5 min; 60°C, 0.3 min & 72°C, 1 min, with a final extension at 72°C for 10 min. Following amplification, the amplicons were purified with QIAquick PCR purification kit (Qiagen, Hilden, Germany) and sequenced at ACGT (Wheeling, IL, USA). After analyzing with BioEdit software and BLAST algorithm for similarity searches, rhomboid sequences were deposited in the GenBank database (see table 3 for accession numbers). The following primers were used: 0110F, 5′-ATATTCGGCTTCGCCGGAACC-3′ (forward)

and 0110R, 5′-ACGCGAAGACAAGCGGCTATC-3′ (reverse) for MTC Rv0110 orthologs; 1337F, 5′ ACGCCGGGTGGAAGTATCTG-3′ (forward) and 1337R, 5′-CCGACGCCGGAATCAAAGACTC-3′ (reverse) for MTC Rv1337 orthologs. For MAC species, primer pair 1554F, 5′-TCGACGGTGACACCGTGTTC-3′ (forward) and 1554R, 5′-TGCCGAGCTCATGTCTTGGG-3′ (reverse) was used. For M. smegmatis, primer pairs 5036F, Belnacasan clinical trial 5′-ACGGCCGGGTGAGACAAATC-3′ (forward) and 5036R, 5′-TGGACCCGGACAACATCCTG-3′ (reverse) for homolog MSMEG_5036; 4904F, 5′-ACGCCGGATGGAAGTATCTG-3′ (forward) and 4904R, 5′-ACACCGGAATCGAAGATCCC-3′ (reverse) for homolog MSMEG_4904 were used. Primers were synthesized by IDT (Leuven, Belgium). Transcription assays mRNA was purified from mycobacteria with the Oligotex mRNA mini kit ATR inhibitor (Qiagen, Hilden, Germany) and ~60 ng/μl (in 15 μl) mRNA used as template for cDNA synthesis. Reverse Transcriptase-PCRs

were performed with the Titan One Tube RT-PCR System (Roche Applied Science, Mannheim, Germany) to amplify Rv0110 and Rv1337 cDNAs in separate reactions. Except for the initial cDNA synthesis step (50°C for 30 min), PCR conditions

were similar to those described above. RT-PCRs were repeated with primers (1337int1: TGGACGTCAACGGCATCAG, MCC950 supplier forward, and 1337int2: CCAGCCCAATGACGATATCCC, reverse) that amplify an internal fragment (~350 bp) of Rv1337 orthologs. Bioinformatic analyses Identification of rhomboids in mycobacteria Rhomboid sequences for rho-7 [GenBank: NP_523704.1] of D. melanogaster, PARL [GenBank: NP_061092.3] of human, glpG [GenBank: AAA23890] of E. coli and aarA [GenBank: L28755] of P. stuartii were obtained from GenBank [62]. These sequences were used as queries in BLAST-searches Tyrosine-protein kinase BLK for rhomboid homologs from an array of mycobacterial genome databases: “”tuberculist”" [63], GIB-DDBJ [64] and J. Craig Venter institute [65]. Sequence analysis The similarity between mycobacterial rhomboids was determined using specialized BLAST bl2seq for comparing two or more sequences [66]. Multiple sequence alignments were performed with ClustalW [67] or MUSCLE [68]. Mycobacterial rhomboids were examined for the presence of rhomboid family domains and catalytic signatures (GxSx). The TMH predictions were done using the TMHMM Server v. 2.0 [69]. The data generated was fed into the TMRPres2D [70] database to generate high resolution images. Cellular localization signals were predicted using TargetP 1.