Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement

Abstract

Progress in the field of mycobacterial research has been hindered by the inability to readily generate defined mutant strains of the slow-growing mycobacteria to investigate the function of specific genes. An efficient method is described that has been used to generate several mutants, including the first double unmarked deletion strain of Mycobacterium tuberculosis. Four mutants were constructed : a marked deletion of the plcABC cluster, which encodes three phospholipases C ; separate unmarked deletions in plcABC and tlyA (encoding a haemolysin) ; and a double unmarked mutant tlyA∆ plcABC∆. To accomplish this, two series of vectors were designed, the first of which, named pNIL, allows manipulation of the target gene sequence at a variety of convenient restriction sites. The second series, named pGOAL, contains marker cassettes flanked by PacI restriction enzyme sites. The final suicide plasmid vectors were then obtained by cloning a marker cassette from a pGOAL vector into the single PacI site of the pNIL vector with the modified gene of interest. Finally, a two-step strategy was employed whereby single cross-over events were first selected, then screening for the second cross-over was carried out to yield the mutant strains. This technique will now allow the construction of potential vaccine strains without the inclusion of antibiotic resistance markers, the ability to make multiple defined mutations and the possibility of making more subtle defined mutations, such as point mutations.

Characterization of CrgA, a New Partner of the Mycobacterium tuberculosis Peptidoglycan Polymerization Complexes

“…Therefore, to further understand the role of CrgA in mycobacteria, we created an M. smegmatis crgA mutant strain by following a two-step homologous recombination protocol ( Fig. 2A) (38). A gentamicin cassette was inserted 27 bp from the N-terminal end of the crgA coding region to disrupt the reading frame of the protein.…”

“…A crgA mutant strain was constructed using a suicide plasmid, pPP107 (Table 1) and a two-step recombination protocol (38). Briefly, a 996-bp BamHI-HindIII fragment containing the crgA coding region, together with the upstream 398-bp and the downstream 314-bp regions, was cloned into pMAL-c4E (Table 1, pPP102a).…”

“…The insertion led to disruption of the crgA reading frame. The ϳ1,800-bp BamHI-HindIII fragment was subsequently subcloned in the same sites of the p2NIL suicide vector (38) (Table 1, pPP103). Finally, a 6.1-kb PacI fragment carrying the lacZ, aph, and sacB genes was inserted into pPP103 to create the recombination suicide plasmid, pPP107 (Table 1).…”

Cross-species inhibition of dUTPase via the Staphylococcal Stl protein perturbs dNTP pool and colony formation in Mycobacterium

“…The strong promoter of Kanamycin gene from p2NIL plasmid [38] was PCR-amplified and cloned into the vector pGem-T-easy (Promega). The Gm-Int HindIII cassette from the pUC-Gm-Int plasmid [38] was introduced into the resulting construct to yield the integrating vectorpGem-int-empty (used as control).…”

“…The Gm-Int HindIII cassette from the pUC-Gm-Int plasmid [38] was introduced into the resulting construct to yield the integrating vectorpGem-int-empty (used as control). The Stl coding region with AU1-tag was PCR-amplified from pGEX-4T-1 expression vector (generated in [33]) and cloned after the promoter (ending in NheI restriction site) in the vector pGemint-empty with NheI restriction sites to get pGem-int-Stl.…”