, 1991; Kalpana et al., 1991; Chua et al., 2000). To confirm that this was not occurring, we rescued the genomic region flanking the EZ::TN transposon from the mutants and looked for a 9-bp target site duplication in the mutant DNA. Analysis Selleck TSA HDAC of the DNA sequence flanking the EZ::TN transposon at MEL and MER revealed that each insertion was flanked by the 9-bp duplication characteristic of the Tn5 insertion (Table 2) (Berg & Berg, 1983), confirming that the antibiotic-resistant transconjugants arose by transposition of the EZ::TN transposon into the host chromosome. The library was screened for auxotrophic mutants to demonstrate the usefulness of the modified EZ::TN5 transposome in mutant library construction.
Five hundred BF638R transposon mutants were replica plated onto minimal media with ABT-199 in vivo or without Casamino acids (0.5% w/v) (Baughn & Malamy, 2002). One of 500 transposon mutants screened failed to grow on minimal medium without Casamino acids, suggesting
that a gene in an amino acid biosynthesis pathway was disrupted (Mutant EZY6). The disrupted gene in the auxotrophic mutant was identified by the SRP-PCR (Fig. 3). The identification of the 19-bp inverted repeat on the amplified PCR products confirmed that isolated auxotrophic mutant was a ‘true’ transposon insertant. We also identified the transposon-disrupted gene using the alternative rescue cloning method described in ‘Materials and methods’. Both the methods independently indicated that EZY6 had a mutation in argC (acetylglutamyl phosphate reductase, BF638R_0529), a gene in the arginine biosynthesis pathway. We found that the SRP-PCR technique was faster and simpler than the rescue cloning method for identifying the disrupted gene. Selected mutants that grew slowly on minimal medium were also chosen for further study. The mutated genes were identified by SRP-PCR, and results are presented in Fig. 4. PAK5 Mutants had transposon insertions in two-component regulators (EZY7), cell division
proteins (EZY11), aminotransferase (EZY17), GMP biosynthesis pathway (EZY19), transport-related proteins (EZY21), and various other genes. The disrupted genes were scattered throughout the genome of BF638R (Fig. 4), confirming that the custom EZ::TN5 transposome described here can randomly insert the transposon into the B. fragilis chromosome. The utility of the customized EZ::TN5 transposon for generating mutants in BF 9343 (ATCC 25285), BF clinical isolates, and B. thetaiotaomicron (Pumbwe et al., 2006a) was examined. The transposome was prepared from BF638R-modified pYV03. The efficiencies of the transposition in the clinical strain BF14412 and B. thetaiotaomicron were 3.6 ± 0.67 × 103 and 6.3 ± 1.2 × 103, respectively, indicating that the system may be useful for some clinical strains of BF as well as B. thetaiotaomicron. No mutants were generated in BF 9343 or the clinical isolate BF7320.