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http//pc13mi.biologie.uni-greifswald.de/sub2d/sub
2d.htm -
Jörg Bernhardt, Heiko Werner, Knut Büttner, Haike
Antelmann, Uwe Völker,


Introduction
Results and Discussion
The complete sequence of the genome (1) provides
excellent perspectives for further investigation
of B. subtilis. However, it is also fairly clear
that the sequence of the genome with its 4100
genes will only be the first step towards a
thorough understanding of the physiology of this
bacterium. One of the next steps will be the
definition of the proteome of B. subtilis, i.e.
to investigate which parts of the genome are
expressed under a particular experimental
condition, and to determine the relative
synthesis rates of the individual proteins under
these defined conditions. The two-dimensional
(2D) polyacrylamide gel electrophoresis of
protein extracts is well suited for global
studies of protein expression. Recent
developments in the 2-D technology have enhanced
its reproducibility and its capacity for large
scale studies. Using computer based analysis
systems the synthesis rates or the amount of
separated proteins can be quantified with
reasonable effort and the changes in the
protein pattern can be analyzed for a whole
variety of conditions. The information obtained
in such studies would be more valuable if the
proteins investigated were identified.
We have used 2-D gel electrophoresis in
combination with microsequencing and MALDI-TOF-MS
to identify about 200 proteins. We identified
vegetative proteins involved in carbohydrate
metabolism, amino acid metabolism, nucleotide
biosynthesis, translation and other processes (4,
6). The synthesis pattern of the proteins during
a variety of conditions was analyzed using the
analysis systems PDQuest (4, 5) and MELANIE II.
The synthesis of most of the vegetative proteins
remained similar or decreased during exposure to
a variety of stresses such as heat shock, salt-,
ethanol- or oxidative stress and during
starvation for glucose or phosphate. But each of
these stimuli induces its typical set of stress
specific proteins. Heat stress for example
induces the heat specific chaperones GroEL and
DnaK. Other proteins are induced specifically
after starvation for glucose or phosphate or
after oxidative stress. The stress-specific
proteins induced by one stress may have a
protective function against a single stimulus in
order (i) to eliminate the stressing agent or
(ii) to adapt to the stress action or (iii) to
repair damages induced by the stress. The
chaperones GroESL
or DnaK for instance might assist in the proper
protein folding, the oxidative stress specific
catalase A, the DNA binding protein MrgA, or the
alkyl hydroperoxide reductase protect the cell
against a lethal oxidative challenge or the
phosphate binding lipoprotein PstS may allow the
phosphate-starved cell to be more competitive in
exploiting low phosphate levels (3). The proteins
were grouped according to their synthesis
pattern. Using these and the following results we
begun to establish a response regulation map of
B. subtilis to define the pathways which control
the synthesis of particular proteins. A group of
about 50 proteins was induced by a variety of
stimuli. Therefore, these proteins were
designated general stress proteins (2, 5). Its
tempting to assume that these proteins may
provide the cell with an unspecific protective
resistance regardless of the factor which induced
their synthesis. After having revealed the
induction pattern of a group of proteins, the
dependence of the induction on global regulators
has to be investigated. The comparison of the
protein patterns of appropriate mutants and the
wild type by 2D electrophoresis permits the
simultaneous allocation of a large number of
proteins to specific regulons or to a
modulon. Most of the general stress proteins
(GSPs) described above absolutely required the
alternative sigma factor SigB for their stress
induction. However, some GSPs remained stress
inducible in a sigB-mutant. According to their
induction pattern these proteins could be divided
into at least two groups. The first group (ClpC,
ClpP and TrxA) was induced by various stresses
via the activation of SigB and by a second
SigB-independent mechanism. ahpC and ahpF are
transcribed from a promoter recognized by the
vegetative sigma factor SigA and display a
superimposed induction by oxidative stress. In
future we will complete the proteome map by the
analysis of additional regulators. Furthermore,
we will extend our analysis on the alcalic pH
range and on the separation and identification of
extracellular and membrane proteins.
Fig 1
Dual channel visualization
colour code of the protein labels class I
specific for heat phosphate
starvation glucose starvaton
oxidative stress housekeeping
proteins represents spots which are
not visible in the shown gel
protein spots
Because of the different methionine content and
staining differences the reallocation of protein
spots, which were identified from Coomassie
brilliant blue stained gels to the corresponding
spots of autoradiograms of 35S methionine labeled
gels poses problems.. As a solution we developed
a false color approach to visualize radiolabeled
and stained proteins in one image using dual
color channels. For this method radiolabeled cell
extracts containing enough protein for a staining
process (the gel was loaded with 250 µg protein
of Bacillus subtilis 168 20 min after imposition
to ethanol stress) were separated on the gel (Fig
2). After the gel run the protein was stained
using Coomassie brilliant blue. The dried gel was
scanned with a light scanner and after exposure
of the same gel to a phosphor screen the
autoradogram was scanned with a MD Phosphorimager
SI. After normalization the light scan and the
autoradiogram were transformed to different
coloured monochromatic images (Fig 3). The
combination of both images (sum of the RGB values
where red green yellow) results in Fig 1.
class II class III class IV
synthesis gt amount amount gt synthesis synthesis
amount
2Universität Greifswald Institut für
Mikrobiologie und Molekularbiologie 17487
Greifswald, Germany
stress / 35S addition
whole protein
1Universität Marburg Fachbereich
Mikrobiologie 35032 Marburg, Germany
accumulation of protein
Coomassie stained proteins
radiolabeled proteins
time
3Universität Osnabrück Abteilung
Mikrobiologie 49076 Osnabrück, Germany
Fig 2
Fig 3
2

Sub2D is the 2D protein index of Bacillus subtilis
Homepage
Andrea Völker, Roland Schmid, Michael Hecker
Sub2D database navigation
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KatE
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protein spot . By clicking on a spot from the
database entries a protein data heet for the
corresponding protein will be created. Besides
nomenclatory, functional, biochemical and
bibliographic data the data sheet contains the
links to almost all databases which contain
Bacillus subtilis relevant entries. For the
indication of the spot position on the 2D pattern
the user has the possibility to choose between
different gel images. For highlighting the spot
two indicators will be combined with the gel
image using the gifmerge-program. The blue flag
represents the experimentally obtained position
and the green solid circle the theoretically
calculated coordinates of the protein spot. For
the visualization of regulatory data obtained
from analysis of 2D gel internal links lead the
user to a dynamically created image map, where
the spots belonging to a regulon or stimulon are
indicated. The example shown in this poster
represents the members of the ethanol stress
stimulon. To assist an interpretation of 2D
protein patterns of Bacillus subtilis a feature
was implemented, which is able to highlight all
identified members of a metabolic pathway. For
this feature the scheme of metabolic pathways
from KEGG (Kyoto Encyclopedia of Genes and
Genomes) was integrated into Sub2D. The
mechanism of this function is demonstrated on the
posterfor the glycolytic enzymes.
To handle all the shown proteomic data the ORACLE
based fully federated database Sub2D was
constructed. To allow an easy database access
from all over the world we are using a WWW
interface. With the help of an internet browser
and the URL (see the poster title) a connection
to the homepage of Sub2D will be established.
The proteome mapping of Bacillus subtilis 168 is
still an unfinished project. Therefore access to
a part of the data is restricted by different
access levels. For a better orientation each page
will be generated with an access level dependent
navigation bar on top. The entry page contains
the search form. Making entries in the form
fields enable queries for a protein by name,
function, SwissProt acc number, SubtiList acc
number, by pI and/or MW or within defined
intervals. The search results in the generation
of the corresponding protein data sheet. Apart
from searches via search form a protein can also
be found using the protein list or a list which
additionally contains synonyms for the common
protein names. To identify a protein directly
from the 2D pattern the on the fly generated
image maps should be useful. A page containing an
overview of the availabe image maps allows the
specification of the gel image which should be
used for the identification of the
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via search form
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According to the rules for the construction of a
federated 2D database (7) the accession to Sub2D
entries from other database was ensured via an
unique procedure for every protein. This
procedure can be combined with the gene name,
synonyms known from the literature or with the
accession number of SubtiList or SwissProt.
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5
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spot name
function
synonyms
accession to other databases
11
http//sun-13.math-inf.uni-greifswald.de8880/sub2
d/pub/sub2d.show? proteinltgene or
synonymgt swissprotltswissprot accessiongt subtili
stltsubtilist accessiongt
biochemical data
method author and publication of 2D
identification
inducing env. conditions or regulators
References
experimetally observed postion
theoretically predicted postion
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available 2D gels
2D gel with indicated spot
description for the 2D gel