Molecular Characterization of hasC from an Operon Required for Hyaluronic Acid Synthesis in Group A Streptococci DEMONSTRATION OF UDP-GLUCOSE PYROPHOSPHORYLASE ACTIVITY

Hyaluronic acid is a high molecular weight glycosaminoglycan composed of repeating subunits of glucuronic acid and N-acetylglucosamine. It is synthesized by the group A streptococcal membrane-associated enzyme hyaluronate synthase. In previous reports, the locus required for expression of hyaluronic acid, the has operon, was identified and found to consist of two genes, hasA and hasB encoding hyaluronate synthase and UDP-glucose dehydrogenase, respectively. Since a transcription terminator was not found at the end of hasB, it was the aim of this study to identify the remaining gene(s) in the has operon. By utilizing the Tn1000 method of DNA sequencing and inverse polymerase chain reaction, hasC, the third gene in the has operon was shown to be 915 base pairs in length (304 amino acids) and located 192 base pairs downstream of hasB. Sequence similarities to other genes suggested that hasC encodes UDP-glucose pyrophosphorylase. Overexpression of hasC using isopropyl-1-thio-beta-D-galactopyranoside induction of the T7 promoter in the pET translation system allowed for the production of bacterial extracts from Escherichia coli that possessed increased UDP-glucose pyrophosphorylase activity as compared to nondetectable levels in extracts with vector alone. In addition, expression of HasC resulted in a protein of approximately 36 kDa as shown by SDS-polyacrylamide gel electrophoresis. These data as well as complementation analysis of hasC in an E. coli galU mutant confirmed that hasC encodes UDP-glucose pyrophosphorylase. Finally, since sequence analysis identified a potential rho-independent transcription terminator at the 3-prime terminus of the gene, hasC is the third and probably the final gene in the has operon.