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Furtwängler, Katarina Saskia Lucia (2009): Expression und Funktion der ABC-Transporter für anorganisches Phosphat und Glycerin-3-phosphat in Halobacterium salinarum R1. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

The effective uptake of inorganic phosphate by cells from the environment depends on specific transport systems. In bacteria, these uptake systems are well characterized and most species contain both secondary transporters as well as primary uptake systems belonging to the ABC-family of transporters. Under phosphate starvation, genes coding for high-affinity phosphate transporters are induced in bacteria as well as in eukarya. In E. coli these are genes of the phosphate-specific transport via Pst or the Glycerol-3-phosphate-specific transporter, Ugp. Archaea possess the PHO stimulon, which induces numerous genes in response to phosphate limitation. So far this stimulon has only been described for Halobacterium salinarum R1. The genome of H. salinarum encodes ABC transporters resembling the Pst and Ugp systems of E. coli which are upregulated under Pi-limited conditions. In this study, the gene expression and function of the phosphate dependent operons pst1, pst2 and ugp of H. salinarum were investigated. First, it was shown that the three operons (pst1, pst2 und ugp) are transcribed as one polycistronic unit. The respective promoters are located upstream of the first ORF (open reading frame) of the operons, and transcription start sites (TSS) were mapped. Two TSSs were found for the pst1 operon, and are utilized in a phosphate dependent manner. Through an unknown regulation mechanism, the cell switches transcription of pst1 mRNA to either a transcript with or without a 60 nt long leader sequence. The transcripts of the pst2 and ugp operons have no 5’UTRs, regardless of phosphate concentration. Using a Ppst1-bgaH reporter system, it was observed that the transcripts with or without a leader sequence have different translation efficiencies. The transcript without a 5‘UTR had a 150-fold higher translation efficiency than the transcript with a 5‘UTR. It was concluded that the expression of the pst1 operon is modulated through a post-transcriptional regulation mechanism. To our knowledge, this is the first identified archaeal protein-coding operon that is transcribed by alternative promoters. The differences in phosphate dependent gene expression of the pst1, pst2 and ugp operons were investigated using the bgaH reporter system. Under phosphate saturated conditions, the expression of the pst2 operon is stronger compared to the expression of the pst1 operon, whereas under phosphate limited conditions, pst1 operon expression is highly induced. This assay system also identified the TATA boxes of the pst1 and pst2 promoters as well as AT-rich motifs, named P boxes. Mutations in the P box, with the consensus ATATWWW , reduced the promoter activity of both the pst1 and pst2 promoters. The results indicated that the TATA-2 box of the pst1 promoter has an impact on the phosphate dependent promoter activity under phosphate limited conditions and could be used as a P box. In the second part of the study, the proposed functions of the transporters Pst1, Pst2 and Ugp and the kinetic parameters were determined. Different knockout mutants were constructed, and these showed that the Pst transporters had different phosphate affinities. It was concluded from the results that the binding proteins PstS1 and PstS2 can interact with both transporters. The phosphate transport systems Pst1 and Pst2 are used differently. Under phosphate saturated conditions the cell operates mainly with the lower-affinity Pst2 transporter. If phosphate becomes limiting in the environment, predominantly the high-affinity transporter Pst1 is induced. This process is regulated on the transcriptional as well as on the post-transcriptional level. Furthermore, it was shown that a deletion of the Pst1 transporters leads to a loss of phosphate-directed chemotaxis under Pi-stress. This result confirms the importance of the Pst1 transporter under phosphate limited conditions. In related studies, growth experiments showed that the Ugp transporter is the only transporter for glycerol-3-phosphate in H. salinarum. In addition, the search for a regulatory protein that is involved in the regulation of the genes of the PHO stimulon did not reveal any likely candidates, but it was shown that deletion of the pst1 operon leads to an induction of the pst2 operon.