The Ssp1 and Ssp2 C50A mutants are inactive. (a) Growth of E. coli MG1655 transformed with plasmids expressing OmpAsp-Ssp1 (sp-Ssp1; pSC152), OmpAsp-Ssp1(C50A) [sp-Ssp1(C50A); pSC548], OmpAsp-Ssp2 (sp-Ssp2; pSC138) or OmpAsp-Ssp2(C50A) [sp-Ssp2(C50A); pSC549] from an arabinose-inducible promoter, or with the empty vector (VC, pBAD18-Kn), on LB or M9 medium containing 0.2% arabinose. (b) Recovery of a sensitive Δrap2a mutant as the target strain following co-culture with the different attacking strains indicated, expressed relative to recovery of target when co-cultured with the wild-type strain. Attacking strains are wild-type S. marcescens Db10 (WT), mutant lacking ClpV (ΔclpV), mutant lacking Ssp2 (Δssp2), each carrying either the vector control plasmid (+VC; pSUPROM) or a plasmid expressing wild-type Ssp2 (+Ssp2; pSC541) or the C50A mutant of Ssp2 [+Ssp2(C50A); pSC1230]. (c) Phenotypes of wild-type S. marcescens Db10 and selected single and double mutants carrying plasmids expressing variants of Ssp1 or Ssp2 following growth on solid medium. For each strain, representative images of the morphology of a culture spot (left; scale bar 1 mm), single colonies (middle; scale bar 1 mm) and individual cells [right; scale bar 2 µm in (i) or 5 µm in (ii)] are shown. Plasmids direct the expression of wild-type Ssp1 (+Ssp1; pSC539), the C50A mutant of Ssp1 [+Ssp1(C50A); pSC1229], wild-type Ssp2 (+Ssp2; pSC541), the C50A mutant of Ssp2 [+Ssp2(C50A); pSC1230] or represent the vector control (+VC, pSUPROM). Growth was for 48 h on MM (i) or 24 h on LB (ii). (d) Immunoblot analysis of levels of Ssp1 or Ssp2 proteins from E. coli MG1655 (top two panels) or S. marcescens (bottom two panels) grown on solid medium. Strains and plasmids are as in (a)–(c), except that wild-type OmpAsp-Ssp1 and OmpAsp-Ssp2 were co-expressed with their cognate immunity proteins to allow the strains to grow [sp-Ssp1(WT)Rap1a; pSC160 or sp-Ssp2(WT)Rap2a; pSC144].