These data suggest that N. gonorrhoeae transformation of ssDNA is largely dependent on the presence of the Crick DUS12. Neisseria gonorrhoeae was grown on GC Medium Base (GCB) (Difco) plates with Kellogg’s supplements I and II (Kellogg et al., 1968) and incubated at 37 °C in a 5% CO2 humidified atmosphere. Escherichia coli strain TOP10F′ (Invitrogen) was used to replicate recombinant M13 phage. The F′ episome was maintained in the TOP10F′ cells by addition of tetracycline (15 μg mL−1) in the LB or YT media used to grow the E. coli. Transformation was investigated in the laboratory strains FA1090
(Connell Sirolimus et al., 1988) and MS11 (Meyer et al., 1982). The concentration of Nalidixic acid (Nal) in GCB was 1 μg mL−1 for strain FA1090 and 3 μg mL−1 for strain MS11. We have previously constructed plasmids containing DUS0 and DUS12 gyrB1 DNA [plasmids gyrB1 DUS0 and gyrB1 DUS12 (Duffin & Seifert, 2010)]; these plasmids were digested with EcoRI, and the DNA fragments were cloned into EcoRI digested M13mp18 check details and M13mp19 replicative form (RF) DNA. Positive clones were isolated in TOP10F′ cells (Invitrogen) using blue/white screening on Xgal containing media. Recombinant RF DNA was purified from infected TOP10F′ cells, and gyrB1 inserts were confirmed
by restriction digest analysis and DNA sequencing. M13mp18 and M13mp19, which have opposing orientation of the multiple cloning sites, were utilized so that either the Watson or the Crick strand of the gyrB1 and DUS12 would be encoded by recombinant phage. Recombinant phage harboring both orientations DUS12 gyrB1 DNA and the DUS0 constructs were obtained and used to produce ssDNA. DNA sequencing was carried at the sequencing core of Northwestern University, and the program suite VectorNTI (Invitrogen) was used to analyze DNA sequences. TOP10F′ cells were infected with recombinant M13 phage and grown for 5 h at 37 °C with constant agitation. Qiagen M13 and Qiagen miniprep kits were
used to click here purify ssDNA and RF DNA, respectively, from recombinant phage infection following the manufacturer’s instructions. The amount of contaminating dsDNA (from RF DNA) in the ssDNA preps was assessed by Southern blots probed with oligonucleotide probes (see below). Owing to variability in the quality of the ssDNA preparations (possibly due to cell lysis during phage infection), each individual ssDNA preparation was measured for ssDNA purity by agarose gel electrophoresis and Southern blot analysis (see below). To obtain sufficient ssDNA for the transformation experiments, ssDNA preparations that were deemed pure (< 1 : 10 000 contaminating DNA) were pooled together to create ssDNA stocks.