Development of a markerless gene deletion system for Streptococcus zooepidemicus: functional characterization of hyaluronan synthase gene.

Streptococcus zooepidemicus is a bacterial pathogen used for production of hyaluronan in industry. Intensive research has significantly contributed to our understanding of S. zooepidemicus biology and pathogenesis. However, the lack of an effective targeted gene inactivation system in S. zooepidemicus has notably prevented the functional genomics analysis of this gram-positive bacterium. Here, we report the development of a markerless gene deletion system in S. zooepidemicus. We constructed a sacB expression cassette on the thermosensitive suicide vector pSET4s and demonstrated its use as a counterselection marker in S. zooepidemicus. We validated the efficiency of this system by deletion of hasA, which synthesizes the important virulence factor hyaluronic acid (HA) capsule. The genotype of the resultant hasA mutant was confirmed by polymerase chain reaction and sequencing. Deletion of hasA resulted in non-mucoid morphology, loss of HA capsule formation, and HA production. These defects can be rescued by introduction of a plasmid containing wild-type hasA expression cassette. Moreover, compared with wild type, hasA mutant showed no significant difference in expressions of other members of the hasABCDE operon, further suggesting that the loss of hasA contributed to the defects observed with ΔhasA mutant. Our results describe the first establishment of a sacB-based counterselection system in S. zooepidemicus, along with the first demonstration of hasA that is the only gene encoding a functional hyaluronan synthase in this bacterium.