Bacterial Physiology & Genetics

• Pin Lin ( 凌 斌 ), Ph.D.

Departg ment of Microbiology & Immunology, NCKU

ext 5632

lingpin@mail.ncku.edu.tw

• 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

Nutrients: synthetic vs. nonsynthetic media

Essential Elements

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

Saccharomycetes

E. coliClostridium

Propionebacterium Enterobacter

StreptococcusLactobacillus

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.

Tricarboxylic Acid (TCA) cycle

NADH ===> 3 ATPFADH2 ===> 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

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

Excision repair

Nucleotide excision repair

Base 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 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

Lysogenic phaseLytic phase

Life cyclePhage as an example

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)

Trans-Gram gene transfer

Spread of transposon throughout a bacterial population

Mechanisms of evolution of Vancomycin-resistant Staphylococcus Aureus

Cloning

Cloning vectors

plasmids

phages

Restriction enzymes

Ligase

In vitro phage packaging

Library construction

Genomic library

cDNA library

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.

Nucleic acid synthesis

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