, 2005). The lack of all three LCP proteins may deplete the cells of envelope structures required to correctly localize autolytic enzymes or PBPs, functions partially attributed to wall teichoic

acids (Atilano et EPZ-6438 concentration al., 2010; Schlag et al., 2010). The determination of the cellular localization of the LCP proteins may shed more light on their involvement in autolysis. Further characterization of the cell physiology and envelope structure/composition of all mutants could further define the individual functions of the LCP proteins. The severely defective growth phenotype and marked temperature sensitivity of the triple mutant could be rescued to different degrees by any one of the three proteins, with MsrR being the most efficient at restoring cell separation and decreasing temperature PI3K phosphorylation sensitivity. Partial restoration

of growth rate and improved cell division in SA0908- and SA2103-complemented triple mutants suggested that while cells cannot grow optimally in the absence of MsrR, cell division is considerably enhanced in the presence of at least one of the three proteins. The reintroduction of any LCP protein also restored biofilm formation of the triple mutant and reduced its sedimentation rate. Almost all phenotypes were, however, complemented to markedly different degrees by the three proteins, once again indicating that while there might be some functional overlap between these proteins, they appear to play distinct roles. Although the proteins are not completely redundant, they appear to be able to substitute for one another to varying degrees, which could ensure against

complete loss of function and allow S. aureus greater flexibility in maintaining cell division. Functional diversification among these proteins could be linked to their sequence-based phylogenetic grouping (Hubscher et al., 2008); however, currently, there are not enough data on specific members of this protein family to support this hypothesis. In S. pneumoniae, the deletion of LytR was thought to only be viable because of a suppressor mutation(s) (Johnsborg & Havarstein, 2009). A similar compensatory mutation may have occurred in S. aureus to facilitate viability of the triple mutant, which could explain why phenotypes of triple mutants complemented with individual LCP proteins Cytidine deaminase did not exactly match the genotypically equivalent double mutants; for example, the triple mutant complemented with SA0908 did not have a phenotype identical to the MsrR/SA2103 double mutant. Phenotypic differences could also be due to the altered copy number of genes expressed from multicopy plasmids. Upregulation of these proteins, as part of the S. aureus cell wall stress stimulon, suggests that they either help to protect cells against cell wall damage or help to maintain cell division in the presence of cell wall-active antibiotics.