Molecular characterization of a locus required for hyaluronic acid capsule production in group A streptococci.
To characterize the production of hyaluronate capsule by the membrane-associated enzyme hyaluronate synthase (HAS), group A streptococci from a recent outbreak of acute rheumatic fever were mutagenized via Tn916 insertion. Acapsular transconjugants harboring multiple, nontandem copies of the transposon were identified and found to lack HAS activity (less than 1% of wild-type levels). Generalized transduction was then performed to determine which Tn916 insertion was responsible for the HAS- phenotype. These marker exchange experiments resulted in the isolation of two distinct classes of acapsular transductants, designated WF61 and WF62. Both transductants also lacked significant HAS activity, and excision of the transposon from WF62 restored capsular hyaluronate production. Southern analysis of WF61 DNA demonstrated a large deletion of genomic DNA adjacent to the Tn916 insertion. This deletion event is presumably responsible for the observed stability of the acapsular phenotype of WF61. Further analyses of transductant whole-cell DNA indicated that the transposon insertions of WF61 and WF62 were separated by 2.5 kb. These studies define a locus required for hyaluronate capsule production in group A streptococci. Further genetic analysis of this locus has identified a gene required for HAS activity which wasd inactivated by TN916 in WF62 and deleted in WF61.
Dougherty
B A
BA
Wake Forest University Medical Center, Winston-Salem, North Carolina 27157.
van de Rijn
I
I
D016667:Bacterial Capsules / Q000378:metabolism*; D001483:Base Sequence; D015139:Blotting, Southern; D003227:Conjugation, Genetic; D004269:DNA, Bacterial; D005798:Genes, Bacterial; D014453:Glucuronosyltransferase / Q000235:genetics* / Q000378:metabolism; D016695:Glycosyltransferases; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000096:biosynthesis / Q000235:genetics*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D013297:Streptococcus pyogenes / Q000201:enzymology / Q000235:genetics*; D014166:Transferases; D029867:Xenopus Proteins
D004269:DNA, Bacterial; D008565:Membrane Proteins; D029867:Xenopus Proteins; D006820:Hyaluronic Acid; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases