by phage ϕAB6.
Clear plaques surrounded by translucent halos were observed in the plaque assay of phage ϕAB6 with A. baumannii strain 54149 as the indicator host.
Fig 4.
Overexpression and depolymerization activity test of ORF40ϕAB6 and ORF41ϕAB1 proteins.
(A) SDS-PAGE of the recombinant proteins overexpressed. Lanes: M, protein markers; 1 and 4, BL21(DE3) harboring an empty plasmid; 2, uninduced BL21(DE3)/pET-orf40ϕAB6; 3, BL21(DE3)/pET-orf40ϕAB6 induced with 0.1 mM IPTG; 5, uninduced BL21(DE3)/pET-orf41ϕAB1; 6, BL21(DE3)/pET-orf40ϕAB6 induced with 0.1 mM IPTG. The arrowheads indicate the overexpressed recombinant proteins. (B) Enzyme activity of purified proteins was tested by spot test. Halo surrounding stands for positive activity.
Fig 5.
Confirmation of chimeric phage ϕAB1tf6 by PCR.
(A) Maps of orf41ϕAB1/orf40ϕAB6 and the surrounding genes. (B) The PCR reactions were performed on the genomes of ϕAB1, ϕAB6, and ϕAB1tf6 as the templates with pf1 and pr3 primers and the amplicons were subjected to separation in 0.8% agarose gel.
Fig 6.
Confirmation of chimeric phage ϕAB1tf6 by RFLP.
(A) The SphI-restriction map shows orf41ϕAB1/orf40ϕAB6 and surrounding genes. (B) RFLP was performed on genomic DNA of ϕAB1, ϕAB6, and ϕAB1tf6 with SphI digestion. The digests were subjected to separation in 0.8% agarose gel.
Fig 7.
Spot assay for testing the infection ability of ϕAB1, ϕAB6, and ϕAB1tf6 for A. baumannii strains 54149 and M68316.
Fig 8.
Adsorption assay for testing the specificity of ϕAB1, ϕAB6, and chimeric phage ϕAB1tf6, as described in Materials and Methods, in binding to A. baumannii strains M68316 and 54149.