Genetic Evidence for the Involvement of the S-Layer Protein Gene sap and the Sporulation Genes spo0A, spo0B, and spo0F in Phage AP50c Infection of Bacillus anthracis

• Type of Material: Article
• Authors: Roger D. Plaut, Food and Drug Administration; John W. Beaber, University of California, Berkeley; Jason Zemansky, University of California, Berkeley; Ajinder P. Kaur, Henry M. Jackson Foundation; Matroner George, Henry M. Jackson Foundation; Biswajit Biswas, Henry M. Jackson Foundation; Matthew Henry, Henry M. Jackson Foundation; Kimberly A. Bishop-Lilly, Henry M. Jackson Foundation; Vishwesh Mokashi, NMRC-Frederick; Ryan M. Hannah, National Biodefense Analysis and Countermeasures Center; Robert K. Pope, National Biodefense Analysis and Countermeasures Center; Timothy Read, Emory University; Scott Stibitz, Food and Drug Administration; Richard Calendar, University of California Berkeley; Shanmuga Sozhamannan, Henry M. Jackson Foundation
• Language: English
• Date: 2014-03-01
• Publisher: American Society for Microbiology
• Publication Version: Final Published Version
• Copyright Statement: © 2014, American Society for Microbiology.
• Final Published Version (URL): http://dx.doi.org/10.1128/JB.00739-13
• Title of Journal or Parent Work: Journal of Bacteriology
• ISSN: 0021-9193
• Volume: 196
• Issue: 6
• Start Page: 1143
• End Page: 1154
• Grant/Funding Information: R. K. Pope and R. M. Hannah were funded under agreement no. HSHQDC-07-C-00020 awarded by the U.S. Department of Homeland Security (DHS) for the management and operation of the National Biodefense Analysis and Countermeasures Center (NBACC), a federally funded research and development center.; This work was supported by funds from the Defense Threat Reduction Agency, U.S. Department of Defense, of the U.S. government (proposal no. 8.10084_08_NM_B;) to S. Sozhamannan.
• Keywords: Science & Technology; ESCHERICHIA-COLI; BACTERIA; WALL POLYSACCHARIDE; MOLECULAR CHARACTERIZATION; ADSORPTION; Microbiology; SUBTILIS; THURINGIENSIS; MICROBIOLOGY; CELL; PLASMID; Life Sciences & Biomedicine; CEREUS
• Research Categories: Health Sciences, Epidemiology; Biology, Microbiology; Biology, Genetics

Abstract

In order to better characterize the Bacillus anthracis typing phage AP50c, we designed a genetic screen to identify its bacterial receptor. Insertions of the transposon mariner or targeted deletions of the structural gene for the S-layer protein Sap and the sporulation genes spo0A, spo0B, and spo0F in B. anthracis Sterne resulted in phage resistance with concomitant defects in phage adsorption and infectivity. Electron microscopy of bacteria incubated with AP50c revealed phage particles associated with the surface of bacilli of the Sterne strain but not with the surfaces of Δsap, Δspo0A, Δspo0B, or Δspo0F mutants. The amount of Sap in the S layer of each of the spo0 mutant strains was substantially reduced compared to that of the parent strain, and incubation of AP50c with purified recombinant Sap led to a substantial reduction in phage activity. Phylogenetic analysis based on wholegenome sequences of B. cereus sensu lato strains revealed several closely related B. cereus and B. thuringiensis strains that carry sap genes with very high similarities to the sap gene of B. anthracis. Complementation of the Δsap mutant in trans with the wildtype B. anthracis sap or the sap gene from either of two different B. cereus strains that are sensitive to AP50c infection restored phage sensitivity, and electron microscopy confirmed attachment of phage particles to the surface of each of the complemented strains. Based on these data, we postulate that Sap is involved in AP50c infectivity, most likely acting as the phage receptor, and that the spo0 genes may regulate synthesis of Sap and/or formation of the S layer.