Charakterisierung eines ABC-Transporters für kompatible Solute in dem hyperthermophilen Archaeon Archaeoglobus fulgidus und Untersuchungen zur thermoprotektiven Wirkung kompatibler Solute in Bacillus subtilis
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Philipps-Universität Marburg
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Abstract
Compatible solutes were discovered for the first time in connection with the
osmostress response of different organisms and called osmoprotectants since
that time.
However, recently it became evident that compatible solutes protect organisms
also against other harsh growth conditions (cold, heat, dessication) and
compatible solutes play thus a more general role as stress protectants.
In the domain of Bacteria uptake systems for these compounds are well
characterized, whereas in Archaea there is only one transporter for glycine
betaine identified on molecular level. Topic of the present work was the
biochemical and physiological analysis of a putative transporter (ProU) from
the hyperthermophilic Archaeon Archaeoglobus fulgidus, which shows significant
sequence-identity to already characterized uptake systems for compatible
solutes and belongs to the family of ABC-transporters.
To investigate the substrate specificity and the kinetic parameters of the
extracellular substrate binding protein ProX it was possible to express the
protein heterologously in Escherichia coli and to purify it to homogeneity.
Substrates of ProX are glycine betaine and proline betaine and the protein
displays an affinity in the low nanomolar range for both compounds.
Above this it shows an interesting temperature dependence,
since it binds its substrates already at 25C and is not inactivated by
incubation at 100C.
To get an insight into the structure and the mode of substrate binding,
crystallization experiments were performed with the heterologously expressed
ProX. The structure of ProX was solved by Andr Schiefner
(AG Welte, university of Konstanz).
The properties of the binding protein imply that ProU serves as a high-affinity
uptake system for compatible solutes in A. fulgidus and thus ProU was the
first uptake system for compatible solutes characterized in a
hyperthermophilic Archaeon.
Investigations concerning the physiological role of glycine betaine in A.
fulgidus lead to the unexpected result that glycine betaine does not
serve for osmoprotective purposes in this organism but increases the heat tolerance of A. fulgidus.
Western blot analyses with a ProX specific antibody revealed,
that the amount of ProX depends on heat stress but is not influenced by
osmolarity.
Although it is not possible to generate deletion mutants in A. fulgidus,
the uptake of glycine betaine goes presumably solely back to the ProU system,
because database analyses revealed that the organism does not possess any
additional uptake systems for the uptake of these compounds.
To get a closer look on the role of compatible solutes as thermoprotectants,
the role of compatible solutes as heat protectants was analyzed in the
mesophilic soil bacterium Bacillus subtilis.
Most compatible solutes which are used by B. subtilis as osmoprotectans also
mediate heat protection.
An analysis of the involved uptake system showed that B. subtilis recruits
the same uptake systems under heat stress as under osmotic stress.
By means of northern blot analyses it could be shown that the transcription
of the genes of the involved transporters is increased upon heat stress.
A comparison of the glycine betaine pool of unstressed B. subtilis cells
with B. subtilis grown at 52C revealed to what extend glycine betaine is
accumulated in response to heat stress.
The glycine betaine pool of B. subtilis is identical at 37C as well as at 52C.
Thus compatible solutes are not necessarily accumulated to molar concentrations
under supraoptimal temperatures as under osmotic stress but lower amounts
are apparently sufficient to mediate effective thermoprotection.
Some organisms synthesize upon heat stress the same compatible solutes de novo
like in response to hyperosmotic growth conditions.
Since B. subtilis accumulates proline under hyperosmotic conditions,
the osmotically controlled proline biosynthetic pathway (ProHJ) was analyzed in
connection with heat stress.
Northern blot analyses revealed that the genes for the enzymes of the
osmotically controlled synthesis of proline are also significant induced under
heat stress and primer-extension analyses showed that transcription is
mediated by the same A-dependent promoter as upon high salinity.
A ProHJ mutant strain however did not show a growth defect at 52C in
comparison with the wild type and HPLC-analyses of cell extracts verified a
missing proline accumulation at 52C. B. subtilis uses exogenic compatible
solutes under heat stress and accumulates these through the Opu-transporters,
but an accumulation of compatible solutes through de novo synthesis is absent
at 52C.
Besides the well known synthesis of so-called heat-shock proteins as response
to heat stress, the accumulation of compatible solutes plays a fundamental
role as adaptation strategy to supraoptimal growth temperatures.
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Dates
Created: 2002Issued: 2003-03-27Updated: 2011-08-10
Faculty
Fachbereich Biologie
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
Archaeoglobus fulgidusBacillus subtilis
DDC-Numbers
570
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Holtmann, Gudrun: Charakterisierung eines ABC-Transporters für kompatible Solute in dem hyperthermophilen Archaeon Archaeoglobus fulgidus und Untersuchungen zur thermoprotektiven Wirkung kompatibler Solute in Bacillus subtilis. : Philipps-Universität Marburg 2003-03-27. DOI: https://doi.org/10.17192/z2003.0070.
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