TABLE 1.

Screening for mtrA::Km^r gene replacements in M. tuberculosis H37Rv

Row	Stepa	Parental strain plasmid(s)b	Selection/ screen	Expected genotype	Resulting strain plasmidc	No. of colonies screened	% Legitimate eventsd
A	Integration	pTZ113 (mtrA::KmrxylE+ sacB+)	Kmr/XylE+ Sucs	mtrA::Kmr-mtrA+	Cointegrate	191	2.6
B	Resolution	Cointegrate	Sucr/XylE− Kmr	mtrA::Kmr	—	500	0
C	Resolution	Cointegrate	Sucr/XylE− Kms	mtrA+	—	600	100
D	Resolution	pTZ178 (mtrA+ Hygr)	Sucr/XylE− Kmr	mtrA::Kmr	pTZ178 (mtrA+ Hygr)	200	96
E	pTZ178 loss	pTZ178 (mtrA+ Hygr)	39°C/Hygs	mtrA::Kmr	—	100	0
F	pTZ193 loss	pTZ193 (Hygr)	39°C/Hygs	mtrA::Kmr-mtrA+	—	200	100
G	pTZ178 loss	pTZ178 (mtrA+ Hygr), pTZ199 (Strr)	Strr/Hygs	mtrA::Kmr	pTZ199 (Strr)	100	0
H	pTZ193 loss	pTZ193 (Hygr), pTZ199 (Strr)	Strr/Hygs	mtrA::Kmr-mtrA+	pTZ199 (Strr)	100	100

^aSteps involved in the construction or resolution of the mtrA disruption. Parental strains were M. tuberculosis H37Rv (row A), M. tuberculosis H37Rv mtrA::Km^r-mtrA⁺ sacB⁺ xylE⁺ (rows B and C), M. tuberculosis H37Rv mtrA::Km^r-mtrA⁺(pTZ178) (rows D, E, and G), and M. tuberculosis H37Rv mtrA::Km^r-mtrA⁺(pTZ193) (rows F and H). 

^bPlasmids were pTZ113 (pUC18 mtrA::Km^r sacB⁺ xylE⁺ [Fig. 1]), pTZ178 [pUC12 oriM (ts) Hyg^r mtrA⁺], pTZ193 [pUC12 oriM (ts) Hyg^r], and pTZ199 (pMV261 oriM xylE⁺ Str^r). Cointegrate refers to pTZ113 integrated on the chromosome of H37Rv via single-crossover homologous recombination with the mtrA gene. 

^c—, no plasmid. 

^dA legitimate event is achievement of the expected genotype.