This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.DNA Mismatch repair (MMR) is a key system for maintaining DNA sequence integrity. Surprisingly, many isolates of bacterial pathogens have defects in MMR, and this frequent occurrence in pathogens has been implicated in increased adaptation to host defenses and resistance to antimicrobial therapy. In Streptococcus pyogenes strain SF370, the defective bacteriophage SF370.4 is integrated between mutS and mutL, two genes required for MMR and normally co-transcribed. The presence of prophage 370.4 would be predicted to disrupt the expression the downstream gene, mutL. METHODS: Strain SF370 was grown to mid-logarithmic or stationary phase, the mRNA isolated, and converted to cDNA. The cDNA was used as a template for PCRs specific to mutS, mutL, the intergenic region between them found in prophage-free strains, or the prophage SF370.4 integrase gene. Genomic DNA also was isolated for parallel reactions. The observed products from any of these reactions were analyzed by DNA sequencing. RESULTS: The mutS gene, which retains the native promoter, was expressed in both mid-logarithmic and stationary grown cells. As anticipated, mutL was not expressed in the stationary grown cells; however, mutL cDNA was present in mid-logarithmic grown streptococci. Further, using purified genomic DNA as a template, products could be observed that corresponded to the genomic arrangement both with and without the integrated prophage which was confirmed by DNA sequencing. DISCUSSION: The evidence shows that both mutS and mutL are expressed in rapidly dividing SF370 while in stationary cells mutL is absent, and prophage excision controls mutL expression during active growth. The lack of mutL activity during stationary phase lead us to hypothesize that prophage excision and integration exists in a dynamic state controlled by the growth state of the cell, and resultantly, MMR in S. pyogenes strain SF370 is activated in response to growth phase. This project will determine the frequency and distribution of phage SF370.4 and related phages in a representative collection of world-wide clinical isolates to uncover associations with disease syndrome or streptococcal serotype and elucidate the mechanism of control used by the SF370.4 prophage to regulate its genomic integration and excision and thus alter the S. pyogenes mutation.
Showing the most recent 10 out of 165 publications
