Fig. 4
From: Anti-phage islands force their target phage to directly mediate island excision and spread
PLE exploits phage encoded PexA as the RDF for excision. a Bacterial two hybrid analysis to detect protein–protein interactions. Cells containing fusions of PexA to the T25 subunit of adenylate cyclase CyaA (indicated on left) and Int to the T18 subunit (indicated on top) were spotted on X-gal. Blue colonies indicate a physical interaction between the proteins fused to the CyaA subunits. b Purified 6xHisSUMO-Int (1 mM) and/or PexA (0.25 mM) were incubated for 30 min and then incubated with nickel resin. Unbound and eluted fractions were collected and were run on an SDS-PAGE gel. c Left, cartoon of in vitro excision reaction shows dsDNA fragments containing attL and attR recombining to attC and attP in the presence of Int and PexA. Right, in vitro excision assay shows when purified PexA or Int are incubated alone with attL and attR (lanes 1 and 2, respectively), no recombination is detected. As the concentration of PexA increases, recombination products attC and attP are detected. d Left, cartoon of in vitro integration reaction depicting the ability of PexA to inhibit the activity of Int when recombining attC and attP sites in an integration reaction. Right, in vitro inhibition of integration (right) by PexA can be seen by the loss of the attL and attR recombination products as purified PexA increases in concentration. Uncropped gels are presented in Supplementary Fig. 5
