PCR-Mediated Amplification of Group A Streptococcal Genes Encoding Immunoglobulin-Binding Proteins

Abstract

A majority of group A streptococci (GAS) express immunoglobulin (Ig)-binding proteins. The genes encoding these proteins belong to either the emm or the emm-related (fcrA and enn) gene family. The present study presents oligonucleotide primers and protocols for the specific PCR-mediated amplification of fcrA and enn genes with and without their own promoters and transcription termination sites. The PCR system appears to be applicable to virtually every GAS serotype. Using these assays, some sequence variability in the 5′ untranslated region of fcrA and at the 5′ end of enn was demonstrated for a minority of GAS serotypes. The fcrA and enn genes of GAS serotype M49 were amplified by PCR, cloned into pJLA602, and expressed in Escherichia coli. The fcrA gene of the GAS serotype M49 strain CS101, including its adjacent regulatory sequences, was sequenced using a universal primer set by means of which emm-related genes can be completely sequenced with only five sequencing reactions. The fcrA49 sequence provides further evidence that fcrA genes are a separate entity from emm-related genes and exhibit a variability at their 5′ ends similar to that of proteins encoded by emm genes.