Markerless multiple-gene-deletion system for Streptococcus mutans

Abstract

Inactivation or selective modification is essential to elucidate the putative function of a gene. The present study describes an improved Cre-loxP-based method for markerless multiple gene deletion in Streptococcus mutans, the principal etiological agent of dental caries. This modified method uses two mutant loxP sites, which after recombination creates a double-mutant loxP site that is poorly recognized by Cre recombinase, facilitating multiple gene deletions in a single genetic background. The effectiveness of this modified strategy was demonstrated by the construction of both single and double gene deletions at the htrA and clpP loci on the chromosome of Streptococcus mutans. HtrA and ClpP play key roles in the processing and maturation of several important proteins, including many virulence factors. Deletion of these genes resulted in reducing the organism's ability to withstand exposure to low pH and oxidative agents. The method described here is simple and efficient and can be easily implemented for multiple gene deletions with S. mutans and other streptococci.

FIG. 1.

(A) Schematic diagram of the Cre-loxP* system for gene deletion with S. mutans. The wild-type (WT) gene targeted for inactivation is cloned into a vector and interrupted by the insertion of a lox66-Ab-lox71 cassette. (i) The construct is transformed into S. mutans, and the successful allelic exchange event (IN, integrated product) is selected using appropriate antibiotic-containing plates. (ii) Removal of the lox66-Ab-lox71 cassette from the chromosome is achieved by Cre-mediated excision (EP, excised product) after the strain is transformed with pCrePA and grown at permissive temperature (30°C). (iii) Elevation of the growth temperature to 37°C results in the loss of the temperature-sensitive (Ts) pCrePA plasmid, resulting in a mutant strain carrying a deletion in the target gene without a selectable marker. (B) Schematic representation of the loxP* sites. The loxP site consists of two 13-bp inverted repeats surrounding an 8-bp asymmetric core sequence (spacer). lox71 and lox66 have 5 bp mutated in the left and right 13-bp repeats, respectively. Mutated sequences are in boldface letters and underlined. Cre-mediated recombination between lox66 and lox71 results in the formation of lox72, which has mutations in both 13-bp repeat sequences.

FIG. 2.

PCR analysis of the modified S. mutans strains. The htrA gene amplified with the Smu-HtrA-F2/Smu-HtrA-R2 primers from UA159 (lane 1), strain IBS501 (lane 2), strain IBS502 (lane 3), strain IBS508 (lane 4), strain IBS511 (lane 5), strain IBS510 (lane 6), and strain IBS511 (lane 7). The clpP gene amplified with the Smu-ClpP-F2/Smu-ClpP-R2 primers from UA159 (lane 8), strain IBS509 (lane 9), strain IBS512 (lane 10), strain IBS510 (lane 11), and strain IBS513 (lane 12).

FIG. 3.

Deletions of htrA and clpP affect the stress tolerance of S. mutans. Freshly grown overnight cultures of UA159 wild type (WT) and its derivatives were serially diluted with 0.85% NaCl. Portions of each dilution (7.5 μl) were spotted onto THY agar plates with no additions (control, THY), methyl viologen (10 mM, THY + MV), or puromycin (6 μg ml⁻¹, THY + Pu). The plates were incubated at 37°C under microaerophilic conditions. Experiments were repeated more than three times, and relevant areas of representative plates are shown.

FIG. 4.

Growth characteristics of the protease mutants. Growth of the wild-type and the S. mutans mutant strains, in buffered THY of pH 7.0 (A) and pH 5.5 (B), at 37°C were monitored with a Klett-Summerson colorimeter. The strains used for analysis include UA159 (▪, WT), IBS511 (▴, ΔhtrA), IBS512 (•, ΔclpP), and IBS513 (⧫, ΔhtrA ΔclpP). The experiments were repeated twice, and representative growth curves are shown.