## 2 Results and Discussion
### A Sortase A Mutant Has a Lower MIC against SK-03-92 Than Wild-Type  
Since the putative MW2284/MW2285 TCS appears to repress transcription of the _srtA_ gene, this regulatory effect could be tied to the mechanism of action of the SK-03-92 drug. Sortase A was first described in _S. aureus_ in 1999 [64]. The protein covalently anchors surface proteins (e.g., fibronectin-binding protein, fibrinogen-binding protein, protein A, clumping factors, collagen adhesion protein) to the cell wall of _S. aureus_ and other Gram-positive bacteria [77]. An LPXTG motif [78; 79; 80] is common among these anchored proteins and many are important for phase I of biofilm formation that allows attachment to biotic or abiotic surfaces [81]. A mutation of the _srtA_ gene caused less expression of several cell wall anchored surface proteins [82; 83]. Moreover, _srtA_ mutants are attenuated compared to the wild-type strain in a variety of murine models of infection [82; 84; 85]. Because _srtA_ and _MW2284/MW2285_ transcription were affected by SK-03-92 treatment, MICs were performed using the SK-03-92 lead compound on an _srtA_ mutant (NE1787), _srtB_ mutant (control, NE1363), _MW2284_ mutant (NE671), and _MW2285_ mutant (NE272) compared to the wild-type strain JE2 [38]. The _srtB_, _MW2284_, and _MW2285_ mutants had MICs that were equal to the wild-type strain (Table 3). However, the _srtA_ mutant had an MIC that was 2-fold lower than the wild-type strain. When a _Listeria monocytogenes_ mutant was tested [39], the MIC for the _srtA_ strain was 8-fold lower than the wild-type strain. A _L. monocytogenes_\(srtB\) mutant had the same MIC as the wild-type bacteria. Figure 3: The effects of SK-03-92 drug concentration on 24 h biofilm formation (OD\({}_{570}\)) for _S. aureus_ strains JE2 (black column) and MW2 (white column). All experiments represent the mean + standard deviation of at least 10 runs done in triplicate. Presumably, SK-03-92 treatment causes downregulation of the _MW2285_ gene with an effect that would be similar to a mutation in the MW2285 gene. The regulatory effect could be derepression of _strA_ transcription. Either event would create more StrA protein that in turn would allow greater extracellular presentation of proteins on the surface of _S. aureus_ cells. This result may suggest that something tethered to the cell walls by sortase A that is conserved in both species may be tied to the mechanism of action of the SK-03-92 drug, and we are exploring this possibility. Mutations in the MW2284/MW2285 Two-Component Regulatory Genes Cause an Upregulation of the strA Gene  
Since the microarray results showed significant upregulation of the _strA_ gene and downregulation of the _MW2284_ and _MW2285_ genes, we hypothesized that the MW2284 gene product, a putative transcriptional regulator protein, may be repressing the _strA_ gene. To confirm that the putative two-component regulatory system (MW2284/MW2285) may be involved in repressing the _strA_ gene, we obtained transposon mutant strains from the Nebraska Transposon Mutant Library [38] with insertion mutations in the _MW2284_ and _MW2285_ genes. A qRT-PCR analysis was then undertaken on RNA isolated from the NE272 (_MW2285_ mutation) and NE671 (_MW2284_ mutation) strains compared to the wild-type strain JE2, targeting the _strA_ gene. The results showed that mutations in both the _MW2284_ and _MW2285_ genes led to a 9.2-fold (\(p<0.005\)) and 8.1-fold (\(p<0.0008\)) upregulation of _strA_ transcription, respectively, suggesting that this putative two-component regulatory system may be repressing transcription of the _strA_ gene (Figure 4). \begin{table}
\begin{tabular}{l l c} \hline \hline
**Strain** & **Genotype** & **MIC** \\ \hline _S. aureus_ & & \\ \hline JE2 & Wild-type & \(1\)a  \\ NE272 & _MW2285_ & \(1\) \\ NE671 & _MW2284_ & \(1\) \\ NE1363 & _strB_ & \(1\) \\ NE1787 & _strA_ & \(0.5\) \\ \hline _L. monocytogenes_ & & \\ \hline EGD & Wild-type & \(1\) \\ EGD _strA_ & _strA_ & \(0.125\) \\ EGD _strB_ & _strB_ & \(1\) \\ \hline \end{tabular}
\end{table}