fortuitum. The amino acid sequences of PorM1 among the M. fortuitum strains 10851/03 and 10860/03 R428 price including the type strain were identical (Figure 4). The mature PorM1 from M. fortuitum featured six amino acid substitutions compared to MspA. Figure 4 Alignment of PorM1 and PorM2 from M. fortuitum and MspA and MspC from M. smegmatis. The start codon ATG and the stop codon TGA were chosen according to the sequence of mspA. The cleavage recognition site of the signal peptidase was predicted for PorM1, PorM2 and MspC using the SignalP 3.0 Server at http://www.cbs.dtu.dk/services/SignalP/[11]. The predicted signal peptide cleavage sites corresponded
to the signal peptide cleavage site of MspA [6]. Identical amino acids are dark grey, similar amino acids are light grey and different amino acids are not shaded. For PorM1 and MspA an identity index of 94.8% was calculated, while PorM2 showed an amino acid identity Adriamycin in vitro of 90.7% 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 selleck chemicals 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 Tolmetin 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.