An Improved Xer-cise Technology for the Generation of Multiple Unmarked Mutants in Mycobacteria

Abstract

Xer-cise is a technique using antibiotic resistance cassettes flanked by dif sites allowing spontaneous and accurate excision from bacterial chromosomes with a high frequency through the action of the cellular recombinase XerCD. Here, we report a significant improvement of Xer-cise in Mycobacteria. Zeocin resistance cassettes flanked by variants of the natural Mycobacterium tuberculosis dif site were constructed and shown to be effective tools to construct multiple unmarked mutations in M. tuberculosis and in the model species Mycobacterium smegmatis. The dif site variants harbor mutations in the central region and can therefore not recombine with the wild-type or other variants, resulting in mutants of increased genetic stability. The herein described method should be generalizable to virtually any transformable bacterial species.

A nucleotidyltransferase toxin inhibits growth of Mycobacterium tuberculosis through inactivation of tRNA acceptor stems

“…Mutant strains of M. tuberculosis H37Rv were constructed by allelic exchange using recombineering ( 43 ), as previously described (fig. S2) ( 47 ). Two ~0.5-kb DNA fragments flanking the menA 3 -menT 3 operon were amplified by PCR using PrimeSTAR GXL DNA polymerase (Takara), M. tuberculosis H37Rv genomic DNA, and the primer pairs MenA 3 Am-For/MenA 3 Zc-Am-Rev or MenT 3 Zc-Av-For/MenT 3 Av-Rev, respectively (table S1).…”

Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms

“…To further investigate the specific contribution of the Glu/Ala repeats, we generated Glu 55 >Ala, Glu 59 >Val and Glu 71 >Ala single substitution mutants of PacL1, and a triple mutant in Glu 55 , Glu 59 and Glu 71 , hereafter called “3EA”. Whereas Δ2 expressing PacL1 variants with the single Glu substitutions restored resistance to 100 μM zinc, those expressing 3EA were as sensitive as parental Δ2 ( Figure 5E ) and CtpC levels were severely reduced in the Δ2 strain expressing the PacL1 3EA variant ( Figure 5F ), compared to the wild-type PacL1 or the single substitution PacL1 E 71 A. RT-qPCR analysis confirmed that pacL1 and ctpC were correctly transcribed in these recombinant strains ( Figure S7D ).…”

“…We generated a triple pacL1 -, pacL2 and pacL3 -deficient mutant of M. tuberculosis 55 , together with several complemented strains expressing target genes from the zinc-responsive promoter P pacL1 . As expected, the triple mutant was sensitive to zinc at 100 μM or 250 μM, and zinc resistance was restored in a strain complemented with P pacL1 ::PacL1 ( Figure 6C , upper panels).…”

“…Escherichia coli strains Stellar TM (Takara bio) and Rosetta™ 2 (Novagen) were grown at 37˚C in LBroth (Difco) or on L-agar plates (Difco) supplemented with streptomycin (25 μg.ml -1 ) or ampicillin (100 μg.ml -1 ) when required. Construction of M. tuberculosis and M. smegmatis mutants Mutant strains of M. tuberculosis H37Rv and M. smegmatis mc²155 were constructed by allelic exchange using the recombineering technology 67 , as described 55 . Two ~0.5 kb DNA fragments flanking the genes of interest were amplified by PCR using PrimeSTAR GXL DNA polymerase (Takara bio), M. tuberculosis H37Rv or M. smegmatis mc²155 genomic DNA, and appropriate primer pairs (Table S1).…”

“…Mutant strains of M. tuberculosis H37Rv and M. smegmatis mc 2 155 were constructed by allelic exchange using the recombineering technology 67 , as described 55 . Two ∼0.5□kb DNA fragments flanking the genes of interest were amplified by PCR using PrimeSTAR GXL DNA polymerase (Takara bio), M. tuberculosis H37Rv or M. smegmatis mc 2 155 genomic DNA, and appropriate primer pairs (Table S1).…”