Expression of a Gene for a Porin-Like Protein of the OmpA Family fromMycobacterium tuberculosisH37Rv

Abstract

An open reading frame in the genomic database ofMycobacterium tuberculosis H37Rv was identified as having homology with an outer membrane protein. We found that the gene specified a protein belonging to the OmpA family, which includes some porins of gram-negative organisms. The gene was amplified by PCR and cloned into Escherichia coli. Overexpression of the gene was toxic to the host, but limited amounts could be purified from cells before growth ceased. A truncated gene devoid of the code for a presumed signal sequence was well expressed, but the protein had no pore-forming activity in the liposome swelling assay. However, the intact protein, OmpATb, behaved as a porin of low specific activity, with a pore diameter of 1.4 to 1.8 nm, and was also active in planar lipid bilayers, showing a single-channel conductance of 700 pS. The protein had a molecular mass of about 38 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal rabbit antiserum raised to the truncated protein recognized a protein of similar molecular mass in detergent extracts of broken M. tuberculosis cells. Reverse transcription-PCR confirmed that the gene for OmpATb was expressed in M. tuberculosis cells growing in culture. Comparison of the purified protein with that in the detergent-extracted preparation using liposomes and planar lipid bilayers showed that the two materials had similar pore-forming properties. OmpATb is different from either of the mycobacterial porins described so far. This is the first report of a porin-like molecule from M. tuberculosis; the porin is likely to be important in controlling the access of hydrophilic molecules to the bacterial cell.

Mycobacterial porins – new channel proteins in unique outer membranes

“…Whereas purified recombinant OmpATb without the putative N-terminal signal peptide did not show channel activity, the recombinant protein with signal peptide increased the permeability towards various sugars in liposome swelling experiments and produced channels with conductances of 0.7 nS to 3.5 nS in lipid bilayer experiments (Table 1). These results were interpreted as proof that OmpATb was a porin of M. tuberculosis (Senaratne et al, 1998). However, the recombinant OmpATb protein used in this study was probably not a good mimic of the native protein, because it still contained the putative signal peptide.…”

“…In a different approach to identify porins, a protein of M. tuberculosis with significant homology to the OmpA protein family was produced in E. coli (Senaratne et al, 1998). Whereas purified recombinant OmpATb without the putative N-terminal signal peptide did not show channel activity, the recombinant protein with signal peptide increased the permeability towards various sugars in liposome swelling experiments and produced channels with conductances of 0.7 nS to 3.5 nS in lipid bilayer experiments (Table 1).…”

The low-molecular-mass subunit of the cell wall channel of the Gram-positive Corynebacterium glutamicum . Immunological localization, cloning and sequencing of its gene porA

“…It is noteworthy, that PorA from C. glutamicum shows no homology to the first cloned mycobacterial cell wall channel OmpATb [19] or the subunit MspA of the cell wall channel from M. smegmatis [25]. There the cell wall channel proteins have considerably higher molecular mass than PorA.…”

pH‐dependent pore‐forming activity of OmpATb from Mycobacterium tuberculosis and characterization of the channel by peptidic dissection

“…Several porin-like proteins have been reported in M. tuberculosis and M. bovis BCG (Kartmann et al, 1999;Lichtinger et al, 1999;Niederweis, 2003), which were not characterized due to restricted amounts of proteins present in these cells. A BLAST search analysis on the M. tuberculosis H37Rv genome identified a protein resembling the OmpA porin of E. coli (Senaratne et al, 1998), and was named OmpATb. The similarity percentage was about 44% for their C-terminal part.…”