5% Difco yeast extract, 1% NaCl), and 0.1% arabinose (Sigma) was used to induce traJ in pBAD constructs. Colonies were grown on LB agar (1.5% Difco Bacto agar) or 2% water agar [5 mM MgSO4, 1.5% Difco Bacto agar, double-distilled (dd) water] with 1 × M9 minimal salts (5 mM Na2HPO4·7H2O, 22 mM KH2PO4, 8 mM NaCl, 19 mM NH4Cl) and 0.4% lactose. The final concentrations of the LDE225 ic50 antibiotics were as follows: ampicillin (Ap) 100 μg mL−1, chloramphenicol (Cm) 20 μg mL−1, kanamycin (Km) 25 μg mL−1, streptomycin (Sm) 200 μg mL−1 and spectinomycin (Sp) 100 μg mL−1. All DNA restriction and ligation enzymes were from Fermentas as well as DNA marker ladders. Vent polymerase
was from NEBiolabs. Formaldehyde was purchased from Anachemia, whereas disuccinimidyl suberate (DSS) was purchased from Pierce. Protein A beads were purchased from Roche. DNA (QIAprep Spin Miniprep Kit) and PCR purification (QIAquick PCR purification kit) solutions and columns were purchased from Qiagen. Primer synthesis
and DNA sequencing find more were carried out by The Molecular Biology Services Unit at the University of Alberta. Mutagenesis of selected residues was performed using the QuikChange kit (Stratagene) according to the manufacturer’s directions. All point mutations were constructed using complementary double-stranded oligonucleotides of 30 base pairs with the mutation in the center Bcl-w of the oligonucleotide. Details of the mutagenic primers are available upon request.
Deletions were constructed by entering an ochre codon at the desired location within the mutagenic primer or by a PCR using primers specific to the 5′ and the appropriate 3′ end of traJ (Table 2). Mating assays have been described elsewhere (Frost & Manchak, 1998). Briefly, MC4100/Flac traJ90 containing pBADTraJ or mutants derived from it were used as donor strains and ED24 was used as the recipient. Overnight cultures of both donor and recipient cells (0.15 mL) grown with appropriate antibiotics were diluted (1 : 50) into 3 mL of LB broth with no antibiotics. Cultures were grown at 37 °C, and after 1 h, 0.1% arabinose (final concentration) was added to donor cells in order to induce TraJ production. At an OD600 nm of approximately 1.0, 100 μL each of donor and recipient cells were incubated together for 1 h at 37 °C in 1 mL of fresh LB with 0.1% arabinose. Mating efficiency was determined as the ratio of transconjugants per 100 donors and expressed as a percent. Simultaneously, another 100-μL aliquot of donor cells was pelleted for immunoblot analysis. Immunoblot analysis was performed as described previously (Gubbins et al., 2002). Polyclonal anti-TraJ antiserum and the secondary antibody (horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin G, GE Health Care) were used at a 1 : 20 000 dilution. Anti-H-NS antiserum was used at a 1 : 100 dilution.