bWe previously identified the protein Tet38 as a chromosomally encoded efflux pump of Staphylococcus aureus that confers resistance to tetracycline and certain unsaturated fatty acids. Tet38 also contributes to mouse skin colonization. In this study, we discovered a novel regulator of tet38, named tetracycline regulator 21 (TetR21), that bound specifically to the tet38 promoter and repressed pump expression. A ⌬tetR21 mutant showed a 5-fold increase in tet38 transcripts and an 8-fold increase in resistance to tetracycline and fatty acids. The global regulator MgrA bound to the tetR21 promoter and indirectly repressed the expression of tet38. To further assess the full role of Tet38 in S. aureus adaptability, we tested its effect on host cell invasion using A549 (lung) and HMEC-1 (heart) cell lines. We used S. aureus RN6390, its ⌬tet38, ⌬tetR21, and ⌬mgrA mutants, and a ⌬tet38 ⌬tetR21 double mutant. After 2 h of contact, the ⌬tet38 mutant was internalized in 6-fold-lower numbers than RN6390 in A549 and HMEC-1 cells, and the ⌬tetR21 mutant was internalized in 2-fold-higher numbers than RN6390. A slight increase of 1.5-fold in internalization was found for the ⌬mgrA mutant. The growth patterns of RN6390 and the ⌬mgrA and ⌬tetR21 mutants within A549 cells were similar, while no growth was observed for the ⌬tet38 mutant. These data indicate that the Tet38 efflux pump is regulated by TetR21 and contributes to the ability of S. aureus to internalize and replicate within epithelial cells.

The ATP-dependent transporter gene abcA in Staphylococcus aureus confers resistance to hydrophobic ␤-lactams. In strain ISP794, abcA is regulated by the transcriptional regulators MgrA and NorG and shares a 420-nucleotide intercistronic region with the divergently transcribed pbp4 gene, which encodes the transpeptidase Pbp4. Exposure of exponentially growing cells to iron-limited media, oxidative stress, and acidic pH (5.5) for 0.5 to 2 h had no effect on abcA expression. In contrast, nutrient limitation produced a significant increase in abcA transcripts. We identified three additional regulators (SarA, SarZ, and Rot) that bind to the overlapping promoter region of abcA and pbp4 in strain MW2 and investigated their role in the regulation of abcA expression. Expression of abcA is decreased by 10.0-fold in vivo in a subcutaneous abscess model. In vitro, abcA expression depends on rot and sarZ regulators. Moenomycin A exposure of strain MW2 produced an increase in abcA transcripts. Relative to MW2, the MIC of moenomycin was decreased 8-fold for MW2⌬abcA and increased 10-fold for the MW2 abcA overexpresser, suggesting that moenomycin is a substrate of AbcA.