ssbB and is also comA deficient, so as to avoid any potential growth deficiency that might be caused by prolonged competence in a mutant deficient in exit from competence. The late gene expression pattern in response to CSP in this reporter parent strain was verified as identical to that reported previously. Mutations in genes with known roles in transformation were crossed into the parent strain. Included were cibABC and cbpD, which affect fratricide, and cinA, linked to the cinbox promoter required for induction of recA by CSP. The internal deletion of each gene or operon was replaced by a KnR marker. Altogether, we examined 12 mutants, defective in a total of about 20 late genes: DcbpD, DcibABC, DssbB, DcglEFG, DcoiA, DdprA, DcelAB, DcclA, DcglABCD, DcflAB, DradA and DPc-cinA. Each mutation’s structure was confirmed by PCR, and the border of a Relative transformation rates were calculated by comparing the transformation efficiencies of the mutants to that of CP2000. b The literature sources for the relative transformation rates of the mutants are found in Pneumococcal Exit from Competence each deletion is shown in Fig. 1. Since the transformation efficiency of these mutants was already known to be very low, we verified that transformation rates for the new strains were comparable to the values expected from the literature. To determine the effect of each of the 12 mutations on exit from competence, each mutant reporter strain was treated with CSP under standard conditions and sampled 0, 10, 20, 30, 40, 60, or 80 minutes later for LacZ assay. The enzyme levels, which indicate late gene expression patterns, 1975694 are shown as a function of the time after CSP induction in Fig. 2. Nearly all of these mutants displayed normal patterns of late gene expression, with a burst of LacZ synthesis restricted to the period between 10 and 30 min after addition of CSP. The single exception was the dprA mutant, in which late gene expression continued beyond 60 min The 10973989 pattern shown in Fig. 2 reproduces the growth defect reported by Berge for the R6 strain, and further supports his interpre tation by showing directly that expression of a late gene continues for an unusually long time after exposure of a dprA mutant to CSP; that is, the cells appear to remain in an active X-state for a greatly extended period when DprA is missing. The greatly prolonged expression of a late gene suggests a prolonged presence of ComX and thus that DprA affects not only expression of the comCDE operon, but also expression of the duplicate comX genes. Combined with 
the report of Mirouze 9 Pneumococcal Exit from Competence that premature expression of dprA repairs the transience defect of a comX mutant, these results suggest that DprA is both AS703026 site necessary and sufficient for the shutoff of early gene expression. From the absence of any similar effect of the other late gene mutations on the temporal pattern of ssbB expression we conclude further that none of the other late gene products examined has a strong role in shutoff of early gene expression. Because of the uniquely strong effect of DprA on shutoff, it is of interest to determine its effects on gene expression in more detail, both to know if it is the only shutoff agent, and to identify its regulatory targets. The Kinetics of Exit from Competence is not Altered in a ClpP Protease-deficient Background We were concerned to be able to detect regulation of comX at multiple levels. While the effect of DprA shown above is dramatic, a