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. 2016 Sep 6;7(5):e00412-16. doi: 10.1128/mBio.00412-16

FIG 3 .

FIG 3 

Viability of S. aureus USA300 lacking SpsB is restored by cro/cI mutation in vitro. (A) cro/cI mutation restores in vitro growth of a mutant lacking SpsB. At nonpermissive temperature (42°C), plasmid pNL9164-ltrA is cured from strain USA300 Δmcr spsB::Tgn(pNL9164-ltrA) (strain GNE0190) and the TargeTron insertion in spsB prevents spsB expression, resulting in complete cessation of growth and confirming the essentiality of spsB. Remarkably, the growth of this strain is restored by a mutation in cro/cI [USA300 Δmcr spsB::Tgn cro/cI(M1V) (strain GNE0191)]. At permissive temperature (30°C), plasmid pNL9164-ltrA mediates splicing of the TargeTron insertion that removes it from the spsB transcript, allowing SpsB expression and growth of USA300 Δmcr spsB::Tgn pNL9164-ltrA (strain GNE0190). Circles, USA300 Δmcr (WT; strain GNE0023); triangles, USA300 Δmcr spsB::Tgn pNL9164-ltrA (strain GNE0190); squares, USA300 Δmcr spsB::Tgn cro/cI(M1V) (after curing plasmid pNL9164-ltrA) (strain GNE0191); diamonds, USA300 Δmcr cro/cI(M1V) (strain GNE0117). (B) Western blotting using a polyclonal rabbit anti-SpsB antibody showed a complete lack of SpsB protein in 3 independent whole-cell lysates from USA300 Δmcr spsB::Tgn cro/cI(M1V) (strain GNE0191) (lanes 4 to 6), compared to normal SpsB expression in USA300 Δmcr (WT, strain GNE0023) (lanes 1 to 3). Rabbit IgG anti-GroEL served as the loading control.