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Bacterial Physiology
A Proteomic Approach to
Oral Diseases
Peter Zilm
Microbiology Laboratory
Dental School
The University of Adelaide
Genomics versus Proteomics
• Post Genomic era- Reading of the human genome sequence
• Relatively few medical breakthroughs derived from genetic research
- Can cellular processes be understood by screening genomes?
- The organisation and timing of cellular events is not a projection
of the genome and its transcription.
• Proteomics - relies on genomics to facilitate protein identification
- which genes are important and under which circumstances
-combination of proteomic and genomic information will
likely lead to the understanding of fundamental processes
such as cell development and growth, cell differentiation,
Cell signaling and cell death.
Post-translational
processing
112 Complete Microbial Genomes - Revised March 10, 2003
Bacteria – 96 species
Agrobacterium tumefaciens
Bacillus subtilis
Bifidobacterium longum
Borrelia burgdorferi
Brucella suis
Buchnera aphidicola
Campylobacter jejuni
Caulobacter crescentus
Chlamydophila pneumoniae
Chlamydia trachomatis
Chlorobium tepidum TLS
Clostridium perfringens
Clostridium tetani E88
Corynebacterium efficiens YS-314
Escherichia coli K12
Fusobacterium nucleatum
Haemophilus influenzae
Helicobacter pylori 26695
Lactococcus lactis subsp. lactis
Mycobacterium tuberculosis H37Rv
Mycobacterium leprae
Mycoplasma pneumoniae
Neisseria meningitidis MC58
Pasteurella multocida
Porphyromonas gingivalis
Pseudomonas aeruginosa
Rickettsia conorii
Salmonella typhimurium LT2
Salmonella typhi
Shigella flexneri
Staphylococcus aureus N315
Staphylococcus epidermidis
Streptococcus mutans UA159
Streptococcus pneumoniae
Streptococcus pyogenes
Streptomyces coelicolor
Treponema pallidum
Tropheryma whipplei
Thermotoga maritima
Ureaplasma urealyticum
Vibrio cholerae
Vibrio vulnificus
Xanthomonas campestris
Wigglesworthia brevipalpis
Xanthomonas citri
Xylella fastidiosa Temecula1
Yersinia pestis CO92
Archaea - 16 species
Aeropyrum pernix K1
Archaeoglobus fulgidus
Halobacterium sp.
Methanobacterium thermoautotrophicum
Methanococcus jannaschii
Methanopyrus kandleri AV19
Methanosarcina acetivorans str.C2A
Methanosarcina mazei Goe1
Pyrobaculum aerophilum
Pyrococcus abyssi
Pyrococcus furiosus
Pyrococus horikoshii
Sulfolobus solfataricus
Sulfolobus tokodaii
Thermoplasma acidophilum
Thermoplasma volcanium
Proteomic Applications
Characterisation of specific sub-sets of
the proteome.
Global Characterisation
of proteome
APPROACHES
Profiling
Functional
Structural
• Regulons or stimulons
• Macromolecular complex or sub-cellular
compartment
• Immunogenic proteins
• Problems with specific post-translational
modification
Perturbation (signal)
State1
State 2
(Healthy)
(disease)
Profile 1
Profile 2
Step by step Proteomics
Sample
preparation
Publish
results
2D-P.A.G.E
Image
analysis
Spot
identification
Data analysis
Protein
identification
Spot cutting &
Mass spec.
analysis
2-Dimensional Gel Electrophoresis
Iso- Electric Focusing
• In the 1st dimension, proteins are separated according to their charge.
• Electrophoretic migration is dependent upon
pH charge dependence and “iso-electricity.
• Since the 1990’s the position of proteins within
gels and their position within the pH gradient
could be correlated with the amino acid
composition of polypeptides.
P.A.G.E.
• In the 2nd dimension proteins are separated according to their relative
mass.
• Thousands of proteins can be displayed in a single experiment.
Iso- Electric Focusing
Mol Mass
Protein Staining Techniques
•Sensitive protein identification methods exist which are compatible with
the resolving power of 2D-PAGE.
Protein required for detection
5000
ng prot
4000
3000
2000
1000
0
ponceau amido
S
black
CBB
stain
India ink colloidal
silver
ExPASy Molecular Biology Server
SWISS-2DPAGE Map Selection
Escherichia coli(4.5-5.5)
: P26427
1 protein has been found in the clicked spot (2D-0015D5):
View entry in original SWISS-2DPAGE format
Entry name
AHPC_ECOLI
Primary accession number
P26427
Entered in SWISS-2DPAGE in
Release 02, August 1995
Last modified in
Release 16, May 2003
Description
Alkyl hydroperoxide reductase C22 protein (EC 1.6.4.-) (SCRP-23) (Sulfate starvationinduced protein 8) (SSI8).
Gene name(s)
AHPC OR B0605 OR C0694 OR Z0749 OR ECS0644 OR SF0524
From
Escherichia coli. [TaxID: 562]
Taxonomy
Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae;
Escherichia.
[1]
MAPPING ON GEL.
MEDLINE=96314059; PubMed=8740179;[NCBI, ExPASy, EBI, Israel, Japan]
Pasquali C., Frutiger S., Wilkins M.R., Hughes G.J., Appel R.D., Bairoch A., Schaller D., Sanchez
J.-C., Hochstrasser D.F.;
"Two-dimensional gel electrophoresis of Escherichia coli homogenates: the Escherichia coli
SWISS-2DPAGE database.";
Electrophoresis 17:547-555(1996).
The Mechanism of Plaque Formation
Plaque as a Biofilm
Growth Changes & Cellular Fractionation
• Growth of F. nucleatum by continuous culture- maintain growth parameters while changing a
single factor of interest.
• Growth conditions examined – growth rate
growth temperature
redox potential
growth pH
presence of chlorhexidine (antimicrobial)
nutrient availability
biofilm growth
• Sample preparationa) consideration of mol. Wt. and pI.
b) reduce the complexity of the protein mixture, (cytoplasmic and
cell envelope).
c) degradation of proteins by proteases
d) removal of nucleic acids
e) staining- determined by amount of protein
f) protein contamination
Increasing
solubility
Protein recovery-sequential protein extraction of
the cell envelope of F. nucleatum ATCC 10953
Growth
Protein mg/ml
Condition Extract 1 Extract 2
BHI
8.7
0.53
Oxygen
4.8
0.41
Control
4.8
0.26
CHX
8.7
0.71
39oC
7.7
0.53
pH 8.0
8.3
0.64
SDS
1.27
0.57
0.11
0.12
0.98
0.26
Extract 1 - 8M Urea, 50mM DTT, 4% CHAPS
Extract 2 – 7M Urea, 50mM DTT, 2M Thiourea, 4% CHAPS
Iso-electric focusing - considerations for the novice
• salt, protein solubility and ampholyte concentration
• What size format? – 7cm, 11cm, 17cm
• pH range – 10 possible
• Protein concentration during rehydration.
• Active or passive rehydration
pH range and IPG size
pH 3
pH 10
11cm IPG
pH 3
7cm IPG
pH10
pH 4
11cm IPG
pH7
200 kDa
14.4 kDa
Cytosolic fraction of F. nucleatum pH 4-7
Master
39oC
BHI
11cm
pH 8.0
Control
CHX