Bacteria were plated onto GCK agar plates containing the appropriate antibiotic, and the plates

incubated for 36-48 hrs. When transformations were performed under nonselective conditions, a spot transformation procedure was used [29]. For transformation, two piliated colonies were resuspended in 100:l GCP + 200 mM MgCl2 + 0.42% NaHCO3 Cell Cycle inhibitor + Kellogg’s supplement. The cell suspension was diluted 1:10, and additional two-fold serial dilutions were then carried out 9 times. An aliquot (5:l) of each suspension was spotted onto a GCK agar plate. To each spot, 5:l of DNA were added. After incubation overnight at 37°C with 4% CO2, individual colonies were isolated and streaked for isolation on GCK agar plates. The next day, individual colonies were inoculated onto GCK and C59 clinical trial spectinomycin-containing GCK agar plates. This procedure was repeated until spectinomycin-sensitive colonies were obtained. The correct replacement of the desired DNA fragment by the transformation process was verified by PCR amplification of the desired region, and restriction digestion analysis of the PCR amplicon, or direct DNA sequencing of the PCR amplicon. Sequence modification of nfsB The nfsB gene from strain FA1090 was amplified by PCR using primers NP1 and NP2. The amplicon was purified,

digested with BamHI and cloned into the BamHI site in pK18, resulting in plasmid pNFSB. To alter the poly adenine sequence at the 5′ end of the gene from

AAAAA to AAGAA, PCR primers NfsB-BsmI-F and NfsB-BsmI-R Selleckchem PD173074 were designed. The resulting amplicon was digested with BsmI, ligated, and most introduced into E. coli by transformation, giving pEC1. Plasmid pEC1 was amplified via PCR using the primers dwnstrm-F and dwnstrm-R, allowing for the insertion of a BsrGI site. A spectinomycin resistance cassette was amplified from pMP45Σ using primer Omega-ABC, and ligated into the BsrG1 site, resulting in pEC3. This plasmid was used to transform strain FA1090 to spectinomycin resistance, resulting in strain NfsB-BsmI-Ω. The spectinomycin resistance cassette was removed using the spot transformation procedure [29] with pEC1, producing a strain that had an intact modified nfsB gene(FA1090-NfsB(mod)). The correct construction was verified by DNA sequence analysis of a PCR amplicon. The DNA sequences for nfsB from the various strains have been submitted to GenBank with the following accession numbers: F62, GU112780; MS11, GU112781; FA19, GU112782; and PID2, GU112783. Point mutations in nfsB that resulted in nitrofurantoin resistance are identified in GenBank as accession numbers: GU112770; GU112771; GU112772; GU112773; GU112774; GU112775; GU112776; GU112777; GU112778; and GU112779. MIC determinations The minimum inhibitory concentration (MIC) of nitrofurantoin for several gonococcal strains was determined by a plate dilution method.