Download E. Coli

Jizhong Zhou, Dorothea K. Thompson, Ying Xu
Prepared by: Merced M. Gutierrez
Nano Biomaterials Science Laboratory
Supervisor: Prof. Sung Chul Yoon
Gyeongsang National University
Chapter 11
The Functional Genomics of
Model Organisms:
Addressing Old Questions
from a New Perspective
Dorothea K. Thompson and Jizhong Zhou
Table of Contents
11.1 Introduction
11.2 Escherichia coli: A Model Eubacterium
11.2.1 E. coli genome
11.2.2 E. coli transcriptomics
11.2.3 E. coli proteomics
11.2.4 Modeling E. coli metabolism: in silico metabolomics
11.3 Bacillus subtilis: A Paradigm for Gram-Positive Bacteria
11.3.1 B. subtilis genome
11.3.2 B. subtilis transcriptomics
11.3.3 B. subtilis proteomics
11.4 Saccharomyces cerevisiae: A Model for Higher Eukaryotes
11.4.1 Yeast genome
11.4.2 Yeast transcriptomics
11.4.3 Yeast proteomics
11.4.4 Yeast interactome: mapping protein–protein interaction
11.5 Comparative Genomics of Model Eukaryotic Organisms
Introduction
 E. coli , Bacillus subtilis and S. cerevisiae serves as a model
organisms because of their reduced structural and functional
complexity and their intrinsic advantages as experimental
systems.
 In this chapter, we focused on the impact of structural and
functional genomics on elucidating the dynamics of the
transcriptome, proteome, metabolome and interactome of the
well-studied model organisms.
Dna Microarray
TRANSCRIPTOME
Bioinformatics
METABOLOME
Model
Organisms:
The Genome
Sequence
INTERACTOME
Phage
display
Mass
spectrometry
E.coli
Computer modeling
Two-hybrid
system
B. subtilis
S. cerevisiae
PROTEOME
2D-PAGE
Protein chips
Mass
Spectrometry
Fig 11.1 Elucidation of
the cellular domains of
model organisms using
a functional genomics
approach and
comprehensive
technologies.
Escherichia coli: A MODEL
EUBACTERIUM
 best-characterized free-living, single-celled organism, served
as a biological model for cellular processes.
 38% of the 4,288 protein-coding genes ( no attributed
biological role) and 1,853 previously described (Blatter et.al, 1997)
DNA replication and repair, DNA transcription, Metabolic pathways,
Adaptive stress responses, Signal transduction, Genetic regulation
Escherichia coli: GENOME
Structural Analysis - Genome-Wide Parallel Functional Analysis
6 newly proposed genes
valZ
lysY
lysZ
lyst operon
lysQ
asnW
ileY
Transcriptional units
unit
Bioinformatic analysis- Structural and Regulatory Components of
Various Biochemical pathways or Cellular Machineries
Operon for the degradation
of aromatic compounds
Pathway for degradation
of aromatic compounds
Transcription regulator for
encoding the aromatic
degradation
E. Coli Transcriptomics
A. The Heat Shock Response
(i) Correlation of Gene Expression & Function
* alternate sigma (δ) factor rpoH (δ32) and rpoE (δE)
- heat shock proteins, homeostatic mechanism exhibited by living
cells when exposed to suboptimal elevated temperature
(ii) Connection between Gene Expression & Physiological State
* When complexed with the core RNA polymerase, the E. coli δ32
transcription factor permits the transcription machinery to initiate heat
shock-regulated promoters fro both steady state and stress-activated
levels of heat shocks gene expression.
.
E. Coli Transcriptomics
A. The Heat Shock Response
(iii) Technological Advantages
* DNA microarray technology accurately detect alternatives in
bacterial transcript abundance & illustrated how functional genomics
allow well-characterized cellular processes to be examined from a new
& global perspective.
Large scale culture
Separation
Mass spectrometry
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E. Coli Transcriptomics
B. Transcriptome Analysis of Cellular Metabolism and Growth
(i) Correlation of Gene Expression & Function
* trp operon ( trpE, trpD, trpC, trpB and trpA)
- enzymes required for the conversion of chorismate, a
branch-point intermediate in the aromatic amino acid pathway
to tryptophan.
(ii) Importance of Transcriptional Regulation
* Transcription of the trp operon is governed by repression
control via the repressor protein TrpR and an entirely different
mode of regulation termed transcriptional attenuation.
E. Coli Transcriptomics
B. Transcriptome Analysis of Cellular Metabolism and Growth
(iii) Technological advantages
1. DNA microarray technology monitor global change in mRNA
abundance connected with tryptophan biosynthesis, transport and
regulation.
trp – tryptophan biosynthesis
aroH – aromatic amino acid biosynthesis
mtr- tyrptophan-specific permease
trpR- tryptophan repressor regulon
E. Coli Transcriptomics
B. Transcriptome Analysis of Cellular Metabolism and Growth
(iii) Technological advantages
2. DNA microarray technology provides genomic expression
for cell growth particularly in the differences in rRNA and tRNA.
1. translational apparatus
2. nitrogen metabolism
3. amino acid biosynthesis
4. biosynthesis of vitamins, cofactors,
prosthetic groups and carriers
5. nucleotide biosynthesis
6. fatty acid biosynthesis and degradation
7. carbon and energy metabolism
8. cellular processes and global regulators
E. Coli Transcriptomics
B. Transcriptome Analysis of Cellular Metabolism and Growth
(iii) Technological advantages
3. DNA microarray technology provides genomic expression for cell
growth particularly in the differences protein synthesis and ribosome
abundance.
ilv GMEDA operon
leuABCD operon
trpEDCBA operon
– isoleucine and valine synthesis
– leucine synthesis
- tryptophan synthesis
E. Coli Transcriptomics
B. Transcriptome Analysis of Cellular Metabolism and Growth
(iii) Technological advantages
4. DNA microarray technology provides genomic expression for cell
growth particularly in the number of carbon and energy metabolism genes.
dld – D-lactate utilization tpoS – regulation of poxB expression
poxB- acetate formation
aceA, aceB, gltA, lcd, & mdh- acetate utilization
uspA- (universal stress protein)
coupling of glucose and acetate cometabolism
The step required in a microarray experiment
E. Coli Transcriptomics
B. The NtrC regulon
(i) Correlation of Gene Expression & Function
* NTr (nitrogen-regulated) system & NtrC(nitrogen regulatory protein)
- perform a cellular function in response to information processes about
the physiological state of the cell nd NtrC transcriptional genes response to
external nitrogen limitation.
(ii) Connection between Gene Expression & Physiological States
* NtrC protein activates transcription of sigma 54-dependent genes and
Nac (nitrogen assimilation control) protein serves as an adapter to activate
transcription of sigma 70-dependent genes.
E. Coli Transcriptomics
B. The NtrC regulon
(iii) Technological advantages
* DNA microarray technology shows operons specified ATP-binding
cassette transporters.
potFGHI – putrescine
dppABCDF – dipeptides
oppABCDF- oligopeptides
nupC- secondary ion-coupled transporter for nucleosides
cycA- D-alaline/D-serine/glycine
E. Coli Proteomics
A. Protein identification and analysis
Proteome analysis concern the authencity of the ORF ( sequence
annotation) and physical properties (isoelectric point (pI) and
molecular mass)
* vivo protein abundance , post translational modification and
proteolysis
B. Proteome-wide differential display of protein in
various conditions
2-DE
364 2-DE spots using
amino acid (N) terminal
Edman sequence analysis
- 60% of the 223 identified
loci encoded proteins
- 18% of 2DE-spots, represented
isoforms in a narrow pI range 4 to 7
molecular mass 10 to 100KDa
E. Coli Proteomics
C. Protein-protein interaction
a.
Tryptophanase (TnaA), which catalyzes both the
degradation and synthesis of tryptophan.
b. Galactose –binding protein (MglB), which is involved
in the transport of galatose into the cell.
c. Starvation- inducible protein (Dps), which froms
stable complexes with DNA and thus protects DNA
from oxidative damage.
E. Coli Proteomics
C. Protein-protein interaction
Figure 11.2 : Proteomic analysis
of the outer membrane of E coli.
(a) Proteins in carbonate-treated
membranes of E. coli were
separated by two-dimensional
gel electrophoresis and then
identified using mass
spectrometry.
(b) E. coli cells grown under
conditions of iron limitation
were subjected to carbonate
treatment and then separated
by two-dimensional gel
electrophoresis.
Modeling E. Coli Metabolism: In silico
Metabolomics

Functional analysis by coresponses : Metabolite analysis
Figure 11.3
Reconstruction
of microbial
metabolic
networks from
annotated
genome
sequence,
biochemical
data, and
physiology
data.
B. subtilis : GENOME
Structural Analysis – Genome Mapping Sequence analysis
Helix-turn-helix (HTH) family
transcription factor
18 of 20 GntR
family
Two-component regulatory
system
15of 19 LysR
family
37 sensor
kinases
5 of 12 LacI
family
34 response
regulators
Physiological analysis – Adaptation of B. subtilis
43 temperature shock/
stress proteins
4.2Mb resembles E. coli
(25%) in terms of size
10 of 11 AraC
family
77 ABC transporter –
gene duplication
4% codes for multi
functional enzymes
B. subtilis Transcriptomics
A. Global characterization of Heat Shocks
(i) Correlation of Gene Expression & Function
* alternate sigma transcription factor
- δB stress regulon, HrcA, heat-inducible genes and CtsR,
transcriptional regulators
(ii) Connection between Gene Expression & Physiological State
* transcription of many general stress genes occurs at a
basal level from vegetative δA –dependent promoters, but is
increased dramatically in a δB -dependent manner in response
to stress and starvation.
B. subtilis Transcriptomics
B. Two-Component Regulatory Systems
(a) DegS/DegU two component system
controls
exoprotease
production,
development and motility
competence
(b) ComP/ComA two component system
- cell density signal activator
(i) PhoP/PhoR two component system
- induction of the Pho regulon in response to phosphate
starvation
B. subtilis Transcriptomics
C. DNA Microarray Analysis
(a) aprE, nprE and ispA
- 116 target gene DegU overproduction
(b) bpr, yukl, ycdA and murD
- newly DegU operon
(c.) YdbG/YdbF two component system
- overproduction of YdbF chemostaxis
(d.) YufL/ YufM two component system
- regulating competence controlled by ComK
(e.) YvrG/YvrH two component system
- cell membrane related proteins
Stages of Sporulation in Bacillus
subtilis
Germination
Stage Stage Stage Stage Stage Stage Stage Stage
0
II1
II3
III
IV
V
VI
VII
Asymmetric
Engulfment
PresporeCortex CoatMaturation
septation
protoplastformationformation
Stages of Sporulation in Bacillus
subtilis
How is the process
regulated?
Stage Stage Stage Stage Stage Stage Stage Stage
0
II1
II3
III
IV
V
VI
VII
sp
spoI
spoII
spoI
spo
spo
o0
I
I
V
V
VI
Stages of Sporulation in Bacillus
subtilis
Golden age of Bacillus genetics
• Identify spo genes
• Clone and characterize spo genes
• Determine their interdependencies
Stage Stage Stage Stage Stage Stage Stage Stage
0
II1
II3
III
IV
V
VI
VII
sp
spoI
spoII
spoI
spo
spo
o0
I
I
V
V
VI
Stages of Sporulation in Bacillus
subtilis

• Care in using reporter gene (lacZ)
• How is regulation exerted?
• Dependence of spoIIA expression on other
genes
Stage Stage Stage Stage Stage Stage Stage Stage
0
II1
sp
o0
II3
spoI
I spoII
A
III
IV
V
VI
VII
spoII
spoI
spo
spo
I
V
V
VI
Global Gene Expression During
Growth and Sporulation of
B. subtilis
Figure 11.4 Hierarchical cluster analysis
of microarray-derived transcription
profiles of 586 Bacillus subtilis genes
whose expression levels depended on
Spo0A.
(I) genes whose expression was
dependent on Spo0A but not on sF;
(II) genes whose expression was
inhibited by Spo0A; and
(III) genes whose expression was under
the control of sF or some
downstream transcription factor in
sporulation.
B. subtilis Proteomics
A. The Extracellular Proteome
Two-dimensional map of cytosolic proteins of B. subtilis indicated that
most abundant proteins perfomed mainly housekeeping functions in
glycolysis, TCA cycle, amino acid biosynthesis, translation and
protein quality control.
B. Heat Stress Proteome during Sporulation
60 stress proteins were synthesized de novo and/or
overexpressed in B. subtilis during sporulation concurrent to
acquired thermotolerance in spores.
Saccharomyces cerevisiae :A MODEL FOR
HIGHER EUKARYOTES
A yeast cells is about 4-7mm large, the ”eyes” at the bottom are bud scars
 simple unicellular eukaryote, valuable for studying the basic mechanisms of
cell life particularly in human genetic diseases. Very manipulable, capable of
being easily deleted, mutated, reintroduced, overexpressed, tagged and
comprehensively analyzed.
Yeast GENOME
 The S. cerevisiae nuclear genome has 16
chromosomes
 6, 274 potential ORFs
 Genetical genomes
(1) a much lower gene density and largely
untranscribeed DNA
(2) the presence of several apparent
pseudogenes and a 15-kbp redundant
sequence
(3) the absence of genes essential for
vegetative growth
Yeast Transcriptomics
A. Genetic Basis of Metal Homeostasis
(i) Correlation of Gene Expression & Function
* Zap1p transcription factor
- senses the status of cellular zinc levels and stimulates expression of its
target genes in response to zinc limitation
be examined from a new & global perspective.
Yeast Transcriptomics
A. Genetic Basis of Metal Homeostasis
(ii) Connection between Gene Expression and Physiological State
* copper ions 3 functional cofactors key enzymes
(1)
an active cytochrome oxidase complex, which
enables yeast cells to grow on nonfermentable carbon
sources
(2)
the copper-metalloenzyme superoxide dismutase,
which protects the cell against the detrimental effects
of reactive superoxide dismutase
(3) the copper-metalloenzyme Fet3, a ferro-oxidase that
is critical for fe(II) uptake
Yeast Transcriptomics
B. Functional Genomics of Metabolic Reprogramming
(i) Connection between Gene Expression and Physiological State
* Genome-wide transcription patterns under aerobic and anaerobic
1)
219 genes displayed a greater 3-fold higher transcription
level, while 140 genes showed a greater than 3-fold
increase in transcript level in response to anaerobosis.
2) 34 genes whose expression at the diauxin shift is
dependent on a functional
Cat8p, a zinc clustercontaining transcriptional activator that is essential for
growth on nonfermentable carbon source.
Yeast Transcriptomics
C. Nucleosome Remodeling Complexes in Gene Regulation
(i) Connection between
Gene Expression and
Physiological State
* Nucleosomal
inhibition of gene
transcription can occur
at the stages of
transcription factor
binding, preinitiation
complex formation, or
transcription
elongation.
Yeast Transcriptomics
C. Nucleosome Remodeling Complexes in Gene Regulation
(i) Connection between
Gene Expression and
Physiological State
* Hierarchical
cluster analysis of
whole genome
microarray data from
a study assessing
the genome-wide
effects of Rvb
inactivation.
a. Green and red indicates
a decrease or an
increase in mRNA
abundance
b. Rvb1p and Rvb2p are
essential components
of a chromatin
remodeling complex
that regulates
transcription of over
5% of yeast genes.
Yeast Proteomics
A. The Extracellular Proteome
2,774 yeast proteins was determined by high-throughput
immunolocalization of epitope-tagged gene products
Yeast Proteomics
B. Proteome Microarray
A.) Protein chip fabrication and analysis of yeast protein kinases.
A protein chips were constructed by pouring PDMS over an acrylic
mold, curing, and mounting the wells on a glass slides. The surface
of the wells was then modified, followed by protein attachment.
Yeast Proteomics
B. Proteome Microarray
B.) Protein chip fabrication and analysis of yeast protein kinases.
Kinase activities were detected using protein chips. Images of
phosphorylation signals in the presence of 12 substrates are shown.
Yeast Interactome: Mapping Protein-Protein
Interactions
A. Genomic Two-hybrid Screens
A.) GAL4, protein, a transcriptional activator that control expression
of genes involved in galatose utilization. It contain two separable and
functionally distinct domains that are both essential for activation of
target gene expression:
1. N-terminal domain –responsible for specific DNA-binding activity
2. C-terminal domain –contains acidic regions that are required for
activation of transcription.
Yeast Interactome: Mapping Protein-Protein
Interactions
A. Genomic Two-hybrid Screens
B.) Ydr016 protein, has potential involvement in spindle pole body
function. Three hypothetical proteins, Ydr016c, Ykr083c and Ylr423c,
are implicated in spindle pole body function based on their
interactions with proteins of known function.
Yeast Interactome: Mapping Protein-Protein
Interactions
B. Visualizing Protein-Protein Interaction Network in
silicos
1.) 63% of the assembled connections occurring between proteins
assigned a common functional role and 76% occuring between
proteins residing in the same subcellular compartment.
2.) 21 proteins involved in membrane fusion and the 141 proteins
involved in vesicular transport.
Yeast Interactome: Mapping Protein-Protein
Interactions
B. Visualizing Protein-Protein Interaction Network in
silicos
Protein–protein interaction map for Cdc28 and Fkh1/2 complexes
involved in signaling pathways. Gray dotted arrows indicate new
interactions determined by high-throughput mass spectrometric protein
complex identification (HMS-PCI).
Yeast Interactome: Mapping Protein-Protein
Interactions
C. Analysis of Yeast Multiprotein Complexes by Mass
Spectrometry
1.) High-throughput mass spectrometric protein complex
identification (HMS-PCI) detects 3, 617 associated proteins ,
representing 25% of the yeast proteome.
2.) Immuno-affinity purification based on the Flag epitope tag was
used to capture 100 protein kinases, 36 phosphatases and
regulatory subunits, and 86 proteins functionally implicated in the
cellular response to DNA damage.
COMPARATIVE GENOMICS OF MODEL
EUKARYOTIC ORGANISM
Distribution of
core biological
functions
conserved in
both yeast
and worm
Large scale culture
Transformation
Separation
LIMS system
workflow
Relative Intensity [%]
An integrated
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