bacterial physiology & genetics pin lin ( 凌 斌 ), ph.d. departg ment of microbiology &...
TRANSCRIPT
Bacterial Physiology & Genetics
• Pin Lin ( 凌 斌 ), Ph.D.
Departg ment of Microbiology & Immunology, NCKU
ext 5632
• 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
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 micro
organisms. 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 s
ource.
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.
Metabolic Requirements
Mineral source
Required for enzyme function.
For most microorganisms, it is necessary to provide so
urces 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.
pH value Neutrophiles ( pH 6-8) Acidophiles ( pH 1-5) Alkalophiles ( pH 9-11) Internal pH is regulated by variou
s 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.
Environmental factors
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
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.
Toxicity of O2 for Anaerobes
Excluding oxygen
Reducing agents
Anaerobic jar
Anaerobic glove chamber
Anaerobic cultivation methods
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.
Pyruvate: universal intermediate
Aerobic respiration
Fermentation
Glycolysis (EMP pathway)
Substrate-level phosphorylation
Glycolysis (EMP pathway)
1. Both bacteria and eukaryote use this process
2. One Glucose => 2 ATP 2 NADH 2 Pyruvate
Fermentation: metabolic process in which the final electron acceptor is an organic compound.
Sources of metabolic energy Respiration: chemical reduct
ion of an electron acceptor through a specific series of electron carriers in the membrane. The electron acceptor is commonly O2, but CO2, SO4
2-, and NO3- are 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.
Substrate-level phosphorylation
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.
Fermentation
Function of TCA cycle
1. Generation of ATP
2. Supplies key intermediates for amino acids, li
pids, purines, and pyrimidines
3. The final pathway for the complete oxidation of
amino acids, fatty acids, and carbohydrates.
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
Functions:
1. Provides various suga
rs as precursors of bio
synthesis, and NADP
H for use in biosynthe
sis
2. The various sugars m
ay be shunted back to
the glycolytic pathway.
Pentose phosphate pathway (hexose monophosphate shunt)
Bacterial Cell Division
1. Replication of chromosome
2. Cell wall extension
3. Septum formation
4. Membrane attachment of DNA pulls into a new cell.
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)
Bacterial growth curve
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 Operon- Negative transcriptional regulation
Negative control
Repressor
Inducer
Operator
Lactose operon:
Lactose metabolism
Under positive or negative control
Positive control
Activator: CAP (catabolite gene-activator protein)
CAP RNA pol
Inducer
Lactose Operon- Positive Control
Tryptophan operon
Transcriptional Regulation (Example II)
-Tryptophan operonNegative control- Repressor- Corepressor (Tryptophan)- Operator
Attenuation
Transcription termination signal
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
MutationTypes of mutations1. Base substitutions
Silent vs. neutral; missense vs. nonsense2. Deletions3. Insertions4. Rearrangements: duplication, inversion, transposition
May cause frameshift or null mutation
Induced mutationsPhysical 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
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 bacteriaMediated by plasmids and phages
PlasmidExtrachromosomal
Autonomously replicating
Circular or linear (rarely)
May encode drug resistance or toxins
Various copy numbers
Some are self-transmissible
Bacteriophage (bacterial virus)
Icosahedral tailess
Icosahedral tailed
Filamentous
Structure and genetic materials of phages
Coat (Capsid)
Nucleic acid
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.
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)
Applications of genetic engineering
Cultivation methods
Medium
Basic media
Rich media
Enrichment media
Selective media
Differential media
Agar: an acidic polysaccharide extracted from red algae
For microbiologic examination
Use as many different media and conditions of incubation as is practicable. Solid media are preferred; avoid crowding of colonies.
For isolation of a particular organism
Enrichment cultureDifferential mediumSelective medium
Isolation of microorganisms in pure culture
Pour plate methodStreak method
For growing bacterial cells
Provide nutrients and conditions reproducing the organism's natural environment.
Most bacteria reproduce by binary fission.
Measurement of microbial concentrations:
Cell concentration (no. of cells/unit vol. of culture)
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.
Growth, survival and death of microorganisms
0.1 ml
10-1 10-2 10-3 10-4 10-5 10-6 10-7
> 1000 220 18
Bacterial concentration:
220 x 106 x 10 = 2.2 x 109/ml
Bacterial growth in nature
Interaction of mixed communities
A natural environment may be similar to a continuous culture.
Bacteria grow in close association with other kinds of organisms.
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.
Biofilms
Polysaccharide encased community of bacteria attached to a surface.
Attachment of bacteria to a surface or to each other is mediated by glycocalyx.
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.
1. Ribose-5-P (product of HMP) synthesis of purine r
ing from sugar moiety inosine monophosphate
purine monophosphate
2. Pyrimidine orotate orotidine monophosphate (pyri
midine orotate attaches to ribose phosphate)
cytidine or urine (pyrimidine) monophosphate
3. Reduction of ribonucleotides at the 2’ carbon of the sug
ar portion deoxynucleotides
Nucleic acid synthesis