Identification of mutations in lipoteichoic acid (LTA) negative Staphylococcus aureus             suppressor strains that allow for growth of these strains in the absence of LTA
The lipoteichoic acid (LTA) negative Staphylococcus aureus                 RN4220ΔspaΔltaS can initially only grow under osmotically stabilizing conditions in                 medium containing salt or sucrose. When strain RN4220ΔspaΔltaS is plated on tryptic                 soya agar plates without the addition of sucrose or salt a small number of colonies                 were obtained. These colonies most likely arose from bacteria that have acquired                 compensatory mutation(s) that allow bacteria to grow in the absence of LTA. To                 analyze this further, five independently isolated suppressor colonies were passed                 four times in tryptic soya broth in order to improve growth. Subsequently genomic                 DNA was isolated from the resulting five suppressor strains 4S4, 4S5, 4N1, 4N2 and                 5S4 and used from genome sequence analysis.
Genomic S. aureus DNA was extracted using standard                     molecular biology procedures. Genome Sequences were determined using a SOLiD™ 3                     System (Applied Biosciences) using DNA fragment libraries. Fifty bp fragments                     libraries were generated by mechanical shearing, treated and coupled to beads                     using emulsion PCR and deposited on glass slides following protocols supplied by                     Applied Biosciences.