Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of gram-negative bacterial genomes

Abstract

A series of modular mini-transposon derivatives which permit the rapid cloning and mapping of the DNA flanking the minitransposon's site of insertion has been developed. The basic plasposon, named TnMod, consists of the Tn5 inverted repeats, a conditional origin of replication, rare restriction endonuclease multiple cloning sites, and exchangeable antibiotic resistance cassettes. The broad host range and low target DNA sequence specificity of the Tn5 transposase, in combination with the flexibility afforded by the modular arrangement of TnMod, result in a versatile tool for the mapping of insertional mutations and the rapid recovery of clones from gram-negative bacteria.

FIG. 1

Construction of pTnMod-OGm. Arrows and boxed text indicate the construction manipulations. The pMB1 origin of replication is represented by a shaded box; the gentamicin resistance cassette from p34S-Gm is represented by an open arrow. Restriction sites in parentheses have been eliminated. All plasmids are drawn to scale. All commonly used restriction sites are labeled on the pTnMod-OGm plasposon illustrated at the bottom of the figure. IR, inverted repeat; oligos, oligonucleotides.

FIG. 2

Restriction maps of the antibiotic resistance cassettes in the p34S plasmids. The cassette inserts in the p34S plasmids are drawn to scale. The duplicate multiple cloning sites flanking the antibiotic resistance cassettes are shown for p34S-Cm and are represented by small hexagons for the other p34S plasmids. Common restriction sites which can be used to determine the orientations of the antibiotic cassettes in pTnMod variants are shown above the DNA. The corresponding GenBank accession numbers are shown to the right of the p34S derivatives.

FIG. 3

Schematic maps of the constructed TnMod variants. TnMod variants are listed with their origins of replication, the types and orientations of their antibiotic resistance cassettes, and their GenBank accession numbers. The flanking rare multiple cloning sites immediately inside the inverted repeats are denoted by the boxes on the left and right sides of each plasposon. Restriction sites on the left side of each plasposon are BglII, SfiI, FseI, SgfI, AscI, NsiI, SalI, and KpnI. Restriction sites on the right side of each plasposon are SstI, PacI, SwaI, XbaI, SpeI, and NotI.