Figure S 1 Construction of p AL 70

Figure S 1. Construction of p. AL 70. The Hyg cassette was amplified from p. IJ 963 (Lydiate et al. 1989) using Pfu polymerase (Stratagene) and oligonucleotides RP 594 and RP 595 (Fig. S 5) carrying the dif sequence of M. tuberculosis and a Bgl. II restriction site at their 5’ ends. The resulting fragment was cloned into the single Bcl. I site present in the mycobacterial integrative vector p. SM 316 (unpublished).

A B C Figure S 2. Construction of p. AL 74 and p. AL 75. A) The Hyg cassette was amplified from p. IJ 963 (Lydiate et al. 1989) using Pfu polymerase (Stratagene) and oligonucleotides RP 594 and RP 803 (Fig. S 5) carrying the dif sequence of M. tuberculosis and a Bgl. II restriction site at their 5’ ends. RP 803 was designed to introduce a Stu. I restriction site immediately before the dif sequence at the 3’ of the cassette. This fragment was cloned into in p. CR-Blunt. II-TOPO (Invitrogen) to obtain p. AL 71 (A). The mycobacterial promoters Pmpt 64 and PRv 1818 c were amplified from p. MV 2 -33 (Delogu et al. 2004 a) and p. MV 4 -36 (Delogu et al. 2004 b), respectively using Pfu polymerase using oligonucleotides RP 804/805 and RP 809/805 (Fig. S 5) containing a Dra. I 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 Stu. I site of p. AL 71 to obtain p. AL 74 and p. AL 75 (C).

P dif Hyg Integration P Hyg Xer-cise Figure S 3. 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. m. RNA is shown as broken line.

Figure S 4. Constructin of p. Dario 1 and p. Dario 2. Both vectors are derivative of the integrative plasmid p. MV 306 (Stover et al. 1991). The hygromycin cassettes from p. AL 74 and p. AL 75, extracted by Hind. III/Xba. I digestion and the GFP coding sequence, extracted from p. MV 10 -25 (Delogu et al. 2004 b) by Nhe. I/Kpn. I digestion, were inserted into p. MV 306 digested Hind. III/Kpn. I.

Figure S 5. Fluorescent microscopy (50 X) of M. smegmatis strains SM 140 (A), SM 141 (B), SM 143 (C), and SM 144 (D).

Table S 1. Oligonucleotides used in this study Name Sequencea Bgl. II RP 594 5’-AGATCT T AAG CCG ATA AGC GAC ATT ATG TCA AGT CTG CAG GTC GAG GTC-3’ RP 595 5’-AGATCT A CTT GAC ATA ATG TCG CTT ATC GGC TTA GGA TGC CAG GGC CTT TC-3’ RP 803 5’-AGATCT A CTT GAC ATA ATG TCG CTT ATC GGC TTA AGGCCT GGA TGC CAG GGC CTT TC-3’ Dra. I Stu. I RP 804 5’-TTTAAA TCG AGC ACG CGA CAC-3’ RP 805 5’-TTTAAA ATC TCG CCC TTG CTC ACC AT-3’ RP 809 5’-TTTAAA CTT GCC GGG ACG AGC TTC-3’ Mlu. I RP 818 5’-ACGCGT GCT ACC ACT ACC GCC ACC CG-3’ Bgl. II RP 819 5’-AGATCT ATG GCC CAG AAC AGT TCG G-3’ Bgl. II RP 820 5’-AGATCT CAC AGC GAC GGC AGC GAA GC-3’ Bam. HI RP 821 5’-GGATCC GAC GCA GAT GTT CCA GCC CG-3’ Mfe. I RP 893 5’-AAA CAATTG CCG CAG CTC GAC GCT CT-3’ Bgl. II RP 894 5’-AAA AGATCT TGG TCC GTG CGG GCG TTG-3’ Bgl. II RP 895 5’-AAA AGATCT GTC ATC CCG CAG GTG TCC AT -3’ Spe. I RP 896 5’-AAA ACTAGT GTA CTC GGT GGC CTT GGC AA -3’ a. Putative dif sequences are boxed; restriction sites are underlined.

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