To detect porin genes orthologous to mspA in M. fortuitum, preliminary hybridisation experiments were performed with a probe derived from the main porin gene of M. smegmatis mspA (accession no.: AJ001442) using the primers hpor and npor (Table 1) covering nucleotide 8 to 697. The probe hybridised to the genomic DNA from M. fortuitum strains (data not shown). Thus, orthologs seem to exist in all strains analysed.

To clone porin genes from M. fortuitum, genomic DNA of M. fortuitum 10860/03 was digested with SacII, and a 3000 bp fragment, which hybridised to the probe, was cloned in pIV2 and transformed into E. coli. Two clones (pSSp107 and pSSp108) containing porin sequences were isolated. Both plasmids were found to contain the same genomic region of 2895 bp, harbouring one porin gene. The inserts were sequenced by primer walking. Both strands of the porin genes and 400 bp of surrounding regions were sequenced at least twice. As shown in Figure 2A, the insert of the plasmids contained several open reading frames (ORFs), one of which was an ortholog of mspA. It contained 636 bp, encoding a protein of 211 amino acids with an N-terminal signal sequence of 27 amino acids, which was predicted using the SignalP 3.0 Server at http://www.cbs.dtu.dk/services/SignalP/11. The in silico analysis of the mature PorM1 (protein without signal peptide) showed a calculated molecular weight of the monomer of 19400 Da and an isoelectric point (pI) of 4.31.

A hypothetical -10 region of a promoter and a ribosome binding site (RBS) were identified upstream of the coding sequence. Downstream of the ORF a hairpin sequence was detected, which might function as a terminator (Figure 2A). It has to be noted that the sequence similarity between M. fortuitum and M. smegmatis was only restricted to the coding sequence. According to the designation of other mycobacterial porin genes and to the instructions of EMBL nucleotide sequence database, the gene was named porM1 (see Table 2 for accession no.).

Besides the porin gene, two other complete ORFs and part of another ORF were detected. ORF1 was interrupted by one of the SacII sites and showed a high similarity to a molybdopterin biosynthesis protein of M. tuberculosis CDC 1551 (accession no.: AAK 45260). ORF2 turned out to be a mechanosensitive channel orthologous to the gene mscL from M. avium subsp. paratuberculosis str. 10 (accession no.: NP 959854). ORF3 was similar to the hypothetical protein Rv0990c from M. tuberculosis H37Rv (accession no.: NP 215505). The entire cloned genomic region was blasted against the M. tuberculosis genome from the Sanger Institute database http://www.sanger.ac.uk/cgi-bin/blast/submitblast/m_tuberculosis to examine if the whole region is conserved between M. fortuitum and M. tuberculosis. However, only ORF1 and ORF2 possessed nucleotide identities higher than 60% showing that the region is not conserved among these mycobacteria.

A new probe derived from the porM1 sequence was used to detect porin genes in different M. fortuitum strains. The probe hybridised to two fragments of the SacII-digested genomic DNA of different M. fortuitum strains. However, the fragment size differed among different strains (Figure 3). Hence, the M. fortuitum genomes contain at least two porin genes.

Next, the presence of porM1 in other M. fortuitum strains was analysed. For this purpose, the porM1-specific primers komf-3f and komf-4b (Figure 2A and Table 1) were chosen to amplify a fragment of approximately 1250 bp, comprising the porM1 gene and its flanking regions. PCRs using a polymerase-mix with proofreading activity generated a fragment of the expected size in all strains. Several PCRs were performed and both strands of the different fragments were sequenced. PorM1 was detected in all three M. fortuitum strains, and the nucleotide sequences were submitted to the EMBL nucleotide sequence database (Table 2). The nucleic acid subsequences such as the -10 signal of a promoter, the RBS, the signal peptide of 81 bp and the hairpin structure were also present and were conserved among all strains tested (data not shown). As already indicated in Table 2, the nucleotide sequences of the gene porM1 differed among different strains of M. fortuitum.

The amino acid sequences of PorM1 among the M. fortuitum strains 10851/03 and 10860/03 including the type strain were identical (Figure 4). The mature PorM1 from M. fortuitum featured six amino acid substitutions compared to MspA.

Since the southern blot experiments had indicated the existence of two genes orthologous to mspA in M. fortuitum, we also attempted to clone and characterise the second porin gene. This porin gene, termed porM2, was amplified by PCR and cloned as a 918 bp fragment into the mycobacterial vectors pMV306 and pMV261, as described in the section Methods. The corresponding recombinant plasmids were named pSRa104 and pSRb103, respectively. Positive clones were confirmed by sequencing. As shown in Figure 2B, the insert of the plasmids contained an ORF of 648 bp, which turned out to be paralogous to the gene porM1. The 648 bp ORF encodes a protein of 215 amino acids with an N-terminal signal sequence of 31 amino acids, which was predicted using the SignalP 3.0 Server at http://www.cbs.dtu.dk/services/SignalP/11. The in silico analysis of the mature PorM2 showed a calculated molecular weight of the monomer of 19374 Da and a pI of 4.31, which were very similar to the calculated values of PorM1. A hypothetical -10 promoter sequence and a hypothetical RBS were located upstream of porM2. A hypothetical terminator sequence was, however, not detected (Figure 2B). The similarity between porM1 and porM2 from strains M. fortuitum 10851/03 and 10860/03 on nucleotide level amounted to 94.1% and 95.3%, respectively. The mspA gene revealed to be more similar to porM1 (87.4% to 88.4% similarity) than to porM2 (86.5% similarity). Sequence comparison revealed that porM2 encodes a protein mainly differing from porM1 within the signal sequence. PorM2 from M. fortuitum 10851/03 and 10860/03 exhibits an insertion of four amino acids and additional six amino acid exchanges within the signal peptide compared to PorM1 (Figure 4). Only one amino acid is replaced in the mature polypeptide [proline¹⁶⁵ (PorM1) with alanine¹⁶⁹ (PorM2)]. We sequenced a 1697 bp region comprising porM2, 500 bp of its upstream region as well as 549 bp downstream of porM2. The DNA sequences flanking the porM1 and porM2 genes revealed no similarity with exception of the 13 bp preceding the porM-coding sequences, which were identical in both regions. The 5' terminus of an ORF orthologous to a glycosyl transferase gene from M. tuberculosis CDC1551 (accession no.: AAK 48256) was detected upstream from porM2. An ORF orthologous to the gene for a pyridoxamine 5'-phosphate oxidase-related protein from M. vanbaalenii (accession no.: ZO 01208463) was present in the downstream region of porM2 (Figure 2B).

Using the primer pairs porM2-fw-hind and porM2-bw-hpa or porM2-rna-fw and porM2-rna-bw (Table 1), porM2 was also detected in other strains analysed. No product was obtained using the plasmid pSSp107 carrying porM1 as template, demonstrating the specificity of this PCR approach for porM2.

The expression of the porins PorM1 and PorM2 were examined by 2D-Electrophoresis, Western Blotting, ELISA and qRT-PCR. For porin protein analysis, M. fortuitum pellets were lysed in POP05 (PBS 0.5% (w/v) n-octylpolyoxyethylene/0.2% EDTA) that was shown to selectively extract MspA 12. For enhanced resolution and characterisation of the proteins, porin preparations were subjected to 2D-Electrophoresis. As shown in Figure 5A, about 50 protein spots were detected on the 2D-gel in M. fortuitum POP05 cell extracts. Western blot experiments with identical gels showed only one defined spot detected by the antiserum pAK MspA#813 6 (see Additional file 2). The protein had an apparent molecular mass of approximately 120 kDa, the expected size of the oligomeric porin, and an apparent pI of about 4, which corresponded well to the predicted pI of the mature protein of 4.31. Oligomers of the porin were readily detected in cell extracts of all M. fortuitum strains as well as in extracts from M. smegmatis that served as a positive control. After extended exposition times, the monomer of the porin was also detected on Western Blots (data not shown). The Western Blots showed considerable differences in porin protein expression among the analysed strains (see Additional file 3). Additionally, ELISA experiments with POP05 extracts were performed to quantify the amount of porin in different strains. Different dilutions of cell extracts from the various strains were loaded into the wells of a microtiter plate and porins were detected using the polyclonal antibody pAK MspA#813. Since M. bovis BCG does not possess orthologous porins 6, extracts of M. bovis BCG were employed as a control to detect the background. Amounts higher than 5 μg per well turned out to be inapplicable due to saturation effects, and the detection of porin in cell extracts failed at concentrations of about 0.04 μg per well. Therefore, the most eligible working range turned out to be 1 μg of cell extract per well. Indeed, the amount of porin differed in various strains. The highest amount of porin was detected in the internal control M. smegmatis SMR5. The type strain M. fortuitum DSM 46621 exhibited porin amounts close to those of M. smegmatis, whereas the other two strains showed clearly decreased porin amounts (Figure 5B). Notably, M. fortuitum 10851/03 exhibited the lowest amount of porin very close to the background, which was represented by the control M. bovis BCG.

Comparative expression analysis was also performed by means of quantitative reverse transcription polymerase chain reaction (qRT-PCR) using sequence-specific primers and probes (Table 1). The values were compared to porin expression in M. smegmatis. Because of the high degree of sequence conservation of the two paralogs porM1 and porM2, a qRT-PCR approach was established using primers and a dually labelled probe that hybridised to a region where both genes are identical (porM1 amplicon: nucleotide 132–232 and porM2 amplicon nucleotide 144–244, see also Table 1). This PCR approach enabled the quantification of the overall expression of the paralogs. As was already proven by the ELISA results, the highest porin mRNA expression was measured in M. smegmatis. It showed transcription rates about twice as high as the highest level among the M. fortuitum strains, which was detected in the type strain M. fortuitum DSM 46621. M. fortuitum 10851/03 exhibited the lowest transcription rate among all M. fortuitum strains (Figure 5C). The quantification of porM mRNA as well as the protein isolated from the various strains demonstrated consistence of transcriptional and translational levels and underlined the differential porin expression among the analysed M. fortuitum strains.

MspA was shown to be accessible on the cell surface of M. smegmatis by using pAK MspA#813 8. Since the expression analysis showed a differing amount of porin in M. fortuitum strains, M. fortuitum DSM 46621 and M. fortuitum 10860/03 (the strains with the highest porin expressions) were employed for detection of porins at the surface of intact mycobacteria. Porins were accessible at the surface of intact cells of M. fortuitum and were detected by the porin-specific antibody. Significantly higher absorption at 450 nm (P < 0.001) was measured for M. fortuitum DSM 46621 as well as M. fortuitum 10860/03 when compared to the relative backgrounds (see Additional file 4).

Heterologous expression of porM1 as well as porM2 was performed in the porin mutant strain M. smegmatis ML10 (ΔmspA; ΔmspC) to prove the functionality of encoded porins. For this purpose, the plasmids pSRa102 and pSRa104 harbouring porM1 and porM2, respectively, were introduced to M. smegmatis ML10. The plasmid pSSa100 13 containing mspA was employed as a positive control.

First, the growth on Mycobacteria agar plates was quantified during four days after electroporation. The growth was compared to a strain harbouring only the vector pMV306. As it is shown in Figure 6A to 6D, heterologous expression of mspA, porM1 and porM2 led to enhanced growth of complemented strains compared to the control. Figure 6A and 6B illustrate the growth retardation of strain M. smegmatis ML10 (pMV306) compared to the mspA-complemented strain M. smegmatis ML10 (pSSa100). The growth of M. smegmatis ML10 was ameliorated by both, plasmid pSRa102 as well as plasmid pSRa104 (Figure 6C and 6D), although the complementation of the growth defect by these plasmids was less pronounced than by mspA expression using pSSa100. Heterologous expression of porM1 in the M. smegmatis mutant (Figure 6C) resulted in better growth than heterologous expression of porM2 (Figure 6D).

The quantification of growth rates of the strains by cfu-counting confirmed these conclusions (Figure 6E). The strain complemented with mspA (containing pSSa100) had reached its final number of colonies after 72 hours. The transformant complemented with porM1 (containing pSRa102) also showed visible colonies after 72 hours, it had, however, not yet reached its final number of colonies after this time period. The strains containing pSRa104 (carrying porM2) and the empty vector pMV306 both showed visible colonies not until 96 hours, but ML10 (pSRa104) outnumbered ML10 (pMV306).

Knock-down of porin expression in M. fortuitum was performed by generating the plasmid pSRr106, which carries a porM antisense fragment (see Figure 2A) under the control of the hsp60 promoter. The employed antisense sequence was first tested for non-specific binding performing a blast search at http://blast.ncbi.nlm.nih.gov/Blast.cgi. The analysis ensured that the antisense fragment specifically binds to mspA class porins, such as porMs and did not show any hits to other sequences deposited in the database.

The efficiency of down-regulation via RNA antisense technique was proven by means of SYBR Green qRT-PCR using strain 10860/03. As shown in Additional file 5, the knock-down strain carrying the plasmid pSRr106 showed about four times lower porin expression compared to the control strain harbouring the vector pSHKLx1.

In order to over-express porM genes in M. fortuitum, the coding sequences of porM1 from strain M. fortuitum 10851/03 and of porM2 from strain 10860/03 were inserted downstream of the hsp60 promoter in the vector pMV261 to generate plasmids pSRb101 and pSRb103, respectively.

We first studied the impact of the modified porM expression rates on the growth of bacteria freshly transformed with plasmids pSRr106, pSRb101 and pSRb103 as well as with the empty vectors pSHKLx1 and pMV261, serving as negative controls. Strains transformed with pSHKLx1 or pSRr106 were either selected by adding kanamycin (100 μg ml^-1) or hygromycin (100 μg ml^-1) to the agar, while transformants electroporated with pMV261, pSRb101 or pSRb103 were selected by addition of kanamycin (100 μg ml^-1). The clearest results were obtained with strains 10851/03 and DSM 46621 and are displayed in Figure 7(A, B, C–E, F, G, H–K). Knock-down of porM expression in both strains resulted in considerable growth reduction (Figure 7A, B and 7F, G) substantiating an important role of porins for the growth of M. fortuitum. This was further supported by the growth pattern of the 10851/03 derivatives over-expressing porM1 or porM2 (Figure 7C–E). Compared to 10851/03 containing the empty plasmid pMV261, both derivatives over-expressing porM genes brought about a slight increase in average colony size on plates containing 100 μg ml^-1 kanamycin. This effect was more pronounced in 10851/03 over-expressing porM2 than in the strain over-expressing porM1. In DSM 46621 the porin over-expression had an adverse effect on growth upon plating on 100 μg ml^-1 kanamycin (Figure 7H–K). In order to figure out if this growth decrease was caused by an increased antibiotic uptake, we then plated the over-expressing DSM 46621 derivatives and the control on plates containing only 25 μg ml^-1 kanamycin (Figure 7L–N). Under these conditions, the over-expression of porM genes slightly enhanced the growth. Again the increase in average colony size was more pronounced upon over-expression of porM2.

Mycobacterial strains (Table 3) were grown in Middlebrook 7H9 medium (BD Biosciences, Heidelberg, Germany), supplemented with 0.05% Tween 80 and either ADC (BD Biosciences) or DC (2 g glucose, 0.85 g NaCl, in 100 ml H₂O) at 37°C without shaking, or on Mycobacteria 7H11 agar (BD Biosciences), supplemented with ADC (BD Biosciences). For selection of recombinant mycobacteria, media were supplemented when required with 25 to 100 μg ml^-1 kanamycin or 100 μg ml^-1 hygromycin B. E. coli DH5α was grown in LB medium at 37°C 35. Media were supplemented with 100 μg ml^-1 kanamycin or 200 μg ml^-1 hygromycin B for selection of recombinant E. coli. All plasmids used in this study are described in Table 4.

To compare the growth rates of M. fortuitum strains, Middlebrook 7H9/DC medium was inoculated with preparatory cultures to obtain an initial OD₆₀₀ of 0.02. During 16 days, the optical densities of the cultures were measured daily. Growth of the strains was monitored by quantification of the ATP content of the cultures with the luminescence-based kit BacTiter-Glo™ Microbial Cell Viability Assay (Promega). The luminescence was reported as relative light units (RLU) with the microplate luminometer LB96V (EG&G Berthold) 36.

Common molecular biology techniques were carried out according to standard protocols 35 or according to the recommendations of the manufacturers of kits and enzymes. Transformation of E. coli was performed according to the method of Hanahan 37. PCR reactions were performed with the following kits: Taq DNA Polymerase (MBI Fermentas, St. Leon-Roth, Germany), BC Advantage GC Polymerase Mix (Takara Bio Europe S.A., Gennevilliers, France), BIO-X-ACT Short Mix and BIOTAQ DNA Polymerase (Bioline GmbH, Luckenwalde, Germany). Sequencing reactions were performed by using the Prism Big Dye™ FS Terminator Cycle Sequencing Ready Reaction Kit from PE Applied Biosystems (Darmstadt, Germany).

Protein and nucleotide sequence analysis such as identification of DNA subsequences (e.g. promoters and terminators) was performed using the software packages MacVector™ 7.2.3 (Accelrys, Cambridge, UK) and Lasergene (DNASTAR, Inc., Madison, WI, USA). Signal peptides were predicted using the SignalP 3.0 Server at http://www.cbs.dtu.dk/services/SignalP/11.

Phylogenetic relationships among the RGM were analysed using the program ClustalW in the MacVector™ 7.2.3 package. Before analysing the phylogenetic relationships, sequences were trimmed in order to start and finish at the same nucleotide position for all employed strains. Phylograms were obtained from nucleotide sequences using the neighbour-joining method with Kimura 2-Parameter distance correction 38.

In order to clone porin genes, genomic DNA from M. fortuitum was digested to completion with the restriction enzyme SacII and separated by agarose gel electrophoresis. The DNA was then transferred to the Hybond+ membrane (GE Healthcare, Munich, Germany) as described by Sambrook and Russell 35. Porin genes were detected by means of Fluorescein-labelled probes using the primer pairs hpor and npor or mf-4IV-fw and mf-4-bw (Table 1) and the PCR Fluorescein Labelling Kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. The region around 3000 bp that was shown to hybridise to the probe was isolated out of the gel and was ligated into the unique SacII site of the plasmid pIV2 39. After transformation of E. coli DH5α, clones were screened by Dot Blot analysis. Inserts of two positive recombinant plasmids, pSSp107 and pSSp108, were sequenced. The inserts contained mspA-related sequences referred to as porM1. Identification of orthologous genes among other members of M. fortuitum was performed by PCR using the primers komf-3f and komf-4b (Table 1), which were derived from the cloned genomic region of porM1.

For the cloning of porM2, genomic DNA from M. fortuitum 10851/03 DNA was digested with the restriction enzyme SmaI and a 4200 bp SmaI fragment that had shown to hybridise to the Fluorescein-labelled probe before was eluted from the agarose gel and ligated into the SmaI site of pLITMUS38 (New England Biolabs, Frankfurt, Germany) and clones were screened as mentioned above. The insert of the only positive clone was sequenced. A 181 bp sequence similar to the 3' terminus of the coding sequence of porM1 was identified, while the following 256 bp of the 3' flanking region showed no similarity to the porM1 flank. A PCR primer within the porM2 flanking region (porM2-51-bw) and another primer hybridising to the first 19 bp of the porM1 coding sequence (porM2-51-fw) were used to amplify porM2 sequences (Table 1). After sequencing the PCR product, a reverse PCR approach was adapted to discover the 5' flanking region of porM2 including its start. Therefore, genomic DNA of M. fortuitum 10851/03 was digested with NcoI. The DNA fragments were circularised by ligation. Then a PCR was performed using the reverse primers porM2-rev-1 and porM2-rev-2 (Table 1) and the product was sequenced to obtain a complete sequence of porM2 and its flanking regions. The primers porM2-fw-hind (located 268 bp upstream of the porM2 coding sequence [CDS]) and porM2-bw-hpa (located directly downstream of the porM2 cds) (Table 1) were derived from the sequence mentioned and were chosen to amplify and clone porM2 and its regulatory sequences. The 918 bp product was cloned into the HindIII/HpaI restriction sites of the integrative mycobacterial vector pMV306 40 and the shuttle vector pMV261 40 to generate the recombinant plasmids pSRa104 and pSRb103, respectively. Positive clones were verified by sequencing. PorM2 was detected in other strains using the primer pairs porM2-fw-hind and porM2-bw-hpa or porM2-rna-fw and porM2-rna-bw (Table 1).

M. smegmatis MspA as well as porins from M. fortuitum were extracted in PBS buffer supplemented with 0.5% (w/v) n-octylpolyoxyethylene (nOPOE, Bachem, Heidelberg) and 0.2% EDTA (POP05), slightly modifying the method of Heinz and Niederweis 12. Mycobacteria were grown to an OD₆₀₀ of up to 1. Subsequently, about 150 mg of mycobacteria (wet weight) were washed twice in PBS buffer supplemented with 0.2% EDTA. Pellets were resuspended in POP05 using a ratio of 200 μl POP05 per 100 mg mycobacteria and were incubated at 100°C for 30 min. Afterwards, cell debris was sedimented by centrifugation at 27,000 × g and 4°C and the supernatant was transferred to a new tube. Quantification of protein samples was carried out using the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA). Western Blot analysis was performed using the antiserum pAK MspA#813 as described previously 13. For 2D-analysis, about 75 μg of protein was precipitated by acetone and pellets were washed with 70% acetone to desalt the sample. Afterwards pellets were resuspended in 200 μl Rehydration solution (8 M Urea, 0.5% CHAPS, 0.2% DTT, 0.5% Pharmalyte, 0.002% Bromphenol blue), incubated for 5 h at room temperature and loaded on IPG strips pH 3–5.6 NL, 11 cm (GE Healthcare). The strips were focused on an Ettan pIGphorII unit and the second dimension was run on vertical 10% SDS-PAGE gels using the Ettan Daltsix electrophoresis unit (GE Healthcare) according to the manufacturer's instructions. The gels were silver-stained using Roti-Black P (Carl Roth GmbH, Karlsruhe, Germany). The porin was detected by Western Blotting as mentioned above.

Expression of porin genes in the different strains was determined by means of qRT-PCR using the Mx3000P™ Real-time PCR System (Stratagene, La Jolla, CA, USA) or the StepOnePlus™ Real-Time PCR-System (Applied Biosystems). Mycobacteria were grown to an OD₆₀₀ of 0.8 and RNA was extracted according to the method of Bashyam and Tyagi 41. 1 or 5 μg of the RNA was treated prior to qRT-PCR with RNase-Free DNase (Fermentas GmbH, St. Leon-Roth, Germany). Reverse transcription of mycobacterial RNA was carried out using the RevertAid™ M-MuLV Reverse Transcriptase (Fermentas GmbH) and hexamers or the Access RT-PCR System (Promega, Mannheim, Germany) according to the manufacturer's protocols. The porin cDNA from M. smegmatis SMR5 42 and M. fortuitum was quantified either by amplifying a fragment of about 100 bp using the primers (mspATaqfw, mspATaqbw, mfpqPCRfw and mfpqPCRrev) as well as TaqMan-probes (mspATaqProbe and mfpqPCRprobe) or the primers porM1-51-sybr-fw and bw based on SYBR Green detection chemistry (Table 1). The qPCR reactions were performed using the SensiMix DNA Kit (Quantace Ltd., Berlin, Germany) or the Access RT-PCR System (Promega) according to the manufacturer's protocol. TaqMan quantification was carried out by running a first step at 95°C for 10 min followed by 40 cycles with 30 s at 95°C and 1 min at 58°C. SYBR Green quantification was performed by initial 10 min at 95°C followed by 40 cycles with 15 s at 95°C, 10 s at 58°C and 20 s at 72°C. Afterwards, the amplicon's melting temperature was determined ramping the temperature from 60°C to 90°C by 0.5°C steps and acquiring the fluorescence signal. cDNA amounts were determined by three measurements for each sample using a calibration curve established with known amounts of linearised pSSa100 13 in case of M. smegmatis or pSSp107 in case of M. fortuitum. DNase treated and non-reverse-transcribed controls were performed with the same samples to guarantee the absence of contaminating genomic DNA.

In addition to the qRT-PCR experiments, the amount of porin in isolates of M. fortuitum and M. smegmatis was determined by Enzyme-Linked Immunosorbent Assay (ELISA). Protein was isolated from mycobacteria using the detergent nOPOE as described above. The isolated protein (15 μl corresponding approximately to 25 μg) was diluted in 50 mM NaHCO₃, pH 9.6 to yield a protein concentration of 1 μg/100 μl. Aliquots (100 μl) of the sample and dilutions thereof were loaded to wells of a Nunc-Immuno Maxisorp Module (Nalgene Nunc International, NY, USA). After incubating the samples at 4°C overnight, wells were washed twice with TBS-T (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM MgCl₂ and 0.05% Tween 80). The surface was blocked with 3% powdered skim milk in TBS for 1.5 h at room temperature followed by three steps of washing with TBS-T. Samples were then treated with the primary antibody for 1.5 h at room temperature, using a 1:1500 dilution of the antiserum pAK MspA#813 8 in TBS. The wells were washed five times with TBS-T and were incubated for 1 h at room temperature with a 1:7500 dilution of Peroxidase-conjugated AffiniPure F (ab') 2 Fragment Goat Anti-Rabbit IgG (H+L) (Jackson Immuno Research, Soham, UK) in TBS. After five steps of washing, the reaction was performed using the SureBlue™ TMB Microwell Peroxidase Substrate (KPL, Gaithersburg, MD, USA) according to the instructions of the manufacturer. Absorption at 450 nm was measured with the microplate reader SPECTRA Fluor (TECAN, Crailsheim, Germany).

40 ml of mycobacterial culture was harvested at OD₆₀₀ of 0.8, washed with PBS-T and the pellet was resuspended in 1 ml PBS-T. 200 μl aliquots were then incubated for 30 min on ice with 1 μl of antiserum (pAK MspA#813); for detection of background pre-immune serum was given to the samples. Afterwards 1 ml PBS-T was given to each sample; mycobacteria were harvested by centrifugation and washed once with PBS-T. Pellets were resuspended in 100 μl of PBS-T, 1 μl of the secondary Peroxidase-conjugated AffiniPure F (ab') 2 Fragment Goat Anti-Rabbit IgG (H+L) (Jackson Immuno Research) was added to each sample and bacilli were incubated on ice for 30 min. After addition of 1 ml PBS-T, mycobacteria were pelleted by centrifugation and were washed once with PBS-T. Pellets were then resuspended in 500 μl of PBS-T, and 100 μl of dilutions thereof were transferred to wells of a Nunc-Immuno Polysorp Module (Nalgene Nunc International). After addition of 100 μl SureBlue™ TMB Microwell Peroxidase Substrate (KPL) and stopping the reaction by addition of 50 μl 1 M HCl, the reaction was detected by the reader SPECTRAFluor (TECAN).

The ability of porM1 and porM2 to complement the growth defect of M. smegmatis ML10 (ΔmspA; ΔmspC) 4 was examined by electroporation with the plasmids pSRa102, pSRa104, pSSa100 (Table 4) as well as the control pMV306. 750 ng of each plasmid was electroporated into M. smegmatis ML10 as described in Sharbati-Tehrani et al. 13. After electroporation the cells were diluted and plated onto Mycobacteria 7H11 agar supplemented with kanamycin (25 μg/ml) for the assessment of growth after four days and for the quantification of growth by cfu counting during four days.

In order to accomplish a simultanous knock-down of porM1 and porM2, we generated a plasmid containing a transcriptional fusion of the hsp60 promoter with the 5' region of porM genes. The primers porM1-as-1 and porM1-as-2 were used to amplify a 100 bp PCR amplicon covering the 5' region of porM1 including the Shine-Dalgarno Sequence. The PCR product was cloned into the BamHI site of pSHKLx1 43, and recombinant plasmids containing the insert in antisense orientation with respect to the hsp60 promoter were identified by sequencing. Afterwards, the selected recombinant plasmid pSRr106 was introduced into M. fortuitum by electroporation. The knock-down efficiency of the introduced antisense RNA was analysed at transcriptional level. For this purpose, RNA was isolated from M. fortuitum strains containing either pSRr106 or pSHKLx1, and porin expression was measured by SYBR Green qRT-PCR as described above.

Over-expression of porM1 or porM2, was achieved by introducing plasmids pSRb101 or pSRb103, respectively, into M. fortuitum.