Biochemical and biophysical characterization of various cell wall channel-forming proteins

  • The mycolic-acid layer of certain gram-positive bacteria, the mycolata, represents an additional permeability barrier for the permeation of small water-soluble solutes. Consequently, it was shown in recent years that the mycolic acid layer of individual bacteria of the group mycolata contains pores, called porins, for the passage of hydrophilic solutes. Corynebacterium amycolatum, a pathogenic Corynebacterium species, belongs to the Corynebacteriaceae family but it lacks corynomycolic acids in its cell wall. Despite the absence of corynomycolic acids the cell wall of C. amycolatum contains a cation-selective cell wall channel, which may be responsible for the limited permeability of the cell wall of C. amycolatum. In the first project of this thesis and based on partial sequencing of the protein responsible for channel formation derived from C. amycolatum ATCC 49368 we were able to identify the corresponding gene coram0001_1986 within the known genome sequence of C. amycolatum SK46 that codes for the cell wall channel. The corresponding gene of C. amycolatum ATCC 49368 was cloned into the plasmid pXHis for its expression in Corynebacterium glutamicum. Biophysical characterization of the purified protein (PorAcoram) suggested that coram0001_1986 is indeed the gene coding for the pore-forming protein PorAcoram in C. amycolatum ATCC 49368. The protein belongs to the DUF3068 superfamily of proteins, mainly found in bacteria from the family Corynebacteriaceae. The nearest relative to PorAcoram within this family is an ORF which codes for PorAcres, which was also recognized in reconstitution experiments as a channel-forming protein in Corynebacterium resistens. More general view of the distribution of this pore-forming proteins within the genus Corynebacterium was performed by extending the search for the homologous proteins to PorAcoram to pathogenic representatives of Corynebacteriales, the C. jeikeium strain K411, the C. urealyticum strain DSM 7109 and C. variabile DSM 44702. A further objective of this thesis was to elucidate the presence of the human VDAC I-like protein in the gram-negative pathogen Legionella pneumophila. Genome sequence screening of different strains such as Paris, Lens and Philadelphia has revealed the arrangement of eukaryotic-like proteins which is unique in Legionella. It is hypothesized that these eukaryotic-like proteins likely contribute in adaptation to the eukaryotic host cells. The existence of protein sequences forming a multi-protein complex comparable to the mammalian mitochondrial permeability transition pore (MPTP) of eukaryotes contributing in the cell apoptosis has been recently described. The most abundant protein in the outer membrane of mitochondria forming the core of (MPTP) is the voltage-dependent anion-selective channel (VDAC) which transports metabolites through the outer membrane as the major conduit. Blast alignment research has shown homologous sequence (Lpg1974) in the genome of L. pneumophila with 22% identity and 35% conservative replacement with the human mitochondrial VDAC 1 protein which has unknown function up to now. To consider the hypothesize whether or not the Lpg1974 is a VDAC-like protein, the Lpg1974 was cloned in pGEX-2T vector and expressed in E. coli KS26 which lacks major porins. Biophysical characterization of this protein after purification exhibited that the Lpg1974 has a channel forming activity with a single-channel conductance around 5.5 nS.

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Publishing Institution:IRC-Library, Information Resource Center der Jacobs University Bremen
Granting Institution:Jacobs Univ.
Author:Nafiseh Soltanmohammadi
Referee:Roland Benz, Mathias Winterhalter, Miquel ViƱas Ciordia
Advisor:Roland Benz
Persistent Identifier (URN):urn:nbn:de:101:1-201307119402
Document Type:PhD Thesis
Date of Successful Oral Defense:2013/03/22
Date of First Publication:2013/04/12
PhD Degree:Biochemical Engineering
School:SES School of Engineering and Science
Library of Congress Classification:Q Science / QR Microbiology / QR75-99.5 Bacteria
Call No:Thesis 2013/4

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