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Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis
M Niederweis1, S Ehrt, C Heinz
1Lehrstuhl für Mikrobiologie, Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.
Abstract:
Porins form channels in the mycolic acid layer of mycobacteria and thereby control access of hydrophilic molecules to the cell. We purified a 100 kDa protein from Mycobacterium smegmatis and demonstrated its channel-forming activity by reconstitution in planar lipid bilayers. The mspA gene encodes a mature protein of 184 amino acids and an N-terminal signal sequence. MALDI mass spectrometry of the purified porin revealed a mass of 19 406 Da, in agreement with the predicted mass of mature MspA. Dissociation of the porin by boiling in 80% dimethyl sulphoxide yielded the MspA monomer, which did not form channels any more. Escherichia coli cells expressing the mspA gene produced the MspA monomer and a 100 kDa protein, which had the same channel-forming activity as whole-cell extracts of M. smegmatis with organic solvents. These proteins were specifically detected by a polyclonal antiserum that was raised to purified MspA of M. smegmatis. These results demonstrate that the mspA gene encodes a protein of M. smegmatis, which assembles to an extremely stable oligomer with high channel-forming activity. Database searches did not reveal significant similarities to any other known protein. Southern blots showed that the chromosomes of fast-growing mycobacterial species contain homologous sequences to mspA, whereas no hybridization could be detected with DNA from slow growing mycobacteria. These results suggest that MspA is the prototype of a new class of channel-forming proteins.
Insights
Researchers identified a novel, stable, channel-forming protein oligomer in Mycobacterium smegmatis, named MspA. This protein, encoded by the mspA gene, is crucial for regulating molecule passage through the mycobacterial cell wall.
Area of Science:
- Microbiology
- Structural Biology
- Molecular Biology
Background:
- Porins are essential for controlling hydrophilic molecule transport across the mycobacterial mycolic acid layer.
- Understanding these channels is critical for deciphering nutrient uptake and drug resistance mechanisms in mycobacteria.
Purpose of the Study:
- To purify and characterize a novel channel-forming protein from Mycobacterium smegmatis.
- To elucidate the genetic basis and structural properties of this protein's channel activity.
Main Methods:
- Protein purification and functional reconstitution into planar lipid bilayers.
- Gene identification (mspA) and protein characterization using MALDI mass spectrometry.
- Expression studies in Escherichia coli and immunological detection with specific antiserum.
- Southern blot analysis to investigate gene distribution across mycobacterial species.
Main Results:
- A 100 kDa protein, MspA, was purified from M. smegmatis, exhibiting significant channel-forming activity.
- The mspA gene encodes a 184-amino acid mature protein; the mature MspA forms a stable, active oligomer.
- MspA shows no significant homology to known proteins, suggesting it represents a new class of channel proteins.
- Homologous mspA sequences are present in fast-growing mycobacteria but absent in slow-growing species.
Conclusions:
- MspA is a novel, highly stable, oligomeric channel protein from Mycobacterium smegmatis, essential for regulating molecular transport.
- The mspA gene and its protein product represent a new class of mycobacterial outer membrane proteins.
- The distribution of mspA suggests a role in the adaptation of fast-growing mycobacteria.