Narges Abdali

• It was commonly believed that the cell walls of Gram-positive bacteria do not represent any permeability barrier and do not contain pore-forming proteins. Members of the phylum: Actinobacteria within the order: Corynebacteriales and the family: Corynebacteriaceae as recently defined form a noticeable exception within this rule. These organisms contain in addition to the peptidoglycan layer, large amounts of lipids in their cell wall. Major constituents of their lipid layers are the mycolic acids. Many species within this diverse group of mycolic acid containing bacteria are known either because of their medical or biotechnological relevance. The emergence of drug resistance in the clinical environment has been a constant threat over the past decades. Therefore, design of new antibiotics based on the knowledge of porin properties could be helpful to control pathogenic microorganisms that have a natural resistance against certain antibiotics such as C. jeikeium, a resident of human skin, often associated with multidrug resistant nosocomial infections in immunodepressed patients. C. urealyticum, another pathogenic memberof the mycolata, is known as causative agent of urinary tract infections although it is also a bacterium of the skin flora. These two pathogenic bacteria share with the mycolata the property of having an unusual cell envelope composition and architecture. The work presented in this dissertation describes detailed biochemical and biophysical investigations of recombinant proteins of both pathogenic bacteria. The first project focuses on cell wall channels of C. jeikeium. Of interest is also the genetic engineering of the channel with the goal to alter the permeability properties of the cell wall channels by applying site-directed mutagenesis. A gene coding for a 40 amino acid long polypeptide responsible for the pore-forming activity was identified in the known genome of C. jeikeium by its similar chromosomal localization to known porH and porA genes of other Corynebacterium strains. The gene jk0268 was expressed in a porin deficient C. glutamicum or porin deficient BL21 DE3 Omp8 E. coli strains. After purification with a temporary C-terminal histidine-tag or a GST-tag at the N-terminus, the homogeneous protein caused channel-forming activity in lipid bilayers with an average conductance of 1.25 nS in 1M KCl identical to the channels formed by the detergent extracts of the whole cell wall of C. jeikeium. Zero-current membrane potential measurements performed with the voltage dependent channel implied selectivity for anions, and we demonstrate that the anion selectivity was caused by a strategically located positive charge (K24) in the channel lumen. In the second part of the presented thesis, based on partial sequencing of the protein responsible for the channel formation derived from C. glutamicum, we investigated the homology of PorA and PorH of C. glutamicum and related bacteria to PorACur of C. urealyticum by its similar chromosomal localization to known porH and porA genes of other Corynebacterium strains. The results suggested that a corresponding gene cur_1714 within the known genome sequence of C. urealyticum codes for the cell wall channel. The gene responsible for coding of PorACur in C. urealyticum was cloned into the plasmids pXmj19, pXHis and pGEX-2T for its expression in C. glutamicum ΔporAΔporH and in porin deficient BL21 DE3 Omp8 E. coli strains. Biophysical characterization of the purified protein (PorACur) suggested that cur_1714 is the gene coding for the pore-forming protein in C. urealyticum. The cell wall porin of C. urealyticum was purified to homogeneity using different biochemical methods and had an apparent molecular mass of about 4 kDa on tricine-containing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The 4 kDa protein formed ion-permeable channels in lipid bilayer membranes with a single-channel conductance of 1.5 nS ± 0.25 in 1 M KCl. The channel-forming proteins were present in detergent treated cell walls and in extracts of whole cells using organic solvents. Zero-current membrane potential measurements with different salt solutions suggested that PorACur is slightly cation selective because of negative charges localized at the channel mouth. The study is the first report of a cell wall channel in the pathogenic C. urealyticum.