Universität zu Köln

Bartsch, Anna Margarida (2015). Identification and functional characterization of cation/proton antiport systems in Corynebacterium glutamicum. PhD thesis, Universität zu Köln.

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Abstract

Corynebacterium glutamicum is a Gram-positive soil bacterium of the actinomycetes suborder Corynebacterineae and was first described in 1958 as a glutamic acid secreting bacterium. C. glutamicum is one of the major industrial production organisms for amino acids and related compounds. In addition, C. glutamicum serves as a model organism for closely related, dangerous human pathogens like Mycobacterium tuberculosis and Corynebacterium diphtheriae. This work aimes to get a better insight into Na+- and K+-homeostasis in C. glutamicum whereas the main focus was placed on the export of the cations. Good candidates for this function are cation/proton antiporters which exchange internal Na+ and/or K+ with external protons. Four putative cation/proton antiporter candidates were found in the genome of C. glutamicum: Mrp1, Mrp2, ChaA and NhaP. Their function as Na+/H+- and/or K+/H+- antiporters was proven on a physiological and on a biochemical level. The growth phenotype of C.glutamicum antiporter deficient mutants in presence of high NaCl or KCl concentrations revealed that Mrp1 and Mrp2 are the cation/proton antiporters with highest relevance during salt stress. The sole absence of Mrp1 lead to a Na+ sensitive phenotype whereas the lack of Mrp2 caused K+ sensitivity. ChaA and NhaP were of minor physiological importance. Antiporter deficient mutants also showed increased internal sodium concentrations ([Na+]i). Interestingly, the increase of [Na+]i caused the reduction of the internal potassium concentration ([K+]i). It could be shown that this fact is not caused by an altered membrane potential so that the cells probably actively downregulate [K+]i. Complementation studies with a C. glutamicum antiporter quadruple mutant (AQM) revealed that Mrp1 and ChaA are involved in Na+ ion export over a wide pH range of pH 6.5 to pH 9.0. The expression of mrp2 and nhaP at pH 9.0 also improved growth of the mutant. In addition, Mrp1, ChaA and NhaP were able to reduce the internal Na+ concentration in the C. glutamicum AQM background. The acridine orange fluorescence assay was used for the biochemical characterization of the putative antiporters. The corresponding C. glutamicum genes were expressed in the antiporterdeficient Escherichia coli strain KNabc and everted membrane vesicles were used to determine cation/proton antiport activities. All four antiporters were able to mediate Na+/H+- and K+/H+- antiport whereas Mrp1 showed a preference for sodium and NhaP seemed to prefer potassium.