Enhancement of targeted homologous recombination in Mycoplasma mycoides subsp. capri by inclusion of heterologous recA

Abstract

A suicide plasmid, pExp1-ctpA::tetM-recAec, employing recA from Escherichia coli and tetM as a selection marker, was used to generate ctpA knockout mutants in Mycoplasma mycoides subsp. capri through targeted gene disruption. Inclusion of E. coli recA greatly enhanced both the consistency and the recovery of mutants generated by homologous recombination.

FIG. 1.

Disruption of ctpA in Mycoplasma mycoides subsp. capri by homologous recombination using pExp1-ctpA::tetM-recAec. (A) Diagrammatic information on preparation of pExp1-ctpA::tetM-recAec; (B) diagrammatic representation of genomic DNA of the Mycoplasma mycoides subsp. capri wild type and M. mycoides subsp. capri ctpA::tetM mutant obtained by homologous recombination; (C) relevant genomic locations (GenBank CP001621) of Mycoplasma mycoides subsp. capri strain GM12 (the complete genome is available at http://cbi.labri.fr/outils/molligen/). P, promoter; USEPRO, USE and promoter; PRO_ctpA, ctpA promoter; For, forward; Rev, reverse; RBS, ribosome binding site.

FIG. 2.

Disruption of the ctpA gene through homologous recombination results in loss of the proteolytic phenotype and is enhanced by inclusion of E. coli recA. (A) PCR amplification of genomic DNA using primer pair 4 and the corresponding phenotype from the M. mycoides subsp. capri wild type and M. mycoides subsp. capri ctpA::tetM mutant obtained by homologous recombination using pExp1-ctpA::tetM-recAec. (B) PCR amplification of genomic DNA using primer pair 4 from the M. mycoides subsp. capri wild type and six ctpA::tetM mutants obtained through homologous recombination using pExp1-ctpA::tetM-recAec. Numbers at the left of the blots are molecular weights. (C) Consistent recovery of mutants with disruption in ctpA was enhanced by inclusion of exogenous recA. #, number of; RIM, random insertional mutagenesis; HR, homologous recombination; Mcap, Mycoplasma capricolum; Mmcap, M. capricolum.