Improved genetic transformation by disarmament of type II Restriction–Modification system in Streptococcus zooepidemicus.

Streptococcus zooepidemicus, group C Streptococci, is currently used for the industrial production of hyaluronic acid (HA). However, genetic manipulation of S. zooepidemicus is severely limited by its low transformation efficiency, which might be in part due to the Restriction–Modification (R–M) systems. The complete genome sequence of S. zooepidemicus ATCC39920 revealed the presence of two putative R–M systems, type I and type II. The putative type I R–M system is encoded by three closely linked genes: hsdR (SeseC_01315), hsdS, hsdM (SeseC_01318), and the putative type II R–M system consists of two closely linked genes: SeseC_02360 and yhdJ (SeseC_02362). Inactivation of hsdR, encoding the restriction endonuclease (REase) of the type I R–M system, showed no apparent effects on transformation efficiency, implying that disarmament of the type I R–M system alone is not sufficient for increasing transformation efficiency. However, inactivation of SeseC_02360, encoding the REase of the type II R–M system, improved transformation efficiency by 4.97 folds, indicating that type II R–M system is the major barrier that restricts genetic transformation in S. zooepidemicus. Furthermore, S. zooepidemicus strains lacking either of the two R–M systems are phenotypically indistinguishable from the wild-type in terms of cell growth and HA production. In summary, our study revealed that the type II R–M system is the main barrier to genetic transformation in S. zooepidemicus ATCC39920, and that the deletion of the type II R–M system renders S. zooepidemicus more transformable, thus facilitating metabolic engineering of this industrially important microorganism. [ABSTRACT FROM AUTHOR]