Structure of the has operon promoter and regulation of hyaluronic acid capsule expression in group A Streptococcus

Group A streptococcal strains vary widely in the amount of hyaluronic acid capsule they produce, although the has operon, which encodes the enzymes required for hyaluronic acid synthesis, is highly conserved, The three genes making up the has operon are transcribed from a single promoter located upstream of the first gene in the operon, hasA. To investigate transcriptional regulation of capsule synthesis, we studied the structure and function of the has operon promoter sequences from two strains of group A Streptococcus: a highly encapsulated M-type 18 strain and a poorly encapsulated M-type 3 strain. Transcriptional fusions of the has operon promoter to a promoterless chloramphenicol acetyltransferase gene were constructed in a temperature-sensitive shuttle vector. The influence of promoter structure on has operon transcription was reflected by chloramphenicol acetyl transferase activity in cell lysates of Escherichia coli harbouring the recombinant plasmids and in group A Streptococcus after integration of the promoter fusions into the streptococcal chromosome. Fusions including as few as 12 nucleotides upstream from the -35 site of the has promoter exhibited full activity, indicating that sequences further upstream do not affect has gene transcription. A transcriptional fusion of the has promoter from the highly encapsulated M-type 18 strain was threefold more active than a similar construct from the poorly encapsulated M-type 3 strain. Analysis of the promoter sequences for the two strains revealed differences in three nucleotides in the -35, -10 spacer region of the promoter and in four nucleotides in the +2 to +8 positions relative to the start site of hasA transcription. To determine the relative importance of the two groups of nucleotide substitutions, chimeric promoter sequences were constructed in which either of the two clusters of variant nucleotides from the M18 has promoter was substituted for the corresponding positions in the M3 has promoter. Analysis of these chimeric promoter fusions showed that sequence changes in both regions influenced promoter strength. These results define the limits of cis-acting chromosomal sequences that influence transcription of the has operon and indicate that the fine structure of the promoter is an important determinant of capsule gene expression in group A Streptococcus.