ZMBH J.Bericht 2000 - Zentrum für Molekulare Biologie der ...

of individual proteins. The combination of peptide
mass finger-printing and peptide fragmentation which
matches the masses of in gel proteolytically generated
peptides against theoretically digested proteins from
the M. pneumoniae database, has proved to be very
effective and reliable. Proteome analysis is limited by
the sensitivity of mass spectrometry and the pH range
of the first dimension of the two-dimensional gel electrophoresis.
Presently proteins with isoelectric points
Figure 1: 2-D-gel of a total protein extract from M. pneumoniae.
First dimension (1-D) immobilized pH gradient,
second dimension (2-D) 12,5% SDS-polyacrylamide gel.
All stained protein spots (≈ 200) were assigned to the corresponding
genes, but only the strongest spots are marked
with a gene number.
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Figure 2: Mock 2-D-gel of all proposed proteins of M.
pneumoniae. Based on the predicted molecular mass and
isoelectric point, each protein could be localized in this
mock 2-D-gel. The yellow and blue dots represent the sum
of all proposed proteins; the „blue“ proteins have been
assigned to a gene.
between 3-10 can only be separated routinely by
immobilized pH gradients (Fig. 1), but the annotation
of the genome sequence of M. pneumoniae predicts
236 proteins with pIs greater than 10 (Fig. 2). When
we analyze the protein extract of entire M. pneumoniae
cells by two-dimensional gel electrophoresis
and visualize the proteins by silver staining, about 450
proteins can be detected and about 200 proteins by
staining with the less sensitive Coomassie blue (Fig.
1). So far, 200 proteins have been identified by mass
spectrometry and assigned to the corresponding genes.
We found in most cases a good correlation between
DNA sequence based prediction of pI and mass of a
protein and its final position within a two-dimensional
gel. However, exceptions have been found where the
experimentally derived values were different. We suggest
that in many of these exceptions posttranslational
modification might have occurred. Furthermore,
a comparison of transcriptome and proteome analysis
shows that a relatively high copy number of a given
transcript does not always correlate with a strong pro-
tein signal and vice versa. Those genes could be interesting
examples for regulation of gene expression at
different levels. The proteome analysis is being done
in cooperation with R. Frank, Heidelberg and A. Görg,
München.
IV. Is there a cytoskeleton-like structure in M.
pneumoniae?
J. Regula, A. Boonmee, W. Schaller
The M. pneumoniae genome does not encode for a
single gene known to be involved in the synthesis
of a bacterial cell wall. M. pneumoniae is only surrounded
by a cytoplasmic membrane that, exceptionally
among bacteria, contains cholesterol as an essential
component. Over the past twenty years evidence
has accumulated that M. pneumoniae possesses a cytoskeleton-like
structure, probably as a substitute for the
missing cell wall. In analogy to the cytoskeleton of
eukaryotic cells, such a structure could provide the
necessary framework for maintaining and stabilizing
the shape of M. pneumoniae, for motility and for the
formation of an asymmetric cell.
The first experimental evidence for a cytoskeleton-like
structure in M. pneumoniae was provided by Meng
and Pfister (1980) who detected fibrous structures
by electron microscopy after treating M. pneumoniae
cells with the nonionic detergent Triton X-100, which
removed the membrane and the cytoplasm. These
observations were confirmed and extended by several
other researchers. These experiments and studies on
the architecture and composition of eucaryotic cytoskeletons
from cells which have been treated with the
detergent Triton X-100 suggested that a cytoskeletonlike
structure would also be enriched in the Triton
X-100 insoluble fraction of M. pneumoniae.
Therefore, we decided to determine the protein com-
position of the Triton X-100 insoluble fraction of
M. pneumoniae by 2-D-gel electrophoresis and mass
spectrometry.
Silver staining of 2-D gels of the Triton X-100 insoluble
fraction revealed about 100 protein spots. By
staining with colloidal Coomassie blue about 50 protein
spots were visualized of which 41 were identified
by determining the mass and the partial sequence of
their tryptic peptides following enzymatic digestion.
The identified proteins belonged to several functional
categories, mainly energy metabolism, translation and
heat shock response. In addition, we found lipoproteins
and most of the proteins involved in cytadherence
which were known to be components of the
Triton X-100 insoluble fraction based on evidence
from previous experiments. There were also 11 functionally
unassigned proteins. The quantitatively most
prevalent proteins were the heat shock protein DnaK,
the elongation factor Tu and the subunits α and ß
of the pyruvate dehydrogenase (PdhA, PdhB). To
prove whether a cytoskeleton-like structure exists
in Mycoplasma pneumoniae, further experiments are
required.
One of the promising newer methods to identify in
vivo interacting proteins is the two-hybrid system in
Saccharomyces cerevisiae. We started in cooperation
with M. Kögel (LION Bioscience, Heidelberg) a twohybrid
analysis with the aim of finding which of the
proposed proteins of the cytoskeleton-like structure
interact directly. We expect that this approach will
also reveal new candidates for structural components.
As starting material for our analysis we used the gene
hmw2. Our selection is based on data from various
laboratories indicating that the gene hmw2 codes for
one of the key components in the formation of the
cytoskeleton-like structures. So far, we have identified
several interacting proteins. These results are now
being verified by a second independent method.
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