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 i s synthesized by the group A streptococcal membrane-associated enzyme hyaluronate synthase. In previous reports, the locus required for expr ession 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 termin ator 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 Tn 1000 method of DNA sequencing and inverse polymerase chain reaction, h asC, the third gene in the has operon was shown to be 915 base pairs i n length (304 amino acids) and located 192 base pairs downstream of ha sB. Sequence similarities to other genes suggested that hasC encodes U DP-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 f rom Escherichia coli that possessed increased UDP-glucose pyrophosphor ylase activity as compared to nondetectable levels in extracts with ve ctor alone, In addition, expression of HasC resulted in a protein of a pproximately 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-independ ent transcription terminator at the 3-prime terminus of the gene, hasC is the third and probably the final gene in the has operon.