Time course of competence gene expression in Streptococcus pneumoniae strains expressing different csRNAs. The strains were grown in brain heart infusion medium and β-galactosidase activities were determined in 30 min. intervals. β-galactosidase activities of representative experiments are shown along with the growth curve determined by measuring OD600. All strains contained a genomic integration of a competence promoter fused to the promoterless β-galactosidase gene lacZ of Escherichia coli and a disrupted protease gene htrA (htrA::aphIII). Relevant genetic characteristics are indicated. (A) Activity of early and late competence promoters in strains without csRNAs. The early competence gene promoter of comX and the late competence gene promoter of cibA were fused to lacZ and integrated into RKL 558 (ccnA:: lox72, ccnB:: lox72, ccnC::lox72, ccnD::lox72, ccnE::lox72, htrA::aphIII). The promoter fusion integrations were: bgaA::tetM-PcomX-lacZ (left), bgaA::tetM-PcibA-lacZ (right). (B) Activity of the early comX promoter in strains with csRNA1 or csRNA5. The early competence gene promoter of comX was fused to lacZ and integrated into RKL688 (ccnB::ermAM, ccnC::lox72, ccnD::lox72, ccnE::lox72, htrA::aphIII, left) or RKL692 (ccnAB::lox72, ccnC::lox72, ccnD::lox72, htrA::aphIII, right). Both strains contained the integrated PcomX-lacZ fusion: bgaA::tetM-PcomX-lacZ. (C) Activity of the early comC promoter in strains with csRNA1 or csRNA5. The early competence gene promoter of comC was fused to lacZ and integrated into the same strains as in (B). (RKL688; ccnB::ermAM, ccnC::lox72, ccnD::lox72, ccnE::lox72, htrA::aphIII, left, or RKL692; ccnAB::lox72, ccnC::lox72, ccnD::lox72, htrA::aphIII, right). Both strains contained the integrated PcomC-lacZ fusion: bgaA::tetM-PcomX-lacZ. (D) Activity of the late cibA promoter in strains with csRNA1 or csRNA5. The late competence gene promoter of cibA was fused to lacZ and integrated into the same strains as in (B,C). (RKL688; ccnB::ermAM, ccnC::lox72, ccnD::lox72, ccnE::lox72, htrA::aphIII, left, or RKL692; ccnAB::lox72, ccnC::lox72, ccnD::lox72, htrA::aphIII, right). Both strains contained the integrated PcibA-lacZ fusion: bgaA::tetM-PcomX-lacZ.