Identification and Disruption of Two Discrete Loci Encoding Hyaluronic Acid Capsule Biosynthesis GeneshasA, hasB, and hasC inStreptococcus uberis

ABSTRACT

The hyaluronic acid capsule of Streptococcus uberis has been implicated in conferring resistance to phagocytosis by bovine neutrophils. Construction of a bank of random insertion mutants ofS. uberis (strain 0140J) was achieved using the pGh9::ISS1 mutagenesis system (22). Phenotypic screening of approximately 5,000 clones enabled the isolation of 11 acapsular mutants. Southern hybridization indicated that two mutants carried a lesion within a group of genes similar to those involved in the assembly of the hyaluronic acid capsule found in the group AStreptococcus (GAS) has operon. The DNA sequence flanking the points of insertion confirmed the presence of homologues of GAS hasA and hasB in S. uberis. The DNA sequence flanking the ISS1 insertion in another mutant identified a homologue of hasC inS. uberis. The GAS hasABC operon structure was not conserved in S. uberis, and two discrete loci comprising homologues of either hasAB or hasCwere identified. Disruption of S. uberis hasA orhasC resulted in the complete cessation of hyaluronic acid capsule production. Correspondingly, these mutants were found to have lost their resistance to phagocytosis by bovine neutrophils. The bactericidal action of bovine neutrophils on S. uberis0140J was shown unequivocally to depend upon the capsule status of the bacterium.