Figure 1

Transposon TrpA-cat-Mu and overview of the transposon mutagenesis strategy. (A) The transposon was made from Cat-Mu [33] by the addition of a gene cassette containing trpA and Pfdx. Orange rectangles indicate 50 bp of Mu R-end DNA. The trpA gene (pink arrow) under the control of Pfdx promoter (red arrow) was used for the selection in H. volcanii; the cat gene (green arrow) under the cat promoter (blue arrow) was used for the selection in E. coli. The transposon was released from its carrier plasmid by BglII digestion. (B) H. volcanii H53 DNA was partially digested with HpaII, AciI, and TaqI and used as a target in an in vitro Mu transposition reaction with TrpA-cat-Mu as a donor DNA. From transposition products, 4 to 6 kb fragments were isolated and cloned into the ClaI site of pBlueSript SK+ to yield a plasmid library. Inserts were released by digesting with XhoI + HindIII or KpnI + EcoRV, gel-purified, and used to transform H. volcanii H295 cells to generate a transposon insertion mutant library.