Cloning and expression of the gene for group B streptococcal hyaluronate lyase.
Group B streptococci (GBS) are a major cause of serious human perinatal 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 the purified enzyme, permitted the polymerase chain reaction amplification from GBS chromosomal DNA of a 363-base pair internal DNA fragment of the GBS hyaluronate lyase gene (hylB). This DNA fragment was used as a probe to screen a lambda phage library of GBS chromosomal DNA fragments. Sequence analysis of positive clones identified an open reading frame capable of coding for a 111-kDa protein. Since no single clone was found to contain the entire gene it was necessary to reconstruct the gene from two plasmids containing inserts with suitable overlapping sequences. When this reconstructed gene was transformed into Escherichia coli, high level expression of hyaluronate lyase activity was obtained.
Lin
B
B
Department of Microbiology, University of Alabama at Birmingham 35294.
Hollingshead
S K
SK
Coligan
J E
JE
Egan
M L
ML
Baker
J R
JR
Pritchard
D G
DG
D000595:Amino Acid Sequence; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000254:growth & development; D015870:Gene Expression; D005798:Genes, Bacterial; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011133:Polysaccharide-Lyases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013292:Streptococcus agalactiae / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D013296:Streptococcus pneumoniae / Q000201:enzymology / Q000235:genetics
D011133:Polysaccharide-Lyases; C100002:hyaluronate lyase