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Transcriptional regulation and physiological importance of the kdp-system from the halophilic archaeon Halobacterium salinarumKixmüller, Dorthe 03 April 2012 (has links)
The high affinity, ATP-dependent K+ uptake system KdpFABC of Halobacterium salinarum, is highly induced under K+ limitation. In contrast to the well-characterized Kdp system in Escherichia coli, in which the kdpFABC genes are transcriptionally regulated by the sensor kinase/response regulator system KdpD/KdpE, transcriptional regulation of the kdp genes in H. salinarum was unknown due to the absence of halobacterial homologues of KdpD/KdpE. Furthermore, the physiological relevance of the KdpFABC K+ uptake system of H. salinarum was puzzling, since hypersaline habitats usually comprise K+ concentrations which do not induce kdp expression. In order to analyze the regulation of kdp gene expression, it was essential to gain information about the transcriptional unit(s) involved. Northern blotting, primer extension analysis and real-time RT-PCR revealed the presence of a polycistronic leaderless kdpFABCQ transcript with a putative kdp terminator or at least a potential mRNA processing site downstream of kdpQ. Furthermore, promoter truncation studies verified the so far only predicted basal transcription elements together with an upstream-located operator sequence. Since deletions of this putative operator sequence did not lead to a constitutive expression, a further component has to be involved in the regulation of the kdpFABCQ genes. However, truncation and scanning mutagenesis analyses of the kdp promoter as well as translational fusions of a halophilic beta-galactosidase to the kdp promoter excluded an additional regulatory element up- or downstream of the basal transcription elements and in the kdp-coding region. These results lead to speculations of multiple basal transcription factors to be involved. Furthermore, an inducible expression vector (shuttle vector) was constructed based on the promoter of the kdpFABCQ operon due to its, K+-sensitive features. Inducible expression systems are yet not available for H. salinarum. The resulting, replicating vector pKIX is functional and enables a K+-dependent expression from the kdp promoter with rather high induction ratios of 50-fold. Expression levels could further be improved by plasmid- and additional chromosomally encoded kdpQ and mutations generated in the kdp promoter. Since transcript levels from pKIX were found to be independent of differential target genes, the general application of pKIX as an inducible expression system is strongly supported and pKIX could, thus, be made accessible to the scientific community. To decipher the physiological relevance of the halobacterial Kdp system, H. salinarum was encountered to desiccation stress and salt crystal (halite) entombment. Halite crystals grown under non-inducing K+ concentrations with entombed strains of H. salinarum and H. salinarum deleted in the kdpFABCQ genes revealed a significantly reduced survival rate of the deletion strain upon recultivation. Additionally, a kdpFABCQ-inducing desiccation stress could already be determined on agar plates under non-limiting K+ concentrations. Furthermore, the cell morphology of H. salinarum entrapped in halite crystals resembled that of H. salinarum grown under K+-limiting conditions. Therefore, the Kdp system promotes survival of H. salinarum under desiccation stress. Furthermore, the Kdp system could be identified as at least one of the systems important for long-term survival of H. salinarum in halite.
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