Isolation of a Streptococcus pyogenes gene locus that directs hyaluronan biosynthesis in acapsular mutants and in heterologous bacteria

Abstract

A contiguous 3-kilobase pair region of DNA was isolated from Group A Streptococcus pyogenes (GAS) that can direct hyaluronic acid (HA) capsule biosynthesis in acapsular mutants as well as heterologous bacteria. The DNA was identified by transposon 916 insertional mutagenesis and subcloned into a plasmid shuttle vector. Mutant acapsular GAS or Enterococcus faecalis containing this plasmid, but not vector alone, displayed a mucoid phenotype on agar plates, possessed a capsule as seen by light microscopy, and produced HA in quantities comparable with wild-type GAS. The polysaccharide was shown to be authentic HA based on its recognition by a specific proteoglycan and its degradation by Streptomyces hyaluronate lyase. Escherichia coli with the complementing plasmid also produced HA but at only 10% of the level made by the above cells. E. coli minicell analysis showed that two proteins, 42 and 45 kDa, are expressed by the functional DNA insert. Deletion analysis of the insert in the minicells revealed that the 42-kDa protein is essential for HA production. This is the first demonstration of reconstitution of HA capsule biosynthesis in vivo.