Transcript Pentose phosphate pathway

Bacterial Physiology
& Genetics
• Pin Lin (凌 斌), Ph.D.
Departg ment of Microbiology & Immunology, NCKU
ext 5632
[email protected]
• References:
1. Chapters 4 & 5 in Medical Microbiology (Murray, P.
R. et al; 5th edition)
2. 醫用微生物學 (王聖予 等編譯, 4th edition)
Outline of Physiology
•
Metabolic Requirements
•
Metabolism & the Conversion of Energy
- Glucose: Glycolysis (Embden-Meyerhof-Parnas
pathway)
TCA cycles
Pentose phosphate pathway
- Nucleic acid synthesis
•
Bacterial Growth
Outline
•
Metabolic Requirements
•
Metabolism & the Conversion of Energy
- Glucose: Glycolysis (Embden-Meyerhof-Parnas
pathway)
TCA cycles
Pentose phosphate pathway
- Nucleic acid synthesis
•
Bacterial Growth
Metabolic Requirements
1. Bacteria must obtain or synthesize Amino acids,
Carbohydrates, & Lipids => build up the cell.
2. Minimum requirements for bacterial growth
– C, N, H2O, Ion & energy
3. Growth requirements & metabolic by-products
=> Classify different bacteria
4. O2 is essential for animal cells but not for all bacteria.
- Obligate aerobes: Mycobacterium tuberculosis
- Obligate anaerobes: Clostridium perfringens
- Facultative anaerobes: Most bacteria
Essential Elements
Nutrients: synthetic vs. nonsynthetic media
Metabolic Requirements
Carbon source
Autotrophs (lithotrophs): use CO2 as the C source
Photosynthetic autotrophs: use light energy
Chemolithotrophs: use inorganics
Heterotrophs (organotrophs): use organic carbon (eg. glucose)
for growth.
Nitrogen source
Ammonium (NH4+) is used as the sole N source by most
microorganisms. Ammonium could be produced from N2 by
nitrogen fixation, or from reduction of nitrate and nitrite.
Metabolic Requirements
Sulfur source
A component of several coenzymes and amino acids.
Most microorganisms can use sulfate (SO42-) as the S
source.
Phosphorus source
A component of ATP, nucleic acids, coenzymes, lipids,
teichoic acid, capsular polysaccharides; also is required
for signal transduction.
Phosphate (PO43-) is usually used as the P source.
Mineral source
Required for enzyme function.
For most microorganisms, it is necessary to provide
sources of K+, Mg2+, Ca2+, Fe2+, Na+ and Cl-. Many
other minerals (e.g., Mn2+, Mo2+, Co2+, Cu2+ and Zn2+)
can be provided in tap water or as contaminants of
other medium ingredients.
Uptake of Fe is facilitated by production of
siderophores (hydroxamates and catechol derivatives).
Growth factors: organic compounds (e.g., amino
acids, sugars, nucleotides) a cell must contain in order
to grow but which it is unable to synthesize.
Environmental factors
pH value
Neutrophiles ( pH 6-8)
Acidophiles ( pH 1-5)
Alkalophiles ( pH 9-11)
Internal pH is regulated by
various proton transport systems
in the cytoplasmic membrane.
Temperature
Psychrophiles (<15 or 15-20 oC)
Mesophiles ( 30-37 oC)
Thermophiles ( at 50-60 oC)
Heat-shock response is induced
to stabilize the heat-sensitive
proteins of the cell.
Aeration
Obligate aerobes
Facultative anaerobes
Microaerophilics
Obligate anaerobes
(Capnophilics: bacteria that
do not produce enough CO2
and, therefore, require
additional CO2 for growth.)
Ionic strength and osmotic
pressure
Halophilic
Toxicity of O2 for Anaerobes
1. O2 reduced to H2O2 by enzymes.
2. O2 reduced to O2- by ferrous ion.
3. In aerobes and aerotolerant anaerobes, O2- is removed
by superoxide dismutase, while H2O2 is removed by
catalase.
4. Strict anaerobes lack both catalase and superoxide
dismutase.
Anaerobic cultivation methods
Excluding oxygen
Reducing agents
Anaerobic jar
Anaerobic glove chamber
Microbial metabolism
Intermediary metabolism-Integrate two processes
1. Catabolism (Dissimilation)
- Pathways that yield metabolic energy for growth and
maintenance.
2. Anabolism (Assimilation)
- Assimilatory pathways for the formation of key intermediates.
- Biosynthetic sequences for the conversion of key
intermediates to end products.
Substrate-level
phosphorylation
Fermentation
Glycolysis
(EMP pathway)
Aerobic
respiration
Pyruvate: universal intermediate
Glycolysis
(EMP pathway)
1. Both bacteria and eukaryote
use this process
2. One Glucose =>
2 ATP
2 NADH
2 Pyruvate
Sources of metabolic energy
Substrate-level phosphorylation
Fermentation: metabolic process in
which the final electron acceptor is
an organic compound.
Respiration: chemical
reduction of an electron
acceptor through a specific
series of electron carriers in
the membrane. The electron
acceptor is commonly O2,
but CO2, SO42-, and NO3are employed by some
microorganisms.
Photosynthesis: similar to
respiration except that the
reductant and oxidant are
created by light energy.
Respiration can provide
photosynthetic organisms
with energy in the absence
of light.
Fermentation
In fermentation, the NADH
produced during glycolysis
is recycled to NAD.
Many bacteria are identified
on the basis of their
fermentative end products.
Fermentation of bacteria
produces yogurt, sauerkraut,
flavors to various cheeses
and wines.
Alcoholic fermentation is
uncommon in bacteria.
Saccharomycetes
Clostridium
Propionebacterium
E. coli
Enterobacter
Streptococcus
Lactobacillus
Function of TCA cycle
1. Generation of ATP
2. Supplies key intermediates for amino acids,
lipids, purines, and pyrimidines
3. The final pathway for the complete oxidation of
amino acids, fatty acids, and carbohydrates.
Tricarboxylic Acid (TCA) cycle
NADH ===> 3 ATP
FADH2 ===> 2 ATP
Electron transport chain
Electron transport
chain
1. Electrons carried by NADH (FADH2)
 A series of donor-acceptor pairs
 Oxygen
 Aerobic respiration
2. Some bacteria use other compounds
(CO2, NO3-) as terminal acceptor
=> Anaerobic respiration
Aerobic Glucose
Metabolism
Pentose phosphate pathway
(hexose monophosphate shunt)
Functions:
1. Provides various
sugars as precursors
of biosynthesis, and
NADPH for use in
biosynthesis
2. The various sugars
may be shunted back
to the glycolytic
pathway.
Bacterial Cell Division
1. Replication of chromosome
2. Cell wall extension
3. Septum formation
4. Membrane attachment of DNA
pulls into a new cell.
Bacterial growth curve
Lag phase (adaptation)
Exponential phase (Log phase)
Determination of the generation
time (doubling time)
The ending of this phase is due to
exhaustion of nutrients in the
medium and accumulation of toxic
metabolic products.
Stationary phase
A balance between slow loss of
cells through death and formation
of new cells through growth.
Alarmones is induced.
Some bacteria undergo sporulation.
Decline phase (the death phase)
Outline of Genetics
• Introduction
• Replication of DNA
• Bacterial Transcription
• Other Genetic Regulation (Mutation,
Repair, & Recombination)
Introduction
• DNA:the genetic material
• Gene: a segment of DNA (or chromosome),
the fundamental unit of information in a cell
• Genome: the collection of genes
• Chromosome: the large DNA molecule associated
with proteins or other components
Why we study Bacterial Genetics?
• Bacterial genetics is the foundation of the
modern Genetic Engineering & Molecular
Biology.
• The best way to conquer bacterial disease is
to understand bacteria first.
Replication of Bacterial DNA
1. Bacterial DNA is the storehouse of information.
=> It is essential to replicate DNA correctly and pass into the
daughter cells.
2. Replication of bacterial genome requires several enzymes:
- Replication origin (oriC), a specific sequence in the
chromosome
- Helicase, unwind DNA at the origin
- Primase, synthesize primers to start the process
- DNA polymerase, synthesize a copy of DNA
- DNA ligase, link two DNA fragements
- Topoisomerase, relieve the torsional strain during the
process
Replication of Bacterial DNA
Features:
1.Semiconservative
2. Multiple growing
forks
3. Bidirectional
4. Proofreading
(DNA polymerase)
Transcriptional Regulation in Bacteria
1. Bacteria regulate expression of a set of genes coordinately &
quickly in response to environmental changes.
2. Operon: the organization of a set of genes in a biochemical
pathway.
3. Transcription of the gene is regulated directly by RNA
polymerase and “repressors” or “inducers” .
4. The Ribosome bind to the mRNA while it is being transcribed
from the DNA.
Lactose Operon
1. E Coli can use either Glucose or other sugars (ex: lactose) as the
source of carbon & energy.
2. In Glu-medium, the activity of the enzymes need to metabolize
Lactose is very low.
3. Switching to the Lac-medium, the Lac-metabolizing enzymes
become increased for this change .
4. These enzymes encoded by Lac operon:
Z gene => b-galactosidase => split disaccharide Lac into
monosaccharide Glu & Gal
Y gene => lactose permease => pumping Lac into the cell
A gene => Acetylase
Lactose OperonNegative transcriptional regulation
Lactose operon:
Lactose
metabolism
Under positive or
negative control
Negative control
Repressor
Inducer
Operator
Lactose Operon- Positive Control
Positive control
Activator: CAP
(catabolite
gene-activator
protein)
CAP RNA
pol
Inducer
Transcriptional Regulation (Example II)
-Tryptophan operon
Negative control
- Repressor
- Corepressor
(Tryptophan)
- Operator
Tryptophan operon
Attenuation
Couple Translation w/
Transcription
Sequence 3:4 pair
-G-C rich stem loop
- Called attenuator
-Like transcriptional
terminator
Sequence2: 3 pair
- weak loop won’t
block translation
Transcription
termination signal
Mutation
Types of mutations
1. Base substitutions
Silent vs. neutral; missense vs. nonsense
2. Deletions
3. Insertions May cause frameshift or null mutation
4. Rearrangements: duplication, inversion, transposition
Induced mutations
Physical mutagens:
e.g., UV irradiation
(heat, ionizing radiation)
Chemical mutagens
Base analog
Frameshift
intercalating agents
Base modification
Transposable elements
Mutator strains
DNA Repair
1. Direct DNA repair
(e.g., photoreactivation)
2. Excision repair
Base excision repair
Nucleotide excision repair
3. Postreplication repair
4. SOS response: induce
many genes
5. Error-prone repair: fill gaps
with random sequences
Thymine-thymine dimer
formed by UV radiation
Excision
repair
Base excision
repair
Nucleotide
excision
repair
Double-strand
break repair
(postreplication
repair)
SOS repair in bacteria
1. Inducible system used only when error-free
mechanisms of repair cannot cope with
damage
2. Insert random nucleotides in place of the
damaged ones
3. Error-prone
Gene exchange in bacteria
Mediated by plasmids and phages
Plasmid
Extrachromosomal
Autonomously replicating
Circular or linear (rarely)
May encode drug resistance
or toxins
Various copy numbers
Some are self-transmissible
Bacteriophage (bacterial virus)
Structure and genetic materials of phages
Coat (Capsid)
Nucleic acid
Icosahedral
tailess
Icosahedral
tailed
Filamentous
Life cycle
Phage l as an example
Lytic phase
Lysogenic phase
Virulent phages: undergo
only lytic cycle
Temperate phages:
undergo both lytic and
lysogenic cycles
Plaques: a hollow formed
on a bacterial lawn
resulting from infection of
the bacterial cells by
phages.
Mechanisms of gene transfer
Transformation: uptake of naked exogenous DNA by
living cells.
Conjugation: mediated by self-transmissible plasmids.
Transduction: phage-mediated genetic recombination.
Transposons: DNA sequences that move within the
same or between two DNA molecules
Importance of gene transfer to bacteria
• Gene transfer => a source of genetic variation =>
alters the genotype of bacteria.
• The new genetic information acquired allows the
bacteria to adapt to changing environmental
conditions through natural selection.
Drug resistance (R plasmids)
Pathogenicity (bacterial virulence)
• Transposons greatly expand the opportunity for
gene movement.
Demonstration
of
transformation
Avery, MacLeod, and
McCarty (1944)
Spread of transposon
throughout a bacterial
population
Trans-Gram
gene transfer
Mechanisms of evolution of Vancomycinresistant Staphylococcus Aureus
Cloning
Cloning vectors
plasmids
phages
Restriction enzymes
Ligase
In vitro phage packaging
Library
construction
Genomic library
cDNA library
Applications of genetic engineering
1. Construction of industrially important bacteria
2. Genetic engineering of plants and animals
3. Production of useful proteins (e.g. insulin, interferon,
etc.) in bacteria, yeasts, insect and mammalian cells
4. Recombinant vaccines (e.g. HBsAg)
Cultivation methods
Medium
For microbiologic examination
Rich media
Use as many different media and conditions
of incubation as is practicable. Solid media
are preferred; avoid crowding of colonies.
Enrichment media
For isolation of a particular organism
Basic media
Selective media
Differential media
Agar: an acidic
polysaccharide
extracted from red
algae
Enrichment culture
Differential medium
Selective medium
Isolation of microorganisms in pure culture
Pour plate method
Streak method
For growing bacterial cells
Provide nutrients and conditions reproducing
the organism's natural environment.
Growth, survival and death of microorganisms
Most bacteria reproduce by
binary fission.
10-1
10-2
10-3
10-4
10-5
10-6
Measurement of microbial
concentrations:
Cell concentration (no. of
cells/unit vol. of culture)
0.1 ml
Viable cell count
Turbidimetric measurements
Biomass concentration (dry
wt. of cells/unit vol. of culture):
can be estimated by
measuring the amount of
protein or the volume
occupied by cells.
> 1000
220
18
Bacterial concentration:
220 x 106 x 10 = 2.2 x 109/ml
10-7
Bacterial growth in nature
Interaction of mixed
communities
Biofilms
A natural environment may be
similar to a continuous culture.
Polysaccharide encased
community of bacteria attached
to a surface.
Bacteria grow in close
association with other kinds of
organisms.
Attachment of bacteria to a
surface or to each other is
mediated by glycocalyx.
The conditions in bacterial
close association are very
difficult to reproduce in the
laboratory. This is part of the
reason why so few
environmental bacteria have
been isolated in pure culture.
About 65% of human bacterial
infection involve biofilms.
Biofilms also causes problems
in industry.
Bioremediation is enhanced by
biofilms.
Biofilm: a community of microbes embedded in an organic polymeric
matrix (glycocalyx, slime), adhering to an inert or living surface.
Nucleic acid synthesis
Nucleic acid synthesis
1. Ribose-5-P (product of HMP)
synthesis of purine
ring from sugar moiety
inosine monophosphate
purine monophosphate
2. Pyrimidine orotate
orotidine monophosphate
(pyrimidine orotate attaches to ribose phosphate)
cytidine or urine (pyrimidine) monophosphate
3. Reduction of ribonucleotides at the 2’ carbon of the
sugar portion
deoxynucleotides