We identified a mutant, named (which stands for folate utilization enzyme for leucovorin), that is hypersusceptible to antifolates. be targeted to sensitize bacterial pathogens to classical antifolates. pneumonia, and prophylaxis against recurrent and drug-resistant infections (5C7). The absence of enzymes required for complete folate biosynthesis in humans and other mammals makes this pathway an attractive and potential target for antibiotic development (8). Whereas enzymatic activities involved in folate metabolism are rather well known, the current antimicrobial antifolates exclusively target two steps in the folate biosynthetic pathway (8C10). Trimethoprim inhibits the reduction step through the inhibition of dihydrofolate reductases, whereas sulfonamides and sulfone drugs are to classical antifolates and thus allow expanding the use of these already available antibiotics. Here, we report the identification of a novel determinant of intrinsic antifolate resistance that exists in two bacterial species of distantly related phyla, of Gram-positive Actinobacteria and of Gram-negative Proteobacteria. This mechanism thus provides a potential target for antifolate potentiation. EXPERIMENTAL PROCEDURES Chemicals and Reagents All chemicals were of the highest available quality. Unless otherwise stated, chemicals were obtained from Sigma-Aldrich. Difco media and components were obtained from Fisher Scientific. 5-Formyltetrahydrofolate racemates (6(= 1C6), (6strains AG1 (mutant were obtained from the National BioResource Project (National Institute of Genetics, Shizuoka, Japan). ArcticExpress (DE3)RP strain was from Stratagene (La Jolla, CA). Other bacterial strains and plasmids used in this study are listed in supplemental Table S1. Oligo primers were synthesized by Eurofins MWG Operon (Huntsville, AL) and are listed in supplemental Table S2. Luria broth agar was used for maintenance and propagation of transposon was used to construct a mutant library (16, 17). SB-674042 Wild-type mc2155 was transformed with pMycoMar vector. Transformed bacteria were cultivated at 28 C SB-674042 overnight to recover and allow multiplication before plating on LB agar plates containing 50 g ml?1 kanamycin. After incubation for 5 days at 39 C, single colonies were picked and cultured separately in 96-well plates in 7H9 medium and 50 g ml?1 kanamycin for 2 days. These plates were used as master plates to replicate to plates of solid NE medium (16) containing serial concentrations of sulfachloropyridazine (10, 15, 20, 25, and 50 g ml?1) or trimethoprim (1.25, 1.5, 2, SB-674042 2.5, and 3 g ml?1). Five wells at different positions of 96-well plates inoculated with wild-type strain mc2155 were used as growth controls. Colonies that grew on NE-kanamycin plates but failed to grow on plates supplemented with antifolates were subjected to two rounds of additional replication to confirm drug susceptibility patterns. Minimum inhibitory concentrations (MICs) of selected mutants to antifolates were determined by serial dilution assays (see below). Mapping of transposon insertion sites in the mutants by using an arbitrary PCR method was carried out as described previously (16, 18). Targeted Deletion of Genes Encoding 5,10-Methenyltetrahydrofolate Synthase (MTHFS) Homologs The chromosomal gene encoding MTHFS homolog (were used to amplify the kanamycin resistance cassette in pKD13 vector (supplemental Table S1). PCR products were gel-purified and directly electroporated SB-674042 to TB10 cells (MG1655, mutation locus was transferred to the wild-type strain MG1655 by P1 phage-mediated transduction as described previously (21). The entire open reading frame of was deleted using the recombineering method as described previously (18). Rabbit polyclonal to ACADS The 616-bp DNA region upstream of was PCR-amplified using primers fuel-Del1 and fuel-Del2 (supplemental Table S2). Similarly, the 492-bp downstream region was amplified using primers fuel-Del3 and fuel-Del4. These DNA arms were cloned into pYUB854 (22) flanking the built-in hygromycin cassette to create pVN842. The mc2155 cells induced to express the recombineering system from pVN701B (18). Plasmid pVN701B was later removed from mutant as described previously (18). Genetic Complementation The 1223-bp DNA fragment including ORF and.