A user-friendly strategy to engineer tailored intermediate strains for overcoming combined type I and type II restriction-modification barriers in Staphylococcus aureus

A significant proportion of Staphylococcus aureus clinical isolates are refractory to plasmid transformation due to the combined action of type I and type II restriction-modification (RM) systems. Current strategies often depend on specialized engineered strains and may require sequence modifications if foreign DNA harbors host-specific methylation patterns, limiting their general applicability. Here, we developed a user-friendly, sequence-independent strategy that employs customized restriction-deficient intermediate hosts. Using the sequence type 121 (ST121) strain XQ as a proof-of-concept, we first engineered an Escherichia coli strain to produce the requisite type I methylation pattern, enabling low-efficiency plasmid transformation. These plasmids were then used to delete the type I and type II restriction genes in XQ, creating the highly transformable intermediate strain XQ01. Once constructed, even plasmids prepared from common E. coli cloning hosts (e.g., TOP10) could be consistently transformed into XQ01. Plasmids propagated in XQ01 were subsequently transformed into wild-type XQ and other ST121 clinical isolates with high efficiency (>10 4 transformants per μg DNA). This streamlined approach allowed us to successfully generate knockout, insertion, and fusion-expression mutants in XQ or its variants. Collectively, this work establishes a versatile transformation strategy that effectively bypasses type I and II RM barriers, facilitating genetic manipulation of ST121 and other RM-harboring S. aureus strains.