A mariner-based transposition system for Listeria monocytogenes

Abstract

In this study, we developed a new mariner-based transposition system for Listeria monocytogenes. The mariner-based system has a high rate of transposition and a low rate of plasmid retention, and transposition is very random, making it an ideal tool for high-throughput transposon mutagenesis in L. monocytogenes.

FIG. 1.

Physical map of the mariner-based transposon delivery vectors pMC38 and pMC39. The vectors comprise the Escherichia coli p15A low-copy-number replication origin (5), the RP4 ori for conjugative transfer (20), the pE194ts gram-positive temperature-sensitive replication origin (11, 25), a gram-negative chloramphenicol resistance gene (cat) (5), a gram-positive noninducible erythromycin resistance gene (ermC) (25) flanked by the 29-bp ITR of the Himar1 mariner (15), a gram-positive kanamycin resistance gene (kan) (12) as a screening marker for loss of the plasmid, and the Himar1 mariner transposase gene (tpase) (16). The indicated restriction sites represent those used for cloning, but they are not necessarily unique. Gm⁻, gram negative.

FIG. 2.

Locations of 77 sequenced Himar1 insertions in the chromosome of L. monocytogenes. The bars located outside the circle indicate transposons that were oriented in the same direction as the positive strand, whereas the bars located inside the circle indicate transposons that were oriented in the opposite direction. Each quarter of the chromosome is identified with the approximate base pair, except for bp 1, which is identified by an arrow.