Study of the channel proteins in the cell wall of the Actinomycetales

  • This thesis is composed of three major studies about channel proteins of the order Actino-mycetales. The cell wall of Streptomyces coelicolor A(3)2 was found to contain a channel-forming protein in reconstitution experiments using artificial lipid bilayer. Membrane experiments indicated that the channel protein has a single-channel conductance of 1.75 nS in 1 M KCl. The channel presumably represents a general diffusion pore for transport of antibiotics as it binds several antibiotics. To identify the gene coding for this channel within the known genome of S. coelicolor A(3)2, the pore-forming protein was sequenced, the candidate genes were inactivated and each mutant was inspected for channel forming activity. Unfortunately, none of the mutants lacked the described pore-forming activity and the identity of the gene could not be attributed to any of the candidate genes. The cell wall of Dietzia maris contained a pore-forming protein as observed from reconstitution experiments with artificial lipid membranes. The porin had a molecular mass of about 120 kDa and formed ion-permeable channels in lipid bilayer membranes with a single-channel conductance of about 5.8 nS in 1 M KCl. Further biophysical analysis indicated that the porin exhibit selectivity for cations because of negative charges localized at the channel mouth. The 120 kDa porin is voltage dependent and has a diameter of 2.1 nm. The porin was subjected to protein analysis by mass spectrometry but its sequence had no significant homology to any known porin sequences which could be indicative of a novel kind of porin. The existence of a cell wall channel of Rhodococcus erythropolis composed of 8.4 kDa monomers has already been described. Based on partial sequencing of the pure protein, we were able to identify the corresponding gene within the genome of R. erythropolis. In order to confirm whether this gene is responsible for the channel activity of R. erythropolis, it was cloned in different expression vectors including the shuttle vector pXMJ19, pXHis and pGEX-2T. The new constructs were transformed into Corynebacterium glutamicum ΔporHΔporA and Escherichia coli BL21 (DE3) Omp8 host cells. However, the expression was problematic as we used other host rather than Rhodococcus and it is conceivable that the protein degraded due to incorrect folding during or after expression.

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Publishing Institution:IRC-Library, Information Resource Center der Jacobs University Bremen
Granting Institution:Jacobs Univ.
Author:Samaneh Mafakheri
Referee:Roland Benz, Matthias Ullrich, Sven Bergström
Advisor:Roland Benz
Persistent Identifier (URN):urn:nbn:de:gbv:579-opus-1004481
Document Type:PhD Thesis
Date of Successful Oral Defense:2014/09/24
Year of Completion:2014
Date of First Publication:2014/10/01
PhD Degree:Biochemical Engineering
School:SES School of Engineering and Science
Library of Congress Classification:T Technology / TP Chemical technology / TP248.13-248.65 Biotechnology / TP248.3 Biochemical engineering. Bioprocess engineering
Call No:Thesis 2014/25

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