B. anthracis IVIAT Figure 1. mRNA expression profiles during in vitro and in vivo growth of IVIAT-identified and paralog/similar B. anthracis genes. Quantitative RT-PCR was performed on RNA recovered from in vitro grown B. anthracis (Ames strain cells grown in BHI and air; late-log phase) and compared to RNA recovered from mice infected with vegetative Ames strain B. anthracis via intravenous injection. RNA from mice was isolated from spleens 18 hr post-infection. Transcript numbers on the Y-axis are normalized against 16S rRNA. Displayed are gene profiles of B. anthracis genes up- regulated in vivo compared to in vitro. In bar graphs, expression levels represent mean values; error bars represent standard deviations (SD). In summary table, expression given as mRNA transcript copies (6 1025) per copy of 16S rRNA. doi:10.1371/journal.pone.0001824.g001 to detect induction of a number of IVIAT identified genes or paralogs in vivo. Despite these limitations, our results indicate host antibody responses and increased bacterial production during infection of several proteins, including a subset of seven L-alanine amidases, five proteins containing lipoprotein domains, a prophage holin, spore cortex lytic protein SleB, and a penicillin-binding protein that may be involved in cell wall/peptidoglycan synthesis, degradation, and/or remodeling. Fisher et al have recently shown that a B. anthracis locus (dltABCD) involved in cell wall L-alanine esterification is involved in endospore remodeling and is required for full virulence of B. anthracis in vivo [40]. Our results further support prominent cell wall structural remodeling activity during anthrax, may suggest new diagnostic targets to detect B. anthracis infection, and our identification of predicted L-alanine ami- dases with B. anthracis specificity may suggest new potential alternative therapeutic approaches against this important bacterial pathogen. Materials and Methods Bacterial strains and conditions Bacillus species and strains used in these experiments are described in Table 3. E. coli BL21(DE3) and BL21(DE3) Codon PLoS ONE | www.plosone.org 5 March 2008 | Volume 3 | Issue 3 | e1824