of clones containing in-frame protein-coding fragments of BXB0048 and BA2125 and lacking gene fragments of other B. anthracis genes was detected (these protein coding regions are not similar to any alternative B. anthracis proteins); however, immuno- reactivity could not be confirmed in clones containing the complete ORFs of these gene fragments, possibly suggesting inefficient expression or display of full-length products in E. coli. Since a number of identified seroreactive ORF products were predicted to be cell wall active amidases or hydrolases, to evaluate whether immuno-reactivity was unique to a given IVIAT- identified ORF, we also evaluated immuno-reactivity of paralogs of IVIAT-identified genes BA4073 (a predicted amidase) and pXO2-08 (a possible hydrolase and our most frequently identified ORF-product), and identified eight additional gene products with immuno-reactivity (Table 2). Antigens identified by IVIAT With respect to novel B. anthracis antigens identified by IVIAT, we were most intrigued by the observation that a number of identified proteins were predicted to be involved in cell wall/ peptidoglycan synthesis or remodeling. In particular, our applica- tion of IVIAT to B. anthracis identified a number of L-alanine amidases, a family of enzymes that cleaves an amide bond between N-acetylmuramic acid and L-alanine in bacterial cell wall peptidoglycan [17]. Interestingly, L-alanine amidases are also present in a number of phages with lytic capabilities specific against B. anthracis. For instance, within the NCBI database, the highest similarity protein to L-alanine amidase BA4073 identified by IVIAT is the B. anthracis specific gamma phage lysin, PlyG, with 82% identity over the full length of both proteins, and PlyG has been demonstrated to function as a species-specific B. anthracis lysin [18]. We also identified prophage holin BA4074 by IVIAT. Holins act upon bacterial cytoplasmic membranes and, in conjunction with an endolysin, promote lysis of infected bacteria [19]. Akin to our identification of amidase BA4073 and prophage holin BA4074 by IVIAT, we similarly identified amidase BA3767, which is also in a potential operon arrangement with a prophage holin, BA3768. Using IVIAT, we also identified spore cortex lytic protein (BA2748), a protein with marked similarity to characterized B. anthracis IVIAT Bacillus cereus and Bacillus subtilis SleB proteins. The latter function as cell wall hydrolases and are required for complete spore-cortex peptidoglycan hydrolysis during germination [20–22]. BA2748 also contains a putative peptidoglycan-binding domain found in a variety of enzymes involved in bacterial cell wall degradation, and expression is induced late during sporulation in B. anthracis [23], although the enzymatic activity in B. subtilis and B. cereus occurs during germination [20,21]. We were also intrigued by our identification of BA4606 that annotates as a penicillin-binding protein. Penicillin-binding proteins are a broad group of membrane-associated macromolecules involved in peptidoglycan biosynthesis, cell wall development, and cell division. BA4606 contains an FtsI cell division protein/penicillin-binding protein 2 domain, and the FtsI penicillin-binding protein family is required for septal peptidoglycan formation during cell division [24]. BA4606 is also 45% identical to B. subtilis hypothetical protein YrrR (also referred to as PBP4b, pbpI) over the full length of both proteins. B. subtilis YrrR is a mother-cell specific penicillin-binding protein expressed during sporulation [25]. The FtsI domain contains a penicillin-binding protein transpeptidase domain, which putatively cross-links peptidoglycan chains to form a rigid cell wall structure. We also identified four hypothetical proteins. The most frequently identified gene product identified by IVIAT was ORF pXO2-08. pXO2-08 is predicted to be an extracellular protein [26], and contains a P60 family lipoprotein domain. pXO2-08 also has sequence similarity to a murein hydrolase domain from Listeria monocytogenes P60 protein (alternatively referred to as iap-invasion associated protein) [27]. Several proteins containing a P60 domain have been implicated in virulence, including two P60 domain proteins in Mycobacterium marinum involved in invasion and intracellular survival within macrophages [28], and a L. monocy- togenes mutant in P60 is severely attenuated, suggesting a potential role in virulence [29]. Although the function of the P60 lipoprotein domain is not completely understood, it is conserved through a variety of bacterial lineages, and some members of this family have been shown to act as murein hydrolases that cleave peptide linkages within peptidoglycan [30]. Immuno-screening of pXO2- 08 paralog ORF clones identified two additional B. anthracis serologically reactive paralogs in the P60 lipoprotein family: Table 2. Immunoreactive paralogs of B. anthracis gene products identified by IVIAT. Functional category Peptidoglycan architecture Peptidoglycan architecture Peptidoglycan architecture Peptidoglycan architecture Peptidoglycan architecture Peptidoglycan architecture Unknown Peptidoglycan architecture ORF BA0485 BA2805 (plyPH) BA3737 BA1952 BA5427 (lytE) Gene product-description, function (reference) N-acetylmuramoyl-L-alanine amidase Extracellular, cell-associated Predicted cellular location Gene encoded by BA2446 N-acetylmuramoyl-L-alanine amidase Extracellular, cell-associated N-acetylmuramoyl-L-alanine amidase; glycosyl hydrolase [48] Extracellular, cell-associated N-acetylmuramoyl-L-alanine amidase Extracellular, cell-associated BA3767 N-acetylmuramoyl-L-alanine amidase Extracellular, cell-associated pXO2-42 N-acetylmuramoyl-L-alanine amidase [49] (amiA) NLP/P60 lipoprotein family NLP/P60 lipoprotein family Extracellular, cell-associated pXO2 BA2446, BA3737 Extracellular, cell-associated Extracellular, cell-associated Chromosome Chromosome pXO2-08 pXO2-08 Chromosome Chromosome Chromosome Chromosome Chromosome Paralog of IVIAT- identified gene product BA4073 BA4073 BA4073 BA4073 BA4073 doi:10.1371/journal.pone.0001824.t002 PLoS ONE | www.plosone.org 3 March 2008 | Volume 3 | Issue 3 | e1824