Csp 1

Bacterial target strains were grown in acidic Columbia broth until they reached an optical density at 600 nm (OD₆₀₀) of 0.05 and followed by addition of 125 μg/ml of CSP1 (sequence: EMRLSKFFRDFILQRKK; purchased from GenScript, NJ, USA) and 1 μg of transforming DNA. The cultures were incubated at 37°C and 5% CO₂ without shaking for 2 h followed by plating on Columbia agar plates containing the appropriate antibiotic, kanamycin (150 μg/ml) or erythromycin (2 μg/ml), and incubating overnight. Resistant colonies were cultured in selective media, and the colonies were confirmed using PCR. Bacterial strains generated in this study are listed in Table S1.

A previously reported protocol was used to transform S. pneumoniae R6 and its derivatives (49 ). In brief, cells at the mid-log phase were diluted to OD₆₀₀ ∼0.03 in THY containing 1 mM CaCl₂ and 0.2% bovine serum albumin, and competence was stimulated by adding 500 ng/ml competence-stimulating peptide (CSP-1; GenScript). After 15 min incubation, 200 ng DNA product was added to a 1 ml culture, and the resulting culture was grown for 1 h. For transformant selection, 100 μl culture was combined with 5 ml molten 1% nutrient broth agar supplemented with appropriate additives, and the mixture was poured onto a TSAII 5% SB plate. Transformants were recovered after overnight incubation. A previously published method was used to introduce the desired mutation at the native pyk locus (50 ). AT1023 containing lacI and IPTG-inducible pyk was first transformed with a DNA cassette that includes an antibiotic resistance marker and B. subtilis sacB flanked by ∼1 kb upstream and downstream regions of pyk. After antibiotic selection, a DNA cassette containing the desired pyk mutation was transformed, and transformants were selected with 10% sucrose. These steps were performed in the presence of 1 mM IPTG to facilitate ectopic pyk expression. Detailed protocols for strain construction can be found in the Supporting information.