In vivo transposition of mariner-based elements in enteric bacteria and mycobacteria

Abstract

mariner family transposons are widespread among eukaryotic organisms. These transposons are apparently horizontally transmitted among diverse eukaryotes and can also transpose in vitro in the absence of added cofactors. Here we show that transposons derived from the mariner element Himar1 can efficiently transpose in bacteria in vivo. We have developed simple transposition systems by using minitransposons, made up of short inverted repeats flanking antibiotic resistance markers. These elements can efficiently transpose after expression of transposase from an appropriate bacterial promoter. We found that transposition of mariner-based elements in Escherichia coli produces diverse insertion mutations in either a targeted plasmid or a chromosomal gene. With Himar1-derived transposons we were able to isolate phage-resistant mutants of both E. coli and Mycobacterium smegmatis. mariner-based transposons will provide valuable tools for mutagenesis and genetic manipulation of bacteria that currently lack well developed genetic systems.

Figure 1

Location of magellan3 insertions in a plasmid. Transposition from a delivery plasmid to a target plasmid occurred in a donor cell (A) and target plasmid was transferred to a recipient cell by conjugation. The location of insertions in the target plasmid was mapped by PCR and a DNA sequencer that also quantitated fluorescence intensity (B). Arrows indicate the position of TA dinucleotides. Positions with insertions are marked with black arrows and those without insertions are marked with gray arrows.

Figure 2

(A) magellan3 insertions in the E. coli lamB gene. A library of magellan3 insertions in E. coli was constructed by conjugating a suicide vector encoding the mariner transposase and magellan3 into a recipient strain. Mutants that were resistant to lysis with a virulent λ phage were selected. To map those insertions that were within the lamB gene, individual colonies were selected and PCR was performed with a primer that hybridized within the lamB gene and a primer that hybridized to the inverted repeat of magellan3. (B) PCR products were analyzed on an agarose gel.

Figure 3

mariner transposition system for mycobacteria. Plasmid pMycoMar, which encodes the Himar1 transposase and the magellan4 minitransposon, was introduced into M. smegmatis by electroporation. After overnight recovery at 30°C without selection, transformants were plated on medium containing kanamycin and incubated at either 30°C (to determine the total number of transformants) or 39°C (to isolate insertion mutants).

Figure 4

Map of magellan4 insertion in a D29 phage-resistant mutant. (A) The location of the insertion with the orientation of the kanamycin resistance gene (kan) and the chromosomal mpr gene. The inverted repeats of the magellan4 minitransposon are indicated by filled arrowheads. (B) The sequence of the transposon-chromosome junctions. The duplicated TA dinucleotide is indicated with outlined letters.