Molecular characterization of a variant of Bacillus anthracis-specific phage AP50 with improved bacteriolytic activity

Abstract

The genome sequence of a Bacillus anthracis-specific clear plaque mutant phage, AP50c, contains 31 open reading frames spanning 14,398 bp, has two mutations compared to wild-type AP50t, and has a colinear genome architecture highly similar to that of gram-positive Tectiviridae phages. Spontaneous AP50c-resistant B. anthracis mutants exhibit a mucoid colony phenotype.

FIG. 1.

Transmission electron micrograph of AP50c phage particles. (a) Uranyl acetate staining at a magnification of 297,000. Scale bar, 100 nm. (b) Plaque morphology of turbid and clear plaques in a mixed lysate.

FIG. 2.

(a) Genome map of AP50. Three gene clusters based on functional grouping and similarities to other Tectiviridae phages infecting gram-positive bacteria are shown. ORF boxes are color coded to indicate the degree of amino acid identity with proteins of other tectiviral phages. The color of the ORFs and percentage identities are as follows: green, 60 to 80%; red, 40 to 60%; purple, 15 to 40%; blue, <15%. ITR, inverted terminal repeat; HVR, highly variable region. Open arrowheads indicate the locations of the mutations in AP50c phages. (b) Visualization summary of whole-genome nucleotide alignments of gram-positive tectiviral phages. The ClustalW alignment file generated from Multifasta alignment was visualized in Base-By-Base (3). In this type of alignment, if two sequences have insertions or deletions relative to one another, the output looks different depending on which of the two sequences is used as the base sequence. White, perfect nucleotide homology; blue, single nucleotide polymorphism; red, deletions in the indicated phage; green, insertions in the indicated phage. The GenBank accession numbers for the sequences used in the alignment are as follows: Bam35c, NC_005258; pBth35646, NZ_AAJM00000000; Gil01, AJ536073; Gil16c, AY701338; AP50, EU408779; pBclin15, AE01878. (c) Sequence changes in AP50c and AP50t genomes. The mutation in the noncoding region just upstream of ORF 1 at nucleotide position 164 is indicated. The second mutation is in ORF 28 at position 12881 and changes amino acid residue 91 (an isoleucine in AP50c to a valine in AP50t).

FIG. 3.

Colony morphologies of B. anthracis Sterne strain 34F₂ after infection with AP50t and morphology of AP50c-resistant 34F₂ mutants. (a) Uninfected 34F₂ cells diluted and plated on phage assay agar plates. (b) AP50t-infected culture, diluted and plated. The smooth and round white colonies are uninfected nonlysogens (solid arrowhead), and the flat wrinked colonies (open arrowhead) are lysogens. (c) Scanning electron micrograph of a wild-type 34F₂ cell infected with AP50c. The arrowheads indicate AP50c particles attached to the outer surface of the bacterium (magnification, 60,000; Dennis Kunkel Microscopy, Inc.). (d) Scanning electron micrograph of 34F₂ AP50c^r mutant infected with AP50c showing the presence of extracellular material coating the outer cell surface and the absence of attached phage particles (magnification, 60,000; Dennis Kunkel Microscopy, Inc.).