Whole genome sequencing of phage resistant Bacillus anthracismutants reveals an essential role for cell surface anchoring protein CsaB in phage AP50c adsorption

Kimberly A Bishop-Lilly,Shanmuga Sozhamannan,Amy Butani,Truong Luu,Alfred J Mateczun,Vishwesh Mokashi,Peter E Chen,Roger D Plaut,Carol Chapman,Shakia Dorsey,Timothy D Read,Kristin M Willner,Scott Stibitz,Richard Calendar,Matroner George,Arya Akmal

doi:10.1186/1743-422x-9-246

Kimberly A Bishop-Lilly, Shanmuga Sozhamannan + Show 14 more

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https://doi.org/10.1186/1743-422x-9-246

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Abstract

BackgroundSpontaneous Bacillus anthracis mutants resistant to infection by phage AP50c (AP50R) exhibit a mucoid colony phenotype and secrete an extracellular matrix.MethodsHere we utilized a Roche/454-based whole genome sequencing approach to identify mutations that are candidates for conferring AP50c phage resistance, followed by genetic deletion and complementation studies to validate the whole genome sequence data and demonstrate that the implicated gene is necessary for AP50c phage infection.ResultsUsing whole genome sequence data, we mapped the relevant mutations in six AP50R strains to csaB. Eleven additional spontaneous mutants, isolated in two different genetic backgrounds, were screened by PCR followed by Sanger sequencing of the csaB gene. In each spontaneous mutant, we found either a non-synonymous substitution, a nonsense mutation, or a frame-shift mutation caused by single nucleotide polymorphisms or a 5 base pair insertion in csaB. All together, 5 and 12 of the 17 spontaneous mutations are predicted to yield altered full length and truncated CsaB proteins respectively. As expected from these results, a targeted deletion or frame-shift mutations introduced into csaB in a different genetic background, in a strain not exposed to AP50c, resulted in a phage resistant phenotype. Also, substitution of a highly conserved histidine residue with an alanine residue (H270A) in CsaB resulted in phage resistance, suggesting that a functional CsaB is necessary for phage sensitivity. Conversely, introduction of the wild type allele of csaB in cis into the csaB deletion mutant by homologous recombination or supplying the wild type CsaB protein in trans from a plasmid restored phage sensitivity. The csaB mutants accumulated cell wall material and appeared to have a defective S-layer, whereas these phenotypes were reverted in the complemented strains.ConclusionsTaken together, these data suggest an essential role for csaB in AP50c phage infection, most likely in phage adsorption. (The whole genome sequences generated from this study have been submitted to GenBank under SRA project ID: SRA023659.1 and sample IDs: AP50 R1: SRS113675.1, AP50 R2: SRS113676.1, AP50 R3: SRS113728.1, AP50 R4: SRS113733.1, AP50 R6: SRS113734.1, JB220 Parent: SRS150209.1, JB220 Mutant: SRS150211.1).

Highlights

Spontaneous Bacillus anthracis mutants resistant to infection by phage AP50c (AP50R) exhibit a mucoid colony phenotype and secrete an extracellular matrix
We reasoned that the causal variation of phage resistance might occur in the same gene in all five mutants (S-R1, S-R2, S-R3, S-R4, and S-R6), assuming that there is a single pathway to resistance
We engineered the gene to Transmission electron microscopic analyses of csaB mutants and complemented strains In an earlier study we found that the spontaneous AP50R mutants produced an extracellular matrix and that AP50c phage failed to attach to the mutant bacterial surface [23]

Summary

Introduction

Spontaneous Bacillus anthracis mutants resistant to infection by phage AP50c (AP50R) exhibit a mucoid colony phenotype and secrete an extracellular matrix. The evolutionary arms race between bacteria and their phages, giving rise to resistance and counter resistance mechanisms, shapes the bacterial and phage population size and composition as well as the evolutionary success of these two entities [1,2]. The prospect of biothreats using B. anthracis and the possibility of naturally emergent or deliberately created antibiotic resistant B. anthracis call for highly integrated and enhanced technological platforms for diagnosis and detection of this organism. This need is best illustrated in the case of a bacterial bioterror attack, where timely detection and intervention with countermeasures such as antibiotic therapy are paramount in preventing fatal consequences. Bacteriophages have been and still remain useful tools for bacterial species and strain differentiation [3,4,5,6], evidence of successful application of phage therapy is still sparse in Western medicine [7]

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