Identification of a cation-specific channel (TipA) in the cell wall of the gram-positive mycolata Tsukamurella inchonensis: the gene of the channel-forming protein is identical to mspA of Mycobacterium smegmatis and mppA of Mycobacterium phlei.

Abstract Detergent extracts of whole cells of the Gram-positive bacterium Tsukamurella inchonensis ATCC 700082, which belongs to the mycolata, were studied for the presence of ion-permeable channels using lipid bilayer experiments. One channel with a conductance of about 4.5 nS in 1 M KCl was identified in the extracts. The channel-forming protein was purified to homogeneity by preparative SDS-PAGE. The protein responsible for channel-forming activity had an apparent molecular mass of about 33 kDa as judged by SDS-PAGE. Interestingly, the protein showed cross-reactivity with polyclonal antibodies raised against a polypeptide derived from MspA of Mycobacterium smegmatis similarly as the cell wall channel of Mycobacterium phlei . Primers derived from msp A were used to clone and sequence the gene of the cell wall channels of T. inchonensis (named tip A for T. inchonensis porin A) and M. phlei (named mpp A for M. phlei porin A). Surprisingly, both genes, tip A and mpp A, were found to be identical to msp A of M. smegmatis , indicating that the genomes of T. inchonensis , M. phlei and M. smegmatis contain the same genes for the major cell wall channel. RT-PCR revealed that tip A is transcribed in T. inchonensis and mpp A in M. phlei . The results suggest that despite a certain distance between the three organisms, their genomes contain the same gene coding for the major cell wall channel, with a molecular mass of 22 kDa for the monomer.