Northern blot analysis of sarA transcripts in Newman derivative ALC6094 and its isogenic sarA deletion mutant carrying pG164-MazF(His₆)-pEPSA5-sarA. (A) The 1.2-, 0.8-, and 0.6-kb transcripts (long arrows) represent the native sarA transcripts from three distinct sarA promoters, which, as expected, were preserved upon MazF_Sa induction. (B) The ∼0.5-kb transcript (open arrow) represents the sarA transcript as driven from the modified pEPSA5 promoter in the plasmid pG164-MazFsa-pEPSA5-sarA. This transcript was preserved in the parental strain ALC6094 and the isogenic sarA mutant. Bottom, rRNA loading control. Total cellular RNAs were extracted at the indicated time points (minutes).

Northern blot analysis of the in vitro cleavage of sarA and recA mRNAs by MazF_Sa. The sarA and recA mRNAs were transcribed from BamHI-linearized pET14b-sarA and pET14b-recA plasmids by using a T7 large-scale transcription kit (Promega) and digested with purified MazF_Sa as described in Materials and Methods. Amounts of 5 μg of sarA and recA mRNAs were digested with 15 pmol MazF_Sa at times indicated for panels A and B. (A) sarA mRNA cleavage by MazF_Sa. Lane 1, no MazF_Sa; lanes 2 to 4, digestion with MazF_Sa for 60, 90, and 120 min, respectively. (B) recA mRNA cleavage by MazF_Sa. Lane 1, no MazF_Sa; lane 2, digestion with MazF_Sa for 60 min. The cleaved fragments were detected with the ³²P-radiolabeled sarA and recA DNA probes in Northern blots as described in Materials and Methods.

Degradation of mRNAs upon ectopic overexpression of MazF_Sa. A culture of S. aureus ALC6094 harboring pG164-MazF(His₆) was induced at an OD₆₅₀ of 0.4 with IPTG (1 mM), while the noninduced culture contained no IPTG. Total cellular RNAs were extracted at the indicated time points (minutes). Northern blot analysis was carried out with specific probes as described in Materials and Methods. (A to F) Transcripts were detected with hla, sigB, sarA, recA, spa, or gyrB probes. The data represent one typical experiment out of three similar independent experiments. The 23S and 16S bands served as the internal loading controls.

Pulse-chase labeling of de novo-synthesized SarA and SigB with [³⁵S]methionine upon ectopic overexpression of MazF_Sa. A culture of S. aureus ALC6094 harboring pG164-MazF(His₆) was induced at an OD₆₅₀ of 0.4 with IPTG (1 mM), while the noninduced culture received no IPTG. New protein synthesis was then monitored by isotopic labeling with [³⁵S]methionine for 15 min under induced or noninduced conditions at indicated intervals (0 to 90 min) after IPTG induction. The cells were then lysed and immunoprecipitated with anti-SigB monoclonal antibody (A) or anti-SarA monoclonal antibody (B) as described in Materials and Methods. Equivalent amounts of cell lysate, derived from equal culture volumes, were subjected to SDS-PAGE, followed by autoradiography. The labeled unknown protein that was immunoprecipitated by anti-SigB and anti-SarA antibodies served as the internal loading control.

Decay of RNAs upon blocking of de novo RNA synthesis with rifampin. Rifampin (200 μg/ml) was added 30 min after the culture containing S. aureus ALC6094 harboring pG164-MazF(His₆) reached an OD₆₅₀ of 0.4. RNA was extracted from control samples at time zero (time when culture reached an OD₆₅₀ of 0.4) and at 15-min time points, shown in lanes 1 and 2, respectively, in panels A to D. Lanes 3 to 8 in panels A to D show the RNA extracted after the addition of rifampin for 0, 2, 4, 6, 10, and 15 min. (A to D) Transcripts detected with sarA, recA, hla, and sigB probes. 23S and 16S rRNA served as the internal loading controls.

Northwestern blot analysis of cell lysates with sarA, recA, hla, and sigB mRNA probes. A culture of S. aureus ALC6094 harboring pG164-MazF(His₆) was induced at an OD₆₅₀ of 0.4 with IPTG (1 mM), while the control culture contained no IPTG. Samples were taken at 0, 30, 60, and 90 min. Amounts of 20 μg of the total-cell lysates from indicated time points (minutes) were resolved on 16% SDS-polyacrylamide gels and transferred to a PVDF membrane as described in Materials and Methods. The blots were then denatured, renatured, and hybridized with the ³²P-labeled sarA mRNA (A), hla mRNA (B), recA mRNA (C), and sigB mRNA (D) probes. The major protein detected (band 1) is indicated on the right of each panel; the protein size markers are shown on the left.

Reduction of the CFU counts by ectopic overexpression of MazF_Sa. (A) Reduction of CFUs with S. aureus ALC6094 harboring pG164-MazF(His₆) under IPTG induction (1 mM) at an OD₆₅₀ of 0.4. To determine the CFUs, samples were withdrawn at various time points as indicated, diluted, spread on TSA plates supplemented with chloramphenicol (10 μg/ml), and incubated at 37°C overnight. The results are those from one typical experiment out of three similar independent experiments. (B) Relative cell viability. Cultures induced at an OD₆₅₀ of 0.4 with or without IPTG (1 mM) were assessed for percentage of cell viability with a Live/Dead BacLight bacterial viability kit at indicated time points, as described in Materials and Methods. The percentage of viable cells from the induced culture at each time point was further compared to that of the noninduced culture to arrive at the relative cell viability. The results represent those of three similar independent experiments. Error bars show standard deviations. (C) Western blot analysis of MazF_Sa expression after IPTG (1 mM) induction at an OD₆₅₀ of 0.4. Twenty micrograms of cell lysate proteins from each time point was resolved by SDS-PAGE, transferred to PVDF membrane, and probed with the anti-MazF_Sa antibodies as described in Materials and Methods. Lanes 1 to 3 show cell lysates prepared after induction with IPTG for 0, 30, or 60 min, respectively.