Table 1.

Key bacterial strains and plasmids

Strain or plasmidRelevant characteristic(s)Reference or source
Strains
M. smegmatismc2155Strain harboring all plasmids used herein19
 Mycobacterial lab strainA fast-growing mycobacterium. Source of glfDNA used to prepare pFP101. The identity of this strain is uncertain (it was originally thought to be M. smegmatismc2155). Its sole use in this study was as a source ofglf DNA, and its sequence is essentially the same as that ofM. smegmatis mc2155 glf DNAThis study
M. smegmatis FP101M. smegmatismc2155 with pFP101 integrated into the glf locus (orientation 1 in Fig. 3)This study
M. smegmatisFP102M. smegmatis FP101 which has undergone a second crossover event in the presence of pMVHG1:Rv3808c and pCG76:TBglfThis study
M. smegmatisFP103M. smegmatis FP101 which has undergone a second crossover event in the presence of pMVHG1:TBglf and pCG76:Rv3808c. The genome of FP103 should be identical to the genome of FP102, with the strains differing only in which rescue plasmids they carryThis study
Plasmids
 pPR27Temperature-sensitive mycobacterial origin of replication. Carries the sacB gene,gen, and the E. coli origin of replication17
 pFP101pPR27 derivative carryingglf::kan and the xylE gene (Fig. 2)This study
 pVV16pMV261 (20) with the hygromycin resistance cassette (hyg), a KAN resistance cassette (kan), and Phsp608
 pMVHG1pVV16 derivative with kan deleted, hygThis study
 pMVHG1:Rv3808cRescue plasmid for Rv3808c; Phsp60 hyg (Fig. 2)This study
 pMVHG1:TBglfRescue plasmid for glf; Phsp60 hygThis study
 pCG76E. coliand Mycobacterium shuttle vector carrying a temperature-sensitive mycobacterial origin of replication and the streptomycin resistance cassette (str)8
 pCG76:TBglfTemperature-sensitive plasmid carrying the M. tuberculosis glf gene (under the control of Phsp60) and str (Fig. 2)This study
 pCG76:Rv3808cTemperature-sensitive rescue plasmid carrying the M. tuberculosis Rv3808c gene (under the control of Phsp60) and strThis study