Figure S1. Construction of pAL70 Figure S1. Construction of pAL70. The Hyg cassette was amplified from pIJ963 (Lydiate et al. 1989) using Pfu polymerase (Stratagene) and oligonucleotides RP594 and RP595 (Fig. S5) carrying the dif sequence of M. tuberculosis and a BglII restriction site at their 5’ ends. The resulting fragment was cloned into the single BclI site present in the mycobacterial integrative vector pSM316 (unpublished).

A B C Figure S2. Construction of pAL74 and pAL75. A) The Hyg cassette was amplified from pIJ963 (Lydiate et al. 1989) using Pfu polymerase (Stratagene) and oligonucleotides RP594 and RP803 (Fig. S5) carrying the dif sequence of M. tuberculosis and a BglII restriction site at their 5’ ends. RP803 was designed to introduce a StuI restriction site immediately before the dif sequence at the 3’ of the cassette. This fragment was cloned into in pCR-BluntII-TOPO (Invitrogen) to obtain pAL71 (A). The mycobacterial promoters Pmpt64 and PRv1818c were amplified from pMV2-33 (Delogu et al. 2004a) and pMV4-36 (Delogu et al. 2004b), respectively using Pfu polymerase using oligonucleotides RP804/805 and RP809/805 (Fig. S5) containing a DraI restriction site on their 5’ ends and designed to include the promoter original rbs into the construct (B); these two fragments were cloned into the single StuI site of pAL71 to obtain pAL74 and pAL75 (C).

P dif dif Hyg Integration P Hyg Xer-cise Figure S3. Schematic representation of the effect of integration and excision of the Hyg excisable cassette on transcription and translation of the downstream gene(s). Disruption of the gene of interest (orange) by single-step double crossover does not interrupt transcription of the downstream gene (green) due to the promoter present at the 3’ of the Hyg excisable cassette. After excision of the cassette, transcription from the physiological promoter is resumed. Even if the two genes are translationally coupled the translation of the downstream gene(s) is not interrupted due to the translation of a truncated form of the gene of interest also in the presence of the Hyg cassette. mRNA is shown as broken line.

Figure S4. Constructin of pDario1 and pDario2 Figure S4. Constructin of pDario1 and pDario2. Both vectors are derivative of the integrative plasmid pMV306 (Stover et al. 1991). The hygromycin cassettes from pAL74 and pAL75, extracted by HindIII/XbaI digestion and the GFP coding sequence, extracted from pMV10-25 (Delogu et al. 2004b) by NheI/KpnI digestion, were inserted into pMV306 digested HindIII/KpnI.

Figure S5. Fluorescent microscopy (50X) of M Figure S5. Fluorescent microscopy (50X) of M. smegmatis strains SM140 (A), SM141 (B), SM143 (C), and SM144 (D).

Table S1. Oligonucleotides used in this study Name Sequencea BglII RP594 5’-AGATCT T AAG CCG ATA AGC GAC ATT ATG TCA AGT CTG CAG GTC GTC GAG GTC-3’ RP595 5’-AGATCT A CTT GAC ATA ATG TCG CTT ATC GGC TTA GGA TGC CAG GGC CTT TC-3’ RP803 5’-AGATCT A CTT GAC ATA ATG TCG CTT ATC GGC TTA AGGCCT GGA TGC CAG GGC CTT TC-3’ DraI StuI RP804 5’-TTTAAA TCG AGC ACC ACG CGA CAC-3’ RP805 5’-TTTAAA ATC TCG CCC TTG CTC ACC AT-3’ RP809 5’-TTTAAA CTT GCC GGG ACG AGC TTC-3’ MluI RP818 5’-ACGCGT GCT ACC ACT ACC GCC ACC CG-3’ BglII RP819 5’-AGATCT ATG GTG GCC CAG AAC AGT TCG G-3’ BglII RP820 5’-AGATCT CAC AGC GAC GGC AGC GAA GC-3’ BamHI RP821 5’-GGATCC GAC GCA GAT GTT CCA GCC CG-3’ MfeI RP893 5’-AAA CAATTG CCG CAG CTG CTC GAC GCT CT-3’ BglII RP894 5’-AAA AGATCT TGG TCC GTG CGG GCG TTG-3’ BglII RP895 5’-AAA AGATCT GTC ATC CCG CAG GTG TCC AT -3’ SpeI RP896 5’-AAA ACTAGT GTA CTC GGT GGC CTT GGC AA -3’ aPutative dif sequences are boxed; restriction sites are underlined.

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