FIG. 1.

ClpX is required to relieve the negative regulatory effect of agr on spa transcription. A) Northern blot detection of the spa transcript. RNA was isolated from cells in the transition to stationary phase (OD₆₀₀ = 2.0 ± 0.1). Lane 1 (wt), 8325-4 (wild-type); lane 2 (P), 8325-4 ΔclpP; lane 3 (X), 8325-4 ΔclpX; lane 4 (A), RN6911 (Δagr); lane 5 (AP), DF2268 (ΔclpP Δagr); lane 6 (AX), DF2269 (ΔclpX Δagr); lane 7 (Sa), PC1839 (sarA); lane 8 (PSa), (ΔclpP sarA); lane 9 (Ss), KT201 (sarS); lane 10 (PSs), DF2296 (ΔclpP sarS); lane 11 (XSx), DF2297 (ΔclpX sarS); lane 12 (ASs), KT202 (Δagr sarS); lane 13 (PASs), DF2298 (ΔclpP Δagr sarS); lane 14 (XASs), DF2299 (ΔclpX Δagr sarS). The arrows indicate the migration of the 16S and 23S rRNA. B) Protein A levels measured by Western blot analysis. Cell wall proteins were extracted from an equal number of cells in late-exponential growth phase (OD₆₀₀ = 1.0 ± 0.1). Extracted proteins were analyzed by SDS-PAGE, blotted onto a polyvinylidene difluoride membrane, and probed with anti-Protein A antibody. The two gels were analyzed in parallel. Lane 1, 8325-4 (wild type); lane 2, 8325-4 ΔclpP; lane 3, 8325-4 ΔclpX; lane 4, RN6911 (Δagr); lane 5, DF2268 (ΔclpP Δagr); lane 6, DF2269 (ΔclpX Δagr); lane 7, PC1839 (sarA); lane 8, DF2271 (ΔclpP sarA); lane 9 (AR), DF2432 (Δagr Δrot); lane 10, protein size marker; lane 11 (PR), DF2332 (ΔclpP Δrot); lane 12 (XR), DF2333 (ΔclpX Δrot); lane 13, KT201 (sarS); lane 14, DF2297 (ΔclpX sarS); lane 15, KT202 (Δagr sarS); lane 16, 2299 (ΔclpX Δagr sarS).