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A common strategy of bacteria to adapt to hypo osmotic environmental conditions is the uptake or synthesis of osmotic active substances, referred to as compatible solutes. This thesis deals mainly with aspects based on the synthesis of those compatible solutes. A commonly used compatible solute in bacteria is the tetrahydropyrimidine derivative ectoine, which can be converted enzymatically into the hydroxylated form hydroxyectoine by the ectoine hydroxylase EctD.
In order to understand the hydroxylation reaction in more detail, x-ray structure analysis were used to analyze the crystal structure of the ectoine hydroxylase EctD from V. salexigens in cooperation with the working group of Prof. Dr. K. Reuter (University Marburg). With the intention to select amino acids for mutagenesis studies, three approaches were used: 1) the EctD crystal structure in complex with Fe3+, 2) a comparative analysis of the active site of the EctD structure with putative ectoine binding proteins from ectoine transporters and 3) bioinformatics comparison of putative EctD protein sequences. Results from those mutagenesis studies of highly conserved amino acid revealed important information about their function and the possible synthesis mechanism of EctD enzyme.
As we were not able to crystallize EctD in a complex with its substrate ectoine or its co-substrate 2-oxoglutarate, putative ectoine hydroxylases from a variety of microorganisms were cloned and purified via affinity chromatography for further crystallization studies. All purified ectoine hydroxylases from the bacteria Sphingopyxis alaskensis, Geobacillus. sp. Y412MC10, Pseudomonas stutzeri A1501, Alkalilimnicola ehrlichi, Acidiphilium cryptum and Halomonas elongata as well as from the crenarchaeon N. maritimus were enzymatically active. The activity of each enzyme and their biochemical parameters like Km, vmax and kcat were measured after optimization of the assay conditions regarding pH value, temperature and salt concentration.
Additional bioinformatics of ect genes, which are mainly organized in gene clusters, showed interesting genes inside those gene clusters. Continually a ask gene encoding for an aspartokinase was identified. Aspartokinases synthesize the essential precursor for ectoine synthesis β-aspartate-semialdehyde from L-aspartate in cooperation with the Asd enzyme. The genome of Pseudomonas stutzeri A1501 contains two genes encoding for putative aspartokinases. Whereas one of those genes is located in the ect gene cluster, the other gene is found at another position in the genome. Both genes were cloned, the proteins were heterologously produced in E. coli and characterized by enzymatic approaches. Those analyses show important difference in features between both aspartokinases.
The physiological role of both competitive solutes ectoine and hydroxyectoine was investigated using growth studies with the actinomycete Streptomyces coelicolor A3(2). Ectoine and hydroxyectoine protects S. coelicolor from the effects of high osmotic conditions and high temperatures. Thereby, the mixture of ectoine und hydroxyectoine in a 1:1 ratio leads to the most efficient protection.
In order to investigate the initiation of the uptake of competitive solutes by S. coelicolor at a certain degree of salinity and temperature, transport studies with radioactive labeled [14C]-ectoine were used. The combination of both stress factors, high salt and high temperature, leads to the highest uptake of competitive solutes in the cell.