Because MAP and M. avium are genetically related, initially, we thought MAP2425c and MAP2426c are truncated portions (resulting from genome annotation Z-VAD-FMK solubility dmso errors) and should have been a whole rhomboid of MAP. Thus, we aimed to determine the correct annotation

for the MAP rhomboid. Using MAV_1554 specific primers, we PCR-amplified and sequenced homologs of MAP2425c and MAP2426c (954 bp) from a cattle isolate of MAP (strain 27, see table 3); the amplicon was similar to MAP2426c and MAP2425c (containing an internal stop codon TGA at nucleotide positions 217-219, and 10 bp translating into residues Gln, His and Lys, in similar location as those of MAV1554). Thus, we confirmed the annotations for MAP2425c (hypothetical protein) and MAP2426c (hypothetical protein). It was later revealed that a nonsense mutation at nucleotide positions 217-219 (formerly TGG, the codon for Trp73), substituted guanine at the wobble

position for adenine, creating a stop codon (i.e. TGG[Trp73]→TGA[stop codon]). Usually, nonsense mutations disrupt ORFs resulting in truncated and non-functional proteins; however, this rare scenario resulted into two unique ORFs of MAP, providing the first evidence of a split rhomboid, contrasting whole orthologs of genetically related species. Although the significance of this is currently not known, cDNA was amplified from both ORFs, implying that both hypothetical proteins may be expressed (see figure 6). Table 3 Features of PCR-amplified mycobacterial rhomboids   check details Clomifene Primer Species/Strain Amplicon size (bp) ORF (bp) Amino acids Accession numberh Protein ID Orthologs of Rv0110 (rhomboid protease 1) 0110F 0110R aH37Rv 967 855 284 HM453890 ADO17908     bBCG 967 855 284 HM453894 ADO17912     cJN55 967 855 284 HM453896 ADO17914     dBN44 967 855 284 HM453892 ADO17910   5036F 5036R

eSMR5 1000 891 296 HM453900 ADO17919 Orthologs of Rv1337 (rhomboid protease 2) 1337F 1337R H37Rv 869 723 240 HM453891 ADO17909     BCG 869 723 240 HM453895 ADO17913     JN55 869 723 240 HM453897 ADO17915     BN44 869 723 240 HM453897 ADO17911   1554F 1554R fSU-36800 954 672 223 HM453898 ADO17916     g27 954 291 (MAP2426c) 72 HM453899 ADO17917         444 (MAP2425c) 147 HM453899 ADO17917   4904F 4904R SMR5 845 738 245 HM453901 ADO17920 a : M. tuberculosis b : M. bovis c : M. bovis (cattle isolate) d : M. tuberculosis (patient isolate) e : M. smegmatis (streptomycin resistant derivative of MC2155) f : M. avium (patient isolate) g : M. avium subsp. Paratuberculosis (cattle isolate) h : Accession numbers are for GenBank Primer sequences are described in methods Figure 6 Transcription analysis of mycobacterial rhomboids. A. RT-PCR amplification of Rv0110 cDNA from MTC and M. smegmatis mRNA. Lanes: M, 1 kbp DNA ladder; 1, M. tuberculosis H37Rv; 2, M. tuberculosis BN44; 3, M. bovis BCG; 4, M.