Identification and Disruption of Two Discrete Loci Encoding Hyaluronic Acid Capsule Biosynthesis Genes hasA , hasB , and hasC in Streptococcus 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 of S. 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 A Streptococcus (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 in S. uberis. The GAS hasABC operon structure was not conserved in S. uberis, and two discrete loci comprising homologues of either hasAB or hasC were identified. Disruption of S. uberis hasA or hasC 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. uberis 0140J was shown unequivocally to depend upon the capsule status of the bacterium.

Phospholipid Dependence and Liposome Reconstitution of Purified Hyaluronan Synthase

“…In this mechanism, cleavage of the UDP-HA linkage at the reducing end results in chain termination. The three streptococcal HASs, which are 417-419 amino acids long (7)(8)(9), contain six membrane domains (MDs), with the N-terminal and C-terminal ends inside the cell (10). The eukaryotic HASs contain an additional ϳ140 amino acids at the C-terminal end, which are predicted to include two MDs.…”

Functional Characteristics and Catalytic Mechanisms of the Bacterial Hyaluronan Synthases

“…The UDP-Glc pyrophosphorylase gene (hasC in streptococcus) encodes an enzyme that creates UDP-Glc from UTP and Glc-1-phosphate. In S. pyogenes these has genes are present in the order ABC and a similar organization was reported for S. uberis (20). The has operons in S. equisimilis and P. multocida have not yet been described.…”

Bovine Mastitis Caused By Streptococcus uberis: Virulence Factors and Biofilm

“…Briefly, virulence factors includes: plasminogen activator proteins such as PauA and PauB and SK, resistance to phagocytosis presented by a hyaluronic acid capsule, CAMP factor, a surface dehydrogenase protein GapC, sortases, Opp proteins implicated in dynamic transport of solutes, lactoferrin binding proteins, and adherence and invasion of epithelial cells mediated by SUAM [42][43][44][45][46][47][48][49][50][51].…”