Effects of MazF_Sa and MazE_Sa on cell-free protein synthesis. (A) Inhibition of protein synthesis by MazF_Sa. Protein synthesis was carried out with the plasmid pET14b-ctpA in an E. coli T7 S30 extract system at 37°C for 1 h. Lane 1, control without MazF_Sa; lanes 2 to 6, various amounts of MazF_Sa were added at 3.75 pmol, 7.5 pmol, 11.25 pmol, 15 pmol, and 30 pmol, respectively. (B) Protein synthesis was rescued by the addition of MazE_Sa. Lane 1, control without MazF_Sa; lane 2, 15 pmol of MazF_Sa; lanes 3 to 6, 8 pmol, 16 pmol, 32 pmol, or 64 pmol of MazE_Sa, respectively, was added together with 15 pmol of MazF_Sa. The synthesis of β-lactamase and CtpA are indicated by arrows.

In vitro primer extension analysis of the MazF_Sa cleavage sites in ctpA mRNA. Primer extension was carried out as described in Materials and Methods. Each primer extension product was analyzed on a 6% sequencing gel with the DNA sequencing ladder prepared with the same primer. (A and B) Cleavage sites in ctpA mRNA detected with the primer pEa. (C and D) Cleavage sites in ctpA mRNA detected with the primer pEb. The RNA sequences complementary to the DNA sequence ladder are shown to the right of each figure, and corresponding cleavage sites are indicated by arrows.

In vivo cleavage of ctpA mRNA after induction for MazF_Sa expression. Expression of MazF_Sa in E. coli BL21(DE3)pLysS carrying plasmids pET14b-ctpA and pBAD-MazF(His)₆ was induced by arabinose (0.2%) (A). The expression of MazF_Sa in S. aureus 178RI carrying plasmid pG164-MazF(His)₆/ctpA was induced by 1 mM IPTG (B). Total cellular RNAs were extracted at the indicated time points. Primer extension was then carried out as described in Materials and Methods. The DNA sequencing ladder was prepared using the same primer. The cleavage sites in the ctpA mRNA are indicated by arrows. The two panels represent in vivo primer extension with primer pEa from E. coli and S. aureus, respectively.

Inhibition of cell growth by S. aureus MazF_Sa. (A) Schematic representation of genetic organization of the S. aureus mazEF_Sa and sigB operons. (B) S. aureus ACL6094 cells were transformed with pG164-MazF(His)₆. The cells were then streaked on a 03GL agar plate with or without induction with IPTG (1 mM) followed by incubation overnight at 37°C. (C) Reduction of CFU with S. aureus ACL6094 harboring pG164-MazF(His)₆ under induction. The cultures were induced at an optical density at 650 nm of 0.4 with IPTG (1 mM). To determine the CFU, samples were withdrawn at various time points, spread on TSA plates supplemented with chloramphenicol (10 μg/ml), and incubated at 37°C overnight. (D) Characterization of MazE_Sa/MazF_Sa. The MazE(His)_6Sa and MazF(His)_6Sa proteins were purified as described in Materials and Methods. The proteins were analyzed by 16% SDS-PAGE. (i) lane 1, protein molecular mass markers; lane 2, MazE(His)_6Sa; lane 3, MazF(His)_6Sa; (ii) lane 4, purified MazE-MazF(His)_6Sa complex. The upper band is MazF(His)_6Sa, and the lower band is the copurified MazE_Sa. (E) Native PAGE analysis of MazEF_Sa complex formation. MazE(His)_6Sa was mixed with MazF(His)_6Sa as described in Materials and Methods and subjected to native PAGE analysis. Lane M, molecular mass markers; lane 1, MazE(His)_6Sa (32 pmol); lane 2, MazF(His)_6Sa (30 pmol); lanes 3 to 5, MazE(His)_6Sa (32 pmol). Samples were mixed with 7.5 pmol, 15 pmol, and 30 pmol of MazF(His)_6Sa, respectively. The MazE_Sa/MazF_Sa complex is indicated by the arrow.

Endoribonuclease activity of MazF_Sa. Five μg of ctpA mRNA was digested with 15 pmol of MazF_Sa as described in Materials and Methods. (A) Cleavage of ctpA mRNA by MazF_Sa. Lane 1, control without MazF_Sa; lanes 2 to 4, mRNA substrates were digested for 60 min, 90 min, and 120 min, respectively. (B) Inhibition of cleavage with the addition of MazE_Sa. Lane 1, mRNA with MazF_Sa; lanes 2 to 4, mRNA substrates digested by MazF_Sa together with 4 pmol, 8 pmol, or 16 pmol of MazE_Sa; lane 5, mRNA with 32 pmol of MazE_Sa.

Cleavage of synthetic RNA by MazF_Sa. All RNA substrates labeled at the 5′ end with [γ-³²P]ATP were digested with MazF_Sa and subjected to analysis in a 20% sequencing gel run with an RNA ladder by alkaline hydrolysis as described in Materials and Methods. (A) Cleavage of a synthetic 18-base RNA, AUUC. Lane 1, RNA ladder generated by alkaline hydrolysis; lane 2, RNA substrate without the addition of MazF_Sa; lane 3, RNA substrate digested by15 pmol of MazF_Sa. The corresponding RNA sequence is shown to the right. The cleavage product and site are indicated by arrows. (B) Cleavage specificity of MazF_Sa with synthetic 18-base RNA substrates. Seven RNA substrates with the center AUUC sequence were changed to AGUC, AUGC, GUUG, UUUC, AUUG, GUUC, and AUUU sequences and named correspondingly. The ATTC indicates the same length of DNA substrate. Lanes 1, 3, 5, 7, 9, 11, 13, 15, and 17 contained no MazF_Sa; lanes 2, 4, 6, 8, 10, 12, 14, 16, and 18 are with 15 pmol of MazF_Sa added. The background in the absence of MazF_Sa was attributed to impurities or incomplete synthesis of the full-length RNA substrates. Nevertheless, cleavage can be seen with MazF_Sa, as indicated by the arrows. (C) Predicted secondary structure formed by RB-1 (5′-UGCAAUUCAUAUGAAUUGU-3′) using the RNA secondary prediction website (http://www.genebee.msu.su/services/rna2_reduced.html). (D) Cleavage of highly purified synthetic RNA substrates with different secondary structures. An 18-base sense RNA, AUUC, and AUUC antisense RNA RB-3, were digested separately by MazF_Sa. The 19-base RNAs RB-1 and RB-2 (an RB-1 variant) were also digested with MazF_Sa. Lanes 2, 4, 6, and 8 are with no MazF_Sa addition; lanes 3, 5, 7, and 9 are with 15 pmol of MazF_Sa added. The cleavage products are indicated by arrows. (E) Effects of RNA-RNA duplex formation on cleavage by MazF_Sa. Lane 1, RNA ladder generated by alkaline hydrolysis; lane 2, labeled sense RNA alone; lane 3, labeled sense RNA digested with 15 pmol of MazF_Sa; lanes 4 to 7, the labeled 18-base sense RNA, AUUC, was annealed with AUUC antisense RNA RB-3 in ratios of 1: 0.2, 1:0.4, 1:0.8, and 1:1, respectively, as indicated and then digested with 15 pmol of MazF_Sa at 37°C for 30 min.

DOX stress induced increasing expression of mazEF_Sa transcripts. The S. aureus Newman culture was treated with 50 ng/ml DOX at optical density at 650 nm of 1.0. Total cellular RNAs were extracted at the indicated time points. Northern blot analysis was carried out with the mazEF_Sa probe as described in Materials and Methods. (A) Transcripts detected with the probe. (B) 23S and 16S served as the internal loading control.