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Study of the properties of channel-forming proteins of the cell walls of different Corynebacteriae
(2008)
The genus Corynebacterium belongs, together with Mycobacterium, Nocardia, Rhodococcus and further closely related genera, to the distinctive suprageneric taxon mycolata. Many species within this diverse group of mycolic acid containing actinomycetes are known either because of their medical or biotechnological relevance. For instance, Mycobacterium tuberculosis, Mycobacterium leprae, Corynebacterium diphtheriae and Nocardia farcinica, causer of most dangerous bacterial infectious diseases world-wide, are among this exceptional group of Gram-positive bacteria. Likewise of importance are some harmless mycolata species which find use in industrial settings. Corynebacterium glutamicum and Corynebacterium efficiens are, e.g., potent producers of the flavour enhancer glutamate and the animal feed additive lysine, while several Rhodococcus species are applied in the production of acrylic acids. The cell wall of mycolata species, compared with that of Gram-positive bacteria, exhibits an unusual composition and organization. Besides an arabinogalactan-peptidoglycan complex, the cell walls of most actinomycetes contain large amounts of mycolic acids. Comparable to the outer membrane of Gram-negative bacteria, these long-chained branched fatty acids form a highly impermeable hydrophobic outer layer which provides the basis of the exceptional drug resistance of mycolata species. Like the outer membrane of Gram-negative bacteria, the cell wall of mycolata contains channel-forming proteins that allow the passage of hydrophilic solutes. By permitting and controlling the exchange and communication between the interior of the cell and the environment in which the bacterium lives, the channels play an important role for the function of the bacterial cell envelope. This thesis aimed to extend our knowledge about cell wall channels in corynebacteria. For this purpose, we examined PorA and PorH proteins that have been associated by previous studies with cell wall pores in C. glutamicum, C. efficiens and Corynebacterium callunae in order to resolve unanswered questions and to gain structural knowledge. We also investigated cell walls of pathogenic corynebacteria, in particular of Corynebacterium diphtheriae and Corynebacterium jeikeium, to investigate if these species possessed channels as is the case with their harmless relatives. In this work we provided evidence for the existence of large and water-filled cell wall channels in C. diphtheriae and C. jeikeium. Moreover, we demonstrated that the major cell wall channels of C. glutamicum, C. efficiens and C. diphtheriae consist of two distinctive polypeptides; one of whom belongs to the class of PorH proteins and the other to the class of PorA proteins. This heteromeric structure of channels of corynebacteria represents a novelty for channels of the mycolata. In contrast, the C. jeikeium channel is solely constituted by a single protein, CjPorA, arranged as an oligomer. Although the molecular mass of this protein (4kDa) is comparable to those of PorH and PorA proteins (5-7 kDa), it shares no distinctive homology in its primary sequence with them. However, there is evidence for relationship between CjPorA and PorH/PorA proteins because the gene jk0268, coding for CjPorA, is localized in a chromosomal region of C. jeikeium that corresponds to the genomic region containing the porH/porA genes in the other corynebacteria. This suggests that jk0268 (coding for the homomeric cell wall channel in C. jeikeium) and the porH/porA genes of C. glutamicum, C. efficiens and C. diphtheriae (coding for heteromeric cell wall channels) are presumably descendants of a common ancestor gene. This assumption gets support from data on phylogenetic analysis of the genus Corynebacterium. Moreover, these data suggest that the here investigated cell wall channels are presumably widespread within this genus. A profound knowledge of cell wall channels, building the main passage of solutes through the outer mycolate membrane in corynebacteria and other members of the mycolata, can be of great economical and medical value.
Study of Omp85 Family Proteins YaeT and YtfM and Multidrug Export Machineries in Escherichia coli
(2006)
In this study the Omp85 family proteins YaeT and YtfM of Escherichia coli were investigated by using biochemical and electrophysiological methods as well as bioinformatical and structural analysis. In addition, knock-out strains were constructed to further study the relevance of these proteins in vivo. The prediction that Omp85 proteins are composed of two domains, a periplasmic amino-terminal POTRA (polypeptide translocation associated) domain and a carboxy-terminal domain anchoring these proteins in the outer membrane, was confirmed by the construction of mutants. It could be shown that the carboxy-terminal part of the proteins is able to insert into the outer bacterial membrane, even if the POTRA domain is removed. Furthermore, pore-forming activity in the black-lipid bilayer was observed for both full-length proteins as well as their carboxy-terminal membrane located parts. The channels formed by both proteins in the black lipid bilayer showed variable single channel conductance states rather than a defined value for conductance. In 1M KCl, e.g. YaeT forms pores with a channel conductance of 100 to 600 pS containing a most abundant value at 400 pS. This variability is at least reasonable for YaeT due to a prerequisite flexibility of its channel for OMP insertion. YaeT was identified to form a cation selective, YtfM an anion selective channel, which is less pH dependent than YaeT. Another feature of the YaeT channel is that its selectivity and conductance is influenced by charged detergent molecules indicating an accumulation of these molecules in hydrophobic pockets inside the compact channel. YaeT revealed heat-modifiable mobility in SDS-PAGE which is characteristic for β-barrel OMPs, whereas YtfM did not show this behaviour. This result could be explained by sequence alignment and structural comparison of YaeT and YtfM via CD and FTIR spectra displaying much higher β-strand content for the carboxy-terminal part of YaeT compared to YtfM. Since the carboxy-terminal parts were shown to have pore forming ability and are inserted in the OM in vivo, the substitution of the essential protein YaeT by its carboxy-terminal mutant was attempted in a yaeT knock-out strain. The carboxy-terminal half of YaeT was not sufficient to compensate depletion of the full-length protein indicating an important role of the amino-terminus for cell viability. In contrary, YtfM is shown to be a non-essential protein and lack of YtfM had no effects on the composition and integrity of the OM. However, chromosomal deletion of ytfM remarkably reduced the growth rate of cells. This study provides the first detailed investigation of the structure of YaeT and describes its electrophysiological behaviour, which could be a basis for further studies of YaeT and its substrate proteins. Furthermore, YtfM was characterised and its in vivo function was investigated revealing YtfM as the second Omp85 family protein of importance in E. coli. In a second part of this study assembly and function of multidrug efflux pumps were investigated. Drug efflux pumps are tripartite export machineries in the cell envelope of Gram-negative bacteria conferring multidrug resistance and therefore causing severe problems for medical treatment of diseases. Protein structures of all three efflux pump components are solved, but the exact interaction sites are still unknown. Assembly of a hybrid exporter system composed of the Pseudomonas aeruginosa channel tunnel OprM, the E. coli adaptor protein AcrA and its associated transporter AcrB could be shown by chemical cross-linking, even though this efflux pump is not functional. Exchange of the hairpin domain of AcrA by the corresponding hairpin from the adaptor protein MexA of P. aeruginosa restored functionality tested by antibiotic sensitivity assays. This shows the importance of the MexA hairpin domain for functional interaction with the OprM channel tunnel. Interestingly, the hybrid protein was also able to assemble with TolC as outer membrane component to form a functional efflux pump indicating a higher flexibility of TolC compared to OprM concerning interaction partners. Based on these results, an interaction model of the hairpin domain and the channel tunnel on molecular level for AcrA and TolC as well as MexA and OprM, respectively, is presented. This model provides a basis for directed mutagenesis to reveal the exact contact sites of the hairpin of the adapter protein and the outer membrane component
The genus Borrelia belongs to the spirochete phylum, an ancient evolutionary branch of the domain bacteria that is only afar related to Gram-negative bacteria. Borreliae can be subdivided into the agents of the two borrelian-caused human diseases, Lyme disease and relapsing fever. Both disease patterns are closely related to the peculiar biology of Borrelia species and exhibit a wide spectrum of diverse clinical manifestations. Due to the small 0.91 Mb chromosome, borreliae have a lack of biosynthetic capacity. Thus, all Borrelia species are highly dependent on nutrients provided by their hosts. The transport of nutrients and other molecules across the outer membrane is enabled by pore-forming proteins, so-called porins. Porins are water-filled channels and can be subdivided into two different classes, general diffusion pores and substrate-specific porins. In terms of the Lyme disease agent Borrelia burgdorferi, three putative porins were characterized in previous studies: P13, Oms28 and P66. In contrast to Lyme disease species, the porin knowledge of relapsing fever Borrelia is low, which means that not any porin has actually been described for representatives of these agents. Thus, the general aim of this thesis was to provide insight into the porin content of both, Lyme disease and relapsing fever spirochetes. This aim could be achieved by isolating and identifying porins from Borrelia outer membranes and by biophysically characterizing them in artificial lipid membranes. In one chapter of this study, the first identification and characterization of a relapsing fever porin is presented. The pore-forming protein was isolated from outer membranes of Borrelia duttonii, Borrelia hermsii and Borrelia recurrentis and designated Oms38, for “outer membrane-spanning protein of 38 kDa”. Biophysical characterization of Oms38 was achieved by using the black lipid bilayer method and demonstrated that Oms38 forms small, water-filled channels with a single-channel conductance of 80 pS in 1 M KCl. The Oms38 channel did not exhibit voltage-dependent closure and is slightly selective for anions with a permeability ratio of cations over anions of 0.41 in KCl. Subsequently, a protein homologous to Oms38 was identified in the Lyme disease agents Borrelia burgdorferi, Borrelia garinii and Borrelia afzelii. The pore-forming protein of these species exhibits high sequence homology to Oms38 and similar biophysical properties, i.e. it forms pores of 50 pS in 1 M KCl. Interestingly, titration experiments revealed that this pore could be partly blocked by dicarboxylic anions, which means that this protein does not form a general diffusion pore but a channel with a binding-site specific for those compounds. Consequently, this porin was termed DipA, for “dicarboxylate-specific porin A”. In another set of experiments, it was shown that the porin P66 is present in both Lyme disease and relapsing fever species. Therefor, the outer membranes of the Lyme disease species Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii and the relapsing fever species Borrelia duttonii, Borrelia recurrentis and Borrelia hermsii were closer investigated. Except of the P66 homologue of Borrelia hermsii P66 of all species was highly active in artificial lipid membranes, forming pores with huge single-channel conductances between 9 and 11 nS in 1 M KCl. Moreover, the channel diameter and the constitution of Borrelia burgdorferi P66 were investigated in detail. Therefor, the P66 single-channel conductance in the presence of different nonelectrolytes with known hydrodynamic radii was analyzed in black lipid bilayers. The effective diameter of the P66 channel lumen was determined to be ~1.9 nm. Furthermore, as derived from multi-channel experiments the P66-induced membrane conductance could be blocked by certain nonelectrolytes, such as PEG 400, PEG 600 and maltohexaose. Additional blocking experiments on the single-channel level revealed seven subconducting states and indicated a heptameric constitution of the P66 channel. This indication could be confirmed by Blue native PAGE analysis which demonstrated that P66 units form a complex with a corresponding mass of approximately 440 kDa. Taking together, this thesis describes detailed biochemical and biophysical investigations of both Lyme disease and relapsing fever Borrelia porins and represents an important step forward in understanding the outer membrane pathways for nutrient uptake of these strictly host-dependent, pathogenic spirochetes. Furthermore, it provides some knowledge of the outer-membrane protein composition of Borrelia spirochetes. A profound knowledge of surface-exposed proteins, such as porins, is one precondition for the production of a successful vaccine and the drug design against the two borrelian-caused diseases.
Corynebacterium glutamicum is together with C. callunae and C. efficiens a member of the diverse group of mycolic-acid containing actinomycetes, the mycolata. These bacteria are potent producer of glutamate, lysine and other amino acids on industrial scale. The cell walls of most actinomycetes contain besides an arabinogalactan-peptidoglycan complex large amounts of mycolic acids. This three-layer envelope is called MAP (mycolyl-arabinogalactan-peptidoglycan) complex and it represents a second permeability barrier beside the cytoplasmic membrane similar to the outer membrane of Gram-negative bacteria. In analogy to the situation in the outer membrane of Gram-negative bacteria, channels are present in the mycolic acid layer of the mycobacterial cell wall for the passage of hydrophilic solutes. Molecular studies have provided far-reaching findings on the amino acid flux and its balance in C. glutamicum in general, but the L-glutamate export still remains unknown. The properties of the outer layers, typical of mycolata, seem to be of major importance in this process, and diffusion seems to play a key role for this part of the cell wall. The major aim of this thesis was to identify and study novel channel-forming proteins of the amino acid producers C. glutamicum, C. callunae and C. efficiens. Cell wall extracts of the organisms were investigated and a novel pore-forming protein, named PorH, that is homologue in all three organisms, was detected and characterized. PorHC.glut was isolated from C. glutamicum cells cultivated in minimal medium. The protein was identified in lipid bilayer experiments and purified to homogeneity by fast-protein liquid chromatography across a HiTrap-Q column. The purified protein forms cation-selective channels with a diameter of about 2.2 nm and an average single-channel conductance of about 2.5 nS in 1 M KCl in the lipid bilayer assay. Organic solvent extracts were used to study the permeability properties of the cell wall of C. callunae and C.efficiens. The cell extracts contained channel-forming activity, the corresponding proteins were purified to homogeneity by fast-protein liquid chromatography across a HiTrap-Q column and named PorHC.call and PorHC.eff. Channels formed by PorHC.call are cation-selective with a diameter of about 2.2 nm and an average single-channel conductance of 3 nS, whereas PorHC.eff forms slightly anion selective channels with an average single-channel conductance of 2.3 nS in 1 M KCl in the lipid bilayer assay. The PorH proteins were partially sequenced and the corresponding genes, which were designated as porH, were identified in the published genome sequence of C. glutamicum and C. efficiens. The chromosome of C. callunae is not sequenced, but PorHC.call shows a high homology to PorHC.eff and PorHC.glut. The proteins have no N-terminal extension, only the inducer methionine, which suggests that secretion of the proteins could be very similar to that of PorAC.glut of C. glutamicum. PorHC.glut is coded in the bacterial chromosome by a gene that is localized in the vincinity of the porAC.glut gene, within a putative operon formed by 13 genes that are encoded by the minus strand. Both porins are cotranscribed and coexist in the cell wall, which was demonstrated in RT-PCR and immunological detection experiments. The arrangement of porHC.glut and porAC.glut on the chromosome is similar to that of porBC.glut and porCC.glut and it was found that PorAC.glut, PorHC.glut, PorBC.glut and PorCC.glut coexist in the cell wall of C. glutamicum. The molecular mass of about 6 kDa of the PorH channel forming proteins is rather small and suggests that the cell wall channels are formed by oligomers. A possibly hexameric form was demonstrated for PorHC.glut in Western blot analysis with anti- PorHC.glut antibodies. Secondary structure predictions for PorHC.glut, PorHC.call and PorHC.eff predict that a stretch of about 42 amino acids of PorHC.glut and 28 amino acids of PorHC.call and PorHC.eff forms amphipathic -helices with a total length of 6.3 nm and 4.2 nm respectively. This should be sufficient to cross the mycolic acid layer. Another objective of this work was to establish an heterologous expression system for corynebacterial channel-forming proteins, to investigate the channel-forming properties of the up to now only hypothetical porins PorA, PorB, PorC from C. efficiens and PorC from C. glutamicum. We could demonstrate with recombinant expression experiments in E. coli that porBC.eff and porCC.eff encode for channel-forming proteins. They are, like PorBC.glut, anion-selective with a similar single-channel conductance of 1 nS in 1 M KCl.