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1	Kdp-dependent Kplus homeostasis of the halophilic archaeon Halobacterium salinarum Strahl, Henrik 14 December 2007 (has links) Halobacteria balance high external osmolality by the accumulation of almost equimolar amounts of KCl. Thus, steady Kplus supply is a vital prerequisite for life of these extreme halophiles. So far, Kplus was supposed to enter the cell only passively by use of potential-driven uniporters. However, the genome of the extreme halophilic archaeon Halobacterium sp. NRC-1 comprises one single operon containing the genes kdpFABC coding for homologs of the bacterial ATP-driven Kplus uptake system KdpFABC, together with an additional ORF so far annotated as cat3. Deletion of the kdpFABCcat3 genes led to a reduced ability to grow under limiting Kplus concentrations, whereas real-time RT-PCR measurements revealed both high induction rates and a transcriptional regulation of the Kdp system dependent on external Kplus concentration and growth phase. The synthesis of the high-affinity KdpFABC complex enables H. salinarum to grow under extreme potassium-limiting conditions of down to 20 µM Kplus. These results provide the first experimental evidence of ATP-driven Kplus uptake in halobacteria. The current opinion that Kplus homeostasis of H. salinarum is solely mediated via membrane potential-driven Kplus uniporters is obviously only one aspect of a more complex system. Halobacterium potassium uptake KdpFABC Cat3 universal stress protein 42.13 - Molekularbiologie 42.30 - Mikrobiologie 32 - Biologie ddc:570
2	Structure and functional dynamics of the KdpFABC P-type ATPase from Escherichia coli Heitkamp, Thomas 17 April 2009 (has links) The KdpFABC complex from E. coli functions as a high affinity K uptake system and belongs to the superfamily of P-type ATPases. So far, no information is available about the orientation of the subunits within the complex as well as its oligomeric state. By chemical crosslinking, gel filtration, electron transmission microscopy and single particle FRET analysis this study shows that the KdpFABC complex occurs as a homodimer with a dissociation constant between 30 to 50 nM. Furthermore, by means of single particle analysis of transmission electron micrographs, the solution structure of the complex at 1.9 nm resolution could be solved, thus providing the first structural analysis resolving all subunits of the holoenzyme. Based on crystal structures, it is generally assumed that P-type ATPases undergo large domain movements during catalysis. However, these conformational changes have never been shown directly. By use of single molecule FRET with alternating laser excitation, distance changes could be measured directly within KdpB during ATP hydrolysis. With this technique, distances and dwell times were determined for three conformational states in the working enzyme as well as in the orthovanadate- and the OCS-inhibited state. KdpFABC P-type ATPase potassium transport single particle analysis electron microscopy ALEX-FRET 35.70 - Biochemie: Allgemeines 42.30 - Mikrobiologie 32 - Biologie ddc:570
3	Transcriptional regulation and physiological importance of the kdp-system from the halophilic archaeon Halobacterium salinarum Kixmüller, 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. Halobacterium Halobacterium salinarum H. salinarum halophile halophil archaea archaeon kdp kdpFABC kdpFABCQ kdp-System ATP Kalium Kaliumaufnahme K+ Halit Salzkristall Austrocknung Trockenstress transkriptionelle Regulation Genexpression pKIX induzierbarer Expressionsvektor Expressionsvektor kdpQ Kdp Komplex halobacterial halophilic kdpFABC genes kdp-system high-affinity ATP-dependent potassium potassium pump K+ uptake desiccation desiccation stress halite salt crystal long-term survival transcriptional regulation gene expression inducible expression vector expression vector Kdp complex ddc:570

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