Detailed Genomic Analysis of the Wβ and γ Phages InfectingBacillus anthracis: Implications for Evolution of Environmental Fitness and Antibiotic Resistance

Phage-mediated lysis has been an essential laboratory tool for rapidly identifyingBacillus anthracis for more than 40 years, relying on the γ phage derivative of aBacillus cereus prophage called W. The complete genomic sequences of the temperate W phage, referred to as Wβ, and its lytic variant γ were determined and found to encode 53 open reading frames each, spanning 40,864 bp and 37,373 bp, respectively. Direct comparison of the genomes showed that γ evolved through mutations at key loci controlling host recognition, lysogenic growth, and possibly host phenotypic modification. Included are a cluster of point mutations at thegp14 tail fiber locus of γ, encoding a protein that, when fused to green fluorescent protein, binds specifically toB. anthracis . A large 2,003-bp deletion was also identified at the γ lysogeny module, explaining its shift from a temperate to a lytic lifestyle. Finally, evidence of recombination was observed at a dicistronic Wβ locus, encoding putative bacterial cell surface-modifying proteins, replaced in γ with a locus, likely obtained from aB. anthracis prophage, encoding demonstrable fosfomycin resistance. Reverse transcriptase PCR analysis confirmed strong induction at the dicistronic Wβ locus and at four other phage loci inB. anthracis and/orB. cereus lysogens. In all, this study represents the first genomic and functional description of two historically important phages and is part of a broader investigation into contributions of phage to theB. anthracis life cycle. Initial findings suggest that lysogeny ofB. anthracis promotes ecological adaptation, rather than virulence, as with other gram-positive pathogens.