CLONING AND EXPRESSION OF THE GENE FOR GROUP-B STREPTOCOCCAL HYALURONATE LYASE

Group B streptococci (GBS) are a major cause of serious human perinata l infections. Most clinical isolates of GBS secrete hyaluronate lyase, and production of high levels of the enzyme has been associated with strain virulence. Degenerate oligonucleotide primers, designed on the basis of the amino acid sequences of tryptic peptides prepared from th e purified enzyme, permitted the polymerase chain reaction amplificati on from GBS chromosomal DNA of a 363-base pair internal DNA fragment o f the GES hyaluronate lyase gene (hylB). This DNA fragment was used as a probe to screen a lambda phage library of GBS chromosomal DNA fragm ents. Sequence analysis of positive clones identified an open reading frame capable of coding for a 111-kDa protein. Since no single clone w as found to contain the entire gene it was necessary to reconstruct th e gene from two plasmids containing inserts with suitable overlapping sequences. When this reconstructed gene was transformed into Escherich ia coli, high level expression of hyaluronate lyase activity was obtai ned.