Xer site-specific recombination, an efficient tool to introduce unmarked deletions into mycobacteria

Abstract

Genetic manipulation of mycobacteria still represents a serious challenge due to the lack of tools and selection markers. In this report, we describe the development of an intrinsically unstable excisable cassette for introduction of unmarked mutations in both Mycobacterium smegmatis and Mycobacterium tuberculosis.

FIG. 1.

Putative dif site sequences of M. avium, M. tuberculosis, and M. smegmatis. Dashes represent nucleotides identical to those of the M. avium sequence.

FIG. 2.

Excision of the dif-hyg-dif cassette from the M. smegmatis MS100 and M. tuberculosis TB44 chromosomes. The chromosomes of 4 Hyg-sensitive MS100 derivatives and 4 TB44 Hyg-sensitive derivatives were analyzed by PCR with primers flanking the excisable cassette. The amplification of a 520-bp band indicated the excision of the cassette. (A) Lane 1, 1-kb ladder (New England); lanes 2 to 5, PCR products from TB44 derivatives; lane 6, M. tuberculosis H37Rv. (B) Lanes 1 to 4, PCR products from MS100 derivatives; lane 5, M. smegmatis mc²155; lane 6, marker VIII (Roche).

FIG. 3.

Analysis of mycobacterial mutants. Introduction of a 247-bp deletion in MSMEG_2694. (A) Agarose gel electrophoresis of PCR fragments obtained with primers flanking the regions used for the recombination. Lane 1, 1-kb ladder (New England); lane 2, mutant strain; lane 3, no-DNA control; lane 4, mc²155. (C) Partial sequence of the PCR product shown in lane 2. The first 59 bp of MSMEG_2964 is followed by the dif sequence (in bold) flanked by BglII restriction sites (italicized and underlined) and the last 33 bp of MSMEG_2964. Introduction of a 123-bp deletion in Rv3790. (B) Agarose gel electrophoresis of PCR fragments obtained with primers flanking the regions used for the recombination. Lane 1, merodiploid mutant strain; lane 2, H37Rv; lane 3, 1-kb ladder (New England); lane 4, no-DNA control. (D) Partial sequence of the lower PCR product shown in lane 1. The dif sequence (in bold) flanked by BglII restriction sites (italicized and underlined) was replaced 123 bp of Rv2790.

FIG. 4.

Sequence of the M. tuberculosis putative dif site in the positive strand (A) and in the negative strand (B). Amino acids encoded by the three reading frames are shown in capital letters. Termination codons are indicated by boxes.

FIG. 5.

(A) Schematic representation of the excisable hyg cassette carried by pAL74 (Pmpt64) and pAL75 (PRv1818c); dif sites are shown in gray. (B) Sequence of the open reading frame (orf) on the 3′ side of the cassette. The dif site is boxed, and the restriction sites are underlined. (C) Western blot analysis performed on M. smegmatis. Lane 1, MS140; lane 2, MS143; lane 3, empty; lane 4, MS141; lane 5, MS143. GFP was detected using a monoclonal antibody.