Gene 16S BA0314 BA0669 BA0698 BA2125 BA2748 BA4073 BA4074 BA4606 BA5474 pXO1-110 (pagA) pXO2-08 BXB0048 BA1952 BA5427 BA0485 BA2446 BA2805 BA3737 Table 4. Primers for RT-PCR quantification of cDNA isolated from Ames strain B. anthracis recovered from infected mice or from in vitro grown B. anthracis. Upstream primer Downstream primer TAGGTGGCAAGCGTTATC GGTGTTCCTCCATATCTCTAC TAGGGGTTTCAGCATTAAG GTGTAATCAGAGAACTCAAC TGTTCAATGGAACCACCAGA ATCAATTCAATGCCGCTTTC GCAGAAATACTCGTTGCCATT GCTAGCAGTTCCGAGAAGGA GTGGTAAAGGGGGATTCGTT GCCTGCAGCATGACTTAACA ATCGGGCGAAGATGTTATTG ACGAGCATTTGCTTCGTTTT ATTGCCCACATAGAATGC TATAATCGCCTTACCTGTC GGTGGTGCTTTTTCTTTTGG GTTAAAGCCTGTGGGAGTGG TTAATTGGCAATGTGGGTGA GCCGGTCCACTTGATACAAT GGCGAATTCGTATCTGGTGT TGAGAAGTGCTAGGGCCTGT GCATGCGTCGTTCTTTGATA CCATTGTTTCAGCCCAAGTT TGGAACTTCAGGGCAAATTC GCCGTTTTATACGCCCAATA TTTGGCTGTCAGTTTTGCTG TCACTCCCCATTTCCACATA Primers for selected paralogs/homologs Paralog/similar proteins of pXO2-08 ATCTTCAATCGCTGGATTCG TCCAGCAACTGTTTGACGAG ACAAGAAGCTGCAGCACAAA CTGGTGTCGGTTCTGGTTTT Paralog/similar proteins of BA4073 pXO2-42 (amiA) TACCAAACTGGGCACAACAA GCATCCTGAAATGACGGTTT ACAGTTTGGGTGGCTGATGT CGCCATACCGAAAGGTGTAT AAATCGTGGTGCAAAGGAAC TTTAGATGGCGGAGTTGTCC GCCGCTTTCTTGGTTTACTG AACGCTCCCATAACATCTGG GACAGGTATTGCGTATATTGAGG CCACTGATTGCCACCAA doi:10.1371/journal.pone.0001824.t004 After an initial denaturation at 95uC for 3 min, the RT-PCR cycle was as follows: denaturation at 94uC for 30 sec, extension at 58uC for 30 sec, extension at 72uC for 1 min, followed by a plate read. The cycle was repeated for a total of 40 cycles. C(t) values were set in the low/linear portion of product curves. We quantified gene copy numbers against pET30-based plasmids containing the gene of interest; we calculated control gene copy numbers using plasmid size, and spectrophotometerically determined A260 readings. We normalized gene copy numbers against copies of 16S rRNA cDNA [44]. Controls included samples lacking reverse transcriptase, and a no template control to provide a reference baseline reaction. We assessed singularity of product species and size by Metaphor gel (Cambrex Bio-Science; Rockland, ME) and melting curve analysis. References 1. Handfield M, Brady LJ, Progulske-Fox A, Hillman JD (2000) IVIAT: a novel method to identify microbial genes expressed specifically during human infections. Trends Microbiol 8: 336–339. 2. Rollins SM, Peppercorn A, Hang L, Hillman JD, Calderwood SB, et al. (2005) In vivo induced antigen technology (IVIAT). Cell Microbiol 7: 1–9. 3. Harris JB, Baresch-Bernal A, Rollins SM, Alam A, LaRocque RC, et al. (2006) Identification of in vivo-induced bacterial protein antigens during human infection with Salmonella enterica serovar Typhi. Infect Immun 74: 5161–5168. 4. Handfield M, Hillman JD (2006) In vivo induced antigen technology (IVIAT) and change mediated antigen technology (CMAT). Infect Disord Drug Targets 6: 327–334. 5. Deb DK, Dahiya P, Srivastava KK, Srivastava R, Srivastava BS (2002) Selective identification of new therapeutic targets of Mycobacterium tuberculosis by IVIAT approach. Tuberculosis (Edinb ) 82: 175–182. B. anthracis IVIAT Generation of predicted amidases BA0485 and BA2446 as histidine-tagged fusion proteins A number of antigens identified using IVIAT were predicted to be amidases that could be involved in cell wall synthesis or remodeling. We, therefore, attempted to produce these amidases as recombinant proteins in sufficient quantities to assess physio- logic effects. We were able to successfully express two such proteins. Specifically, we expressed BA0485 and BA2446 proteins containing a carboxyl-terminal hex-histidine tag from pET30- based vectors in E. coli BL21(DE3) Codon Plus-RIL induced with 1 mM IPTG. We recovered these proteins using affinity chromatography, and assessed protein concentration and purity using SDS-PAGE analysis and Coomasie Plus Reagent (Pierce, Rockford, IL). Functional activity of exogenous BA0485 and BA2446 recombinant amidase proteins on vegetative cell suspensions of Bacillus sp. and E. coli To begin to determine the effect of identified amidases on B. anthracis cell wall integrity, we incubated a 1:40 dilution in LB broth of overnight cultures of B. anthracis Sterne strain, B. cereus ATCC 14579, B. subtilis 168, and E. coli DH5a for 2–2.5 hrs in an environmental shaker at 37uC. We centrifuged cultures, resus- pended bacteria to an A595 of 0.4 in 20 mM sodium phosphate buffer (pH 4.7, 7.0, and 8.7), added purified BA0485 or BA2446 protein to a final concentration of 2 uM to cell suspensions, and measured A595 every 2 min. Control samples contained buffer lacking recombinant protein. We quantified viable bacterial CFUs using LB agar plates prior to addition of BA0485 or BA2446 protein, and then again after 20 min of incubation. We assessed cellular morphology via Gram stain and light microscopy, and calculated mean vegetative cell chain length based on ten fields of view. Biosafety All work described in this experiment involving B. anthracis strains listed in the Centers for Disease Control and Prevention Select Agent Program was performed in fully-certified BL2 and BL3 facilities, was fully approved, and met all institutional and local, state, and federal governmental regulations. Procedures that required BL3 animal work involving macaques were performed through the Centers for Disease Control and Prevention; procedures that required BL3 animal work involving mice were performed through the University of New Mexico Health Science Center, Albuquerque, NM. Author Contributions Conceived and designed the experiments: SC TK ER SR AP MD MH JH. Performed the experiments: SR AP JY MD AB MB. Analyzed the data: SC TK ER SR AP JY. Contributed reagents/materials/analysis tools: TK SR CL CQ DA. Wrote the paper: ER SR. 6. Hang L, John M, Asaduzzaman M, Bridges EA, Vanderspurt C, et al. (2003) Use of in vivo-induced antigen technology (IVIAT) to identify genes uniquely expressed during human infection with Vibrio cholerae. Proc Natl Acad Sci U S A 100: 8508–8513. 7. Kim YR, Lee SE, Kim CM, Kim SY, Shin EK, et al. (2003) Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients. Infect Immun 71: 5461–5471. 8. John M, Kudva IT, Griffin RW, Dodson AW, McManus B, et al. (2005) Use of in vivo-induced antigen technology for identification of Escherichia coli O157:H7 proteins expressed during human infection. Infect Immun 73: 2665–2679. 9. Salim KY, Cvitkovitch DG, Chang P, Bast DJ, Handfield M, et al. (2005) Identification of group A Streptococcus antigenic determinants upregulated in vivo. Infect Immun 73: 6026–6038. PLoS ONE | www.plosone.org 8 March 2008 | Volume 3 | Issue 3 | e1824