Identification and disruption of two discrete loci encoding hyaluronic acid capsule biosynthesis genes hasA, hasB, and hasC in Streptococcus uberis

The hyaluronic acid capsule of Streptococcus uberis has been implicated in conferring resistance to phagocytosis by bovine neutrophils. Construction o f a bank of random insertion mutants of S. uberis (strain 0140J) was achiev ed using the pGh9::ISS1 mutagenesis system (22). Phenotypic screening of ap proximately 5,000 clones enabled the isolation of 11 acapsular mutants. Sou thern hybridization indicated that two mutants carried a lesion within a gr oup of genes similar to those involved in the assembly of the hyaluronic ac id capsule found in the group A Streptococcus (GAS) has operon. The DNA seq uence flanking the points of insertion confirmed the presence of homologues of GAS hasA and hasB in S. uberis. The DNA sequence banking the ISS1 inser tion in another mutant identified a homologue of hasC in S. uberis. The GAS has ABC operon structure was not conserved in S. uberis, and two discrete loci comprising homologues of either hasAB or hasC were identified. Disrupt ion of S. uberis hasA or hasC resulted in the complete cessation of hyaluro nic acid capsule production. Correspondingly, these mutants were found to h ave lost their resistance to phagocytosis by bovine neutrophils. The bacter icidal action of bovine neutrophils on S. uberis 0140J was shown unequivoca lly to depend upon the capsule status of the bacterium.