Genexpressions- und Protein-DNA-Interaktions-Studien des Aminosäure-Metabolismus und metallabhängiger Prozesse in Halobacterium salinarum R1

摘要

Archaea combine bacterial with eukaryotic features to regulate cellular processes. While initiation of transcription resembles the eukaryotic RNA-Polymerase II apparatus, transcriptional regulation is predominantly bacteria-like. In this work the gene expression profile of amino acid metabolism and metal dependent processes in Halobacterium salinarum R1 was elucidated. To gain insights into transcriptional regulatory processes, microarray technology was used as a global approach. Genes encoding certain DNA binding proteins were deleted and/or overexpressed, to compare the expression pattern of the deletion- and overexpression strains, respectively, with the parental strain R1. For a better understanding of metal dependent processes H. salinarum was grown under iron starvation and compared to cells grown under normal conditions.For the investigation of metal dependent processes the DNA binding proteins SirR and TroR were chosen. To determine a possible function of the regulator protein, conclusions were drawn from a comparison of the deletion mutants ∆sirR and ∆troR, respectively, with the parental strain. SirR (staphylococcal iron regulator repressor) was shown to repress the expression of a Fe(II)/Mn(II) dependent ABC-transporter in the presence of iron. In accordance with this data the same transport operon was shown to be induced under iron starvation. Furthermore, TroR (transport related operon) was shown to repress the expression of a Mn(II)-dependent ABC-transporter. In addition, TroR induces the gene expression of the metal dependent regulator gene idr2, which represses together with iron siderophor synthesis genes. To study the amino acid metabolism in H. salinarum Lrp (leucine-responsive regulatory protein) proteins were chosen, because in both archaea and bacteria Lrp is connected to the coordination of amino acid metabolism. To take a closer look on Lrp-homologs further investigation were performed with lrp and lrpA1. Both genes are located next to genes, encoding proteins involved in amino acid metabolism. Possible Lrp target genes were identified by either constructing lrp and lrpA1 deletion mutants or overexpressing the two genes. Microarray analysis revealed that Lrp functions as a global regulator of transcription. Lrp activates the gene expression of the glutamine synthetase gene glnA, regulates the peptide- and phosphate transport, as well as the central intermediary metabolism, and activates the expression of the transcriptional regulator sirR. By the control of sirR gene expression through Lrp correlation between amino acid metabolism and metal dependent processes could be demonstrated. In contrast to Lrp, LrpA1 regulates gene expression of less genes, amongst them the aspartate transaminase gene aspB3, so that further studies were focussed on the gene regulation of aspB3. The second part of this work examines with specific protein-DNA interactions. Prior to interaction studies, RACE-analysis was used to determine 5´UTR and 3´UTR of certain transcripts. To perform protein-DNA binding studies LrpA1 and TroR were recombinantly expressed in Escherichia coli. A DNA-binding assay adapted to halophilic conditions revealed manganese dependent binding of TroR to its own promoter region. LrpA1 was also shown to bind to the lrpA1 promoter region, as well as an aspartate dependent binding to the aspB3 promoter region. CD-spectroscopy experiments could prove that the interaction between L-aspartate and LrpA1 stabilizes the secondary structure of the protein. To gain more insights into the LrpA1 and L-aspartate dependent aspB3 gene expression, northern blot analysis were performed, that showed an induction of the aspB3 transcription in the absence of L- aspartate. This occurs either in a medium lacking aspartate or after aspartate is metabolized in the stationary phase. At the same time, an induction of the lrpA1 gene expression was observed. This can be illustrated in a model that postulates a reciprocal regulation of the lrpA1 and aspB3 gene expression.