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Charakterisierung des putativen Außenmembran-Proteins TTC0322 von Thermus thermophilus

Charakterisierung des putativen Außenmembran-Proteins TTC0322 von Thermus thermophilus

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PÖLTL, Dominik, 2006. Charakterisierung des putativen Außenmembran-Proteins TTC0322 von Thermus thermophilus

@mastersthesis{Poltl2006Chara-8811, title={Charakterisierung des putativen Außenmembran-Proteins TTC0322 von Thermus thermophilus}, year={2006}, author={Pöltl, Dominik} }

deposit-license Characterization of the putative outer membrane protein TTC0322 of Thermus thermophilus Pöltl, Dominik The bacterium Thermus thermophilus lives in hot thermal springs and has potential importance for biotechnology, as it contains thermostable proteins.<br />In this study the characterization of the protein TTC0322 was initiated. Bioinformatical research predicts either a localization in the periplasm or in the outer membrane. Considering the localization on the chromosome, upstream genes encode for an ABCtransporter for glucose and an energizing system like ExbB/ExbD or Tol/Pal. This led to the model of a function of TTC0322 in the active uptake of glucose across the outer membrane. In this model TTC0322 is an active outer membrane transporter for glucose, which is activated by the putative energizing proteins encoded by the three direct upstream genes. To test the model a TTC0322-knockout-mutant was constructed by inserting a bleomycin-resistence-cassette into the gene TTC0322. In a following phenotypically characterization it was found that TTC0322 is essential for growth on glucose, but not for other sugars. Nevertheless, our hypothesis was disapproved, because the knockout-mutant continued transporting glucose. Besides, by knocking out TTC0322, the glucose-ABC-transport-system was constitutively present, which normally is regulated through presence of glucose. That implicates a regulatory effect of TTC0322 on induction. Whether this effect is primarily related to this protein itself or carried out by some second protein influenced through it, was not determined. At this point, a conclusion about the exact role of TTC0322 in T. thermophilus is not possible. Furthermore I tried to express a his-tagged version of TTC0322 in E. coli and T. thermophilus in order to purify it and start some localization studies with it. Low expression only worked in E. coli, whereas T. thermophilus showed no expression, making it impossible to purify the protein or localize it in cellular extracts of T. thermophilus. Additionally there were efforts made to localize the protein by cell-fractionating T. thermophilus wild-type and TTC0322 mutant strains. However, this method was unsuggestive of the localization of TTC0322. By using some bioinformatic research tools extensively in order to find out other hints on this proteins function, it could be shown that the encoding gene isn t necessarily located in one operon with the glucose-ABC-transporter-genes, since another<br />promoter in front of these genes was found. It might form an own operon, including only additional the genes for the energizing system.<br />Another interesting observation alongside these experiments is that MalK, which<br />is known as the ATP-hydrolyzing subunit of both ABC-transporters for glucose and<br />maltose, might also be part of a so far unknown fructose-ABC-transporter. Experiments showed that a knockout-mutant of malK lost its ability to grow on fructose. 2006 2011-03-24T17:46:38Z 2011-03-24T17:46:38Z deu Pöltl, Dominik Charakterisierung des putativen Außenmembran-Proteins TTC0322 von Thermus thermophilus application/pdf

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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