FIG 5.

Development of a galactose-selectable genetic system in F. nucleatum subsp. nucleatum 25586. (A) Schematic of the strategy to use five purified F. nucleatum MTases to methylate plasmid pDJSVT13 to transform into F. nucleatum 25586. (B) Schematic of single-crossover and galKT gene deletions using plasmid pDJSVT13, which was first homologously recombined with up- and downstream sequences of the galKT operon. Primers used for PCR verification of positive clones are denoted with yellow or blue arrows, and the labels DJSnnn and TNnnn refer to specific primers found in Table S5 in the supplemental material. (C) PCR verification of the initial chromosomal incorporation (a crossover) as well as the full operon deletion (ΔgalKT). (D) Sanger sequencing verification of a full, clean, deletion of the galKT operon. (E) Selection for A-crossover strains on thiamphenicol (T5) containing plates, and verification that the ΔgalKT strain had removed the vector and antibiotic cassette and no longer grew on thiamphenicol. (F) Proof of survival of ΔgalKT on plates containing deoxygalactose (dGal), which is toxic to wild-type F. nucleatum subsp. nucleatum 25586. (G) Growth curves showed no growth defect for F. nucleatum subsp. nucleatum 25586 ΔgalKT compared to WT F. nucleatum subsp. nucleatum 25586, WT F. nucleatum subsp. nucleatum 23726, and F. nucleatum subsp. nucleatum 23726 ΔgalKT.