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D-gerH Germinant Recognition O OH HO HOCH2 N N N NH2 Adenosine (Purine nucleosides) (Aromatic Amino Acids) gerL gerK gerS gerX gerH Germination Macrophage Associated Germination Target Validation #2 dltA, is the first of four genes in the dlt operon, which controls the incorporation of D-alanine into lipoteichoic acids on the cell surface. Essential for: maintenance of normal cell shape, control of autolytic enzymes, and resistance to cationic anti-microbial peptides in Gram positive organisms. Loss of dlt operon has been shown to decrease the virulence of S. aureus and L. monocytogenes. It has many characteristics of an potential candidate for drug development targeting in B. anthracis. Figure 2. Expression analysis. Integration of pNFd13 into the targeted locus results in the B-galactosidase reporter being placed downstream of the native promoter for expression analysis. Panel A shows a typical growth curve of B. anthracis in modified G medium. Panel B shows the expression patters of dlt and gerH during this growth cycle. Panel C shows the expression patters of these loci based on microarray analysis under identical growth conditions (2). The pNFd13 analysis verifies and quantitates the microarray data. A B C gerH dltA Figure 4. Loss of dlt results in cell shape defects. Wild type (A) or the dlt disruption strain (B) were grown overnight on BHI plates and analyzed by Gram staining. Loss of dlt results in cell shape deformities ( ) and irregular cell size. Additionally, dlt – cells become autolytic during stationary phase resulting in many ‘ghost’ cells ( ) in the culture. A B Figure 5. Resistance to the Cationic Anti-microbial Peptide Nisin depends on the expression level of the dlt locus. 34F2 dltA:pNFd13 or 34F2 gerA:pNFd13 (a control strain with pNFd13 inserted at a silent locus). Cells were grown to early stationary phase then back diluted into fresh media containing the indicated concentration of IPTG and serial dilutions of Nisin.