Gateway bp clonase 2 enzyme mix

The tagN mutant (ΔTN) in S. aureus WH39 was constructed by allelic replacement using the pKOR1 shuttle vector as described previously (41 (link)). The approximately 1-kb upstream and downstream fragments of the tagN gene were amplified separately by PCR using chromosomal DNA of S. aureus WH39 as a template. Primers used for gene deletion are listed in Table S4. The above amplification products were purified and spliced by fusion PCR, and then recombined on vector pKOR1 using the Gateway BP ClonaseTM II enzyme mix (Invitrogen Corp., CA, USA). Recombinant plasmid pKOR1-ΔtagN was transformed into S. aureus WH39 via electroporation to obtain the ΔTN strain. The allelic replacement mutant was checked by PCR and DNA sequencing. The S. aureus-E. coli shuttle vector pTSSCm was used to construct the expression of plasmid pTSSCm-tagN at BamHI and XhoI sites (primers; see Table S4) (42 (link)). The resulting constructs were then transformed into ΔTN via electroporation to obtain the tagN mutant complementation (c-ΔTN). The complementation was identified by PCR and DNA sequencing.

The backbone plasmids p0380-bar and p0380-sur-gateway [42 (link)] were used to construct plasmids for cdc42 deletion and complementation. Briefly, the 5’ and 3´ fragments (1428 and 1819 bp, respectively) of cdc42 comprising partial coding and flanking regions were amplified from the WT DNA with paired primers (Table S1) and inserted into p0380-bar at the XmaI/BamHI and XbaI/SpeI sites, respectively, forming p0380-5´cdc42-bar-3´cdc42. The full-length sequence of cdc42 and its flanking regions (3501 bp in total) were amplified from the WT DNA and inserted into the p0380-sur-gateway to exchange for the gateway fragment under the action of Gateway BP Clonase^TM II Enzyme Mix (Invitrogen), yielding p0380-sur-cdc42 vectoring the sur marker. The two constructed plasmids were propagated in E. coli Top10 and E. coli DH5α and transformed into the WT and the Δcdc42 mutant via Agrobacterium-mediated transformation [18 (link)], respectively. Putative mutants were screened in terms of the bar resistance to phosphinothricin (200 μg/mL) or the sur resistance to chlorimuron ethyl (15 μg/mL) in a selective medium. The expected recombination events (Figure S1A) were verified by PCR (Figure S1B) with paired primers (Table S1). Positive Δcdc42 mutant and its control strains (parental WT and Δcdc42::cdc42) were used in phenotypic experiments including three independent replicates.