Bacterial Physiology

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Bacterial Physiology A Proteomic Approach to
Oral Diseases
Peter Zilm Microbiology Laboratory Dental
School The University of Adelaide
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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.

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112 Complete Microbial Genomes - Revised March
10, 2003
Bacteria 96 species
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
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Proteomic Applications
APPROACHES Profiling Functional Structural
Perturbation (signal)
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Step by step Proteomics
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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 1990s 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.

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Iso- Electric Focusing
Mol Mass
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Protein Staining Techniques

• Sensitive protein identification methods exist
  which are compatible with
• the resolving power of 2D-PAGE.

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ExPASy Molecular Biology Server
SWISS-2DPAGE Map Selection
Escherichia coli(4.5-5.5)
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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 starvation-induced 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. MEDLINE96314059 PubMed8740179NCBI,
ExPASy, EBI, Israel, JapanPasquali 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 17547-555(1996).
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The Mechanism of Plaque Formation
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Plaque as a Biofilm
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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 preparation-
• a) 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

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Increasing solubility
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Protein recovery-sequential protein extraction of
the cell envelope of F. nucleatum ATCC 10953
Extract 1 - 8M Urea, 50mM DTT, 4 CHAPS Extract 2
7M Urea, 50mM DTT, 2M Thiourea, 4 CHAPS
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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

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pH range and IPG size
pH 3
pH 10
11cm IPG
pH 3
pH10
7cm IPG
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11cm IPG
200 kDa
14.4 kDa
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Cytosolic fraction of F. nucleatum pH 4-7
39oC
BHI
Master
11cm
CHX
pH 8.0
Control
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