Supplemental Experimental Procedures Bacterial Strains Used

Pneumococci were grown on blood agar plates overnight at 37ºC in 5 % CO2. The bacteria were subcultured in semisynthetic medium c+y [S1] to log-phase. We used the following strains: TIGR4 (sero-type group 4) [S2] and its isogenic capsular knockout mutant, T4R [S3], as well as several clinical isolates (Type 1 [PJ1354], Type 2 [D39], Type 7F [PJ1446], Type 9V [I95], Type 14 [RP2732], and Type 19F [SME 70]). We used PCR ligation mutagenesis [S4] to knockout endA in TIGR4. Primers used for construction and screening of dele-tion alleles were 50-CTCCATCGTTGTATTAGGGC-30 and 50-TTTGGG CCCAAGGCTGGTTCTCCAGCC-30 flanked with ApaI for the up-stream fragment and 50-TTGGATCCGTAAAAGATAAGCCTAAACT CC-30and 50-TAAGGAGACTCTTGGTGTGA-30 flanked with BamHI for the downstream fragment. The PCR products were ligated with an erythromycin cassette (GenBank: AB057644) containing ApaI and BamHI sites and transformed into the recipient pneumococcal strain (TIGR4) as previously described [S5]. The resulting transform-ants were selected on blood agar plates containing erythromycin (1 mg/ml) and confirmed by PCR and by DNase activity test. To create TIGR4D(endA)V(endA), first, we integrated a kanamycin cassette downstream of endA in TIGR4 by PCR ligation mutagenesis with an upstream fragment linked with a BamHI site and a down-stream fragment linked with an ApaI site. Primers used for the con-struction were 50-CTCCATCGTTGTATTAGGGC-30 and 50-TTGGAT CCTTACTGAGTTACAGTTACTTCT-30 for the upstream fragment and 50-TTGGGCCCAAGATAAGCCTAAACTCCTATG-30 and 50-TAA GGAGACTCTTGGTGTGA-30 for the downstream fragment. The PCR products were ligated with the kanamycin cassette [S6] con-taining ApaI and BamHI sites and transformed into TIGR4. Clones were selected on plates containing kanamycin (400 mg/ml), and the chromosomal DNA was transformed into TIGR4D(endA) to make TIGR4D(endA)V(endA). Shigella flexneri M90T were grown as described [S7]