Bacteria Genetics

&

Exchange of Genetic Information

Di Qu (瞿涤)

MOH&MOE Key Lab of Medical Molecular Virology

School of Basic Medical Sciences

Shanghai Medical College of Fudan University

复旦大学上海医学院分子病毒学

教育部/卫生部重点实验室

Chapter 7

Bacterial Structures

• Cell Wall

-Lipopolysaccharides

-Teichoic Acids

• Cell Membrane & Cytoplasm

-Inclusions

• Ribosomes

• Nucleoid

-Chromosome & Plasmids

• Capsule

• Flagella

• Pili

• Spores

Chapter 2some bacteria

All bacteria

Key Words

Chromsome

Plasmid

Transposable Genetic Elements

Phage

-Lysophage (temperate), virulent phage

prophage, lysogen /lysogeny

Gene transfer

Transformation

Transduction

General transduction

Lysogenic （specific）transduction/conversion

Conjugation (transfer of genetic material between

bacterial cells by direct cell-to-cell contact or by a

bridge-like connection between two cells)

F factor，Hfr， R plasmid

The central dogma of molecular biology

All organisms have DNA and RNA

as genetic material

All organisms use the same

nucleotides

All organisms replicate, transcribe

and translate DNA

Genetics of Bacterial

Chromsome: the prokaryotic genome is circular, haploid

Plasmid： mobile

Transposable Genetic Elements：mobile elements,

integrate into chromsome or plasmid,

-carried genes, …drug resistant

-insertion mutation

Bacteriaphage (phage)

RNA-mRNA, rRNA

-In prokaryotes, an mRNA molecule carry information for

several genes (eukaryotes an mRNA for one gene)

-The ribosomes are 70S in prokaryotes vs 80S in

eukaryotes

-Transcription: synthesis of RNA from a DNA template

-Translation: formation of a protein (amino acid sequence)

from RNA sequence

Chapter 7, p101

Some fundamentals of bacterial genetics

Bacterial DNA can be altered by mutations

Mutations can result in changes in proteins

-diversity

-acquisition of resistance

New traits can be transmitted to other microbes

Mutations in Bacteria

• Mutations arise in bacterial populations

– Point mutation (synonymous/nonsynonymoussubstitution)

Induced or spontaneous

– Genetic recombination

• Rare mutations are expressed

– Bacteria are haploid

– Rapid growth rate

(bacteria generation time/doubling time? E.coli)

• Selective advantage enriches for mutants

antibiotics, nutrients…

• Horizontal gene transfer

Mutations are inheritable

changes in the base

sequence of nucleic acid --

the genetic material. An

organism with these

changes is called a mutant.

Genetic recombination is

the process where genes

from two genomes are

combined together. A

mutant will be different from

its parent, its genotype or

genetic makeup has been

altered.

synonymous/nonsynonymou

s substitution

9

Plasmids

• Plasmids are circular double strand DNA molecules

• Definition:

• Extrachromosomal genetic elements

• Replicate independently of the bacterial chromosome

(replicon) encode a variety of genes usually not essential

bacterial genes but may give bacterium new properties

(antibiotic resistance, virulent, etc.), can lost during culture.

• Size vary widely, mobile and can be transferred between

individuals and among species (host range)

• Plasmids are used in genetic engineering as gene transfer

vectors

• Episome (virology) - a plasmid that can integrate into the

chromosome

Table 7-2, p.105

Table 9-2, p.153

Examples of Metabolic Activities Determined by Plasmids

Organism Activity

Pseudomonas species Degradation of camphor, toluene, octane,

salicylic acid

Bacillus

stearothermophilus

a-Amylase

Alcaligenes eutrophus Utilization of H2 as oxidizable energy source

Escherichia coli Sucrose uptake and metabolism, citrate

uptake

Klebsiella species Nitrogen fixation

Streptococcus (group N) Lactose utilization, galactose

phosphotransferase system, citrate

metabolism

Rhodospirillum rubrum Synthesis of photosynthetic pigment

Flavobacterium species Nylon degradation

Table 7–2 p.105

Classification of Plasmids

• Transfer properties

–Conjugative plasmid (containing tra genes, which perform

the complex process of conjugation, the transfer of plasmids to

another bacterium)

–Nonconjugative plasmid(incapable of initiating conjugation)

• Phenotypic effects

– Fertility (F plasmid)

– Resistance plasmid (R factors)

– Bacteriocinogenic plasmid

- controls the synthesis of bacteriocin

Integration

Extrachromosomal

Bacterial conjugationConjugative plasmid

Transposable Genetic Elements

• Definition: Segments of DNA that are able to move from

one location to another on the chromosome- jumping gene

• Bacteria contain a wide variety of transposable elements

• The smallest and simplest

insertion sequences (IS elements)

1–3 kb in length and encode the transposase protein required for

transposition and one or more additional proteins that regulate the rate

of transposition

• Properties

– “Random” movement “hot spot”

– Not capable of self replication (not a replicon)

– Transposition mediated by site-specific recombination

• Transposase

– Transposition may be accompanied by duplication

Types of Transposable Genetic Elements

• Insertion sequences (IS)

– Definition: Elements that carry no other genes

except those involved in transposition

– Nomenclature - IS1 (ISn)

– Structure (flanking inverted repeats- palindrome)

– Importance

• Insertional Mutation

• Plasmid insertion

• Phase variation

TransposaseABCDEFG GFEDCBA

Integration

17

Phase Variation in Salmonella H Antigens

ISH1 gene H2 gene

H1

flagella

H2

flagella

Types of Transposable Genetic Elements

• Transposons (Tn)

– Definition: Elements that carry other genes in

addition to those involved in transposition, gene

that moves from one DNA molecule to another within

the same cell or from one site on a DNA molecule to

another site on the same molecule

– Nomenclature - Tn10

– Structure

• Composite Tns

– Importance

• Antibiotic resistance

Integration

21

BACTERIOPHAGES

=bacterial virus

infect host cell - bacteria

Phage Composition and Structure

• Composition

– Nucleic acid

genome

ds, ss DNA

- Protein

Capsid or

head

Tail fibers etc.

-Protection

-Infection

Tail

Tail Fibers

Base Plate

Head/Capsid

Contractile

Sheath

Types of Bacteriophage

• Lytic or virulent phage– Phage that multiply within the host

cell, lyse the cell and release progeny phage (e.g. T4)

• Lysogenic or temperate phage: Phage that can either

multiply via the lytic cycle or genome integrating into

chromosome of bacteria, entering a quiescent state in the

bacterial cell.

• In lysogenic status:

– Expression of most phage genes repressed

– Prophage: Phage DNA integrated in chromosome of bacteria

– Lysogen: Bacteria harboring a prophage

PHAGE T4 – lytic

phage

CYTOPLASM

WALL - OUTER MEMBRANE

CYTOPLASMIC MEMBRANE

EXTERIOR

RECEPTOR

PROTEIN

INJECTION -

PENETRATION

NOBEL (1969)

Alfred Hersheydiscovery on the

replication of viruses and

genetic structure

HE

AD

TA

IL

CAPSOMER

CORE

SHEATH

COLLAR

BASE PLATE

TAIL FIBER (6)

SPIKES

• Adsorption

–Tail fibers

– Receptor is LPS for T4

• Irreversible attachment

Base plate

• Sheath Contraction

• Nucleic acid injection

BACTERIOPHAGES - LYTIC GROWTH

LYTIC PHAGE GROWTH (5 steps)

Attachment (adsorption, specificity)

Penetration (injection)

Replication -Transcription, translation

- Host provides: energy, ribosomes, RNA polymerase.

etc. for macromolecular synthesis

- Production of viral proteins and nucleic acids

Assemble (maturation) (packaging) intact progeny

viruses

Release

- cell Lysis - release of progeny

General Phage Life Cycle

Lytic Cycle

total time = ~15 mins

attach

Inject DNA

replication

assemble

cell lysis

releasing ~200 phage

Host cell

TEMPERATE PHAGES AND LYSOGENY

Lambda - Infection : Attachment, Penetration,

genome integrated into chromosome

Repression of lytic genes, Integration, Lysogeny

Prophage, Lysogen (host cell)

Prophage Induction (a high stress environment)

Inducing agent Repression abolished, Lytic gene

expression.

Excision

Lytic growth

29

Lysogenic Cycle

All phage species can undergo a

lytic cycle

Phages capable of only the lytic

cycle are called virulent

lysogenic cycle:

-no new phage produced

-the infected bacterium survives

-a phage DNA is transmitted to each

bacterial progeny cell when the cell

divides

Those phages that are also capable

of the lysogenic cycle are called

temperate

Integration

lysogenic

cycle

Lysogen- bacteria

Lysogenic phaseLytic phase

Events Leading to Lysogeny

• Site-specific recombination

Phage coded enzyme

• Repression of the phage genome

– Repressor protein

– Specific

– Immunity to superinfection

•Induction

–Adverse conditions

•Role of proteases

–recA protein

–Destruction of repressor

• Gene expression

• Excision

• Lytic growth

Lysogenic

phase

Lytic

phase

Exchange of Genetic Information

-horizontal gene transfer

General Features of Gene Transfer in

Bacteria

• Unidirectional

– Donor to recipient

• Donor does not give an entire chromosome

• Gene transfer can occur between species

• Transformation- uptake of “naked” DNA

• Transduction- by bacteriophages

• Lysogenic conversion

• Conjugation- bacterial cells come in direct contact

with each other. Plasmid is often transferred (Hfr)

Transformation

• Definition: Gene transfer resulting from the uptake

of DNA from a donor.

• Factors affecting transformation

– DNA size and state

• Sensitive to nucleases

– Competence of the recipient

(Bacillus, Haemophilus, Neisseria, Streptococcus)

• Competence factors

• Induced competence

Transformation

Significance

– Phase variation in Neiseseria

– Recombinant DNA technology

Steps

- Donor DNA

- Uptake of DNA• Gram +

• Gram –

- Competence of the recipient

- RecombinationLegitimate, homologous or general

recA, recB and recC genes

Transformant

identified

by selection

Recombination

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Griffith’s transformation experiment (1928)

R form

S form

Avery, MacLeod, McCarty Experiment

(1944)

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Experiment showed that DNA, not RNA, was the

transforming principle

Avery, MacLeod, McCarty experiment

Recipient bacteria must be “competent” to take up and

incorporate DNA

Few strains of bacteria are naturally competent

Bacteria can be made artificially competent

- calcium solutions

- electric current

Transduction

• Definition: Gene transfer from a donor to a

recipient by a bacteriophage

• Resistant to environmental nucleases

• Bacteriophage (phage): a virus that infects bacteria

can incorporate genetic material into chromosomal

DNA. Bacterial cell can change characteristics and

pathogenic factors:

– Diphtheria toxin

– Botulinum neurotoxin

– Staphylococcal enterotoxin

– Cholera toxin

Table 9-2

P. 153

Transduction

• Types of transduction

– Generalized Transduction in which potentially

any dornor bacterial gene can be transferred.

– Specialized Transduction in which only certain

donor genes can be transferred

• Significance

– Common in Gram+ bacteria

– Lysogenic (phage) conversion

• e.g. Corynebacterium diptheriae toxin

– Toxin derived from lysogenic phage

Generalized Transduction

• Infection of Donor (phage)

• Phage replication and degradation of host DNA

• Assembly of phages particles

• Release of phage

• Infection of recipient (cell-bacterium)

• Homologous recombination

Potentially any donor gene can be transferred

Transduction

Recombination

47

General Transduction

A bacteriophage transfers

the DNA from one bacterial

cell to another

During a LYTIC infection, a

transducing phage, such as P1

infecting E. coli, accidentally

packages a piece of the

bacterial chromosome into a

virus particle instead of its own

viral DNA.

The phage carrying the

bacterial DNA then delivers it

to the recipient cell when it

tried to infect again.

The injected bacterial DNA

may then be integrated into

recipient chromosome by

homologous recombination

Specialized Transduction

Lysogenic Phage

• Excision of the prophage (carrying diphtheria toxin gene etc.)

• Replication and release of phage

• Infection of the recipient

• Lysogenization of the recipient

– Homologous recombination also possible

Lytic

Cycle

Lyso

gen

icC

ycle

Lysogen- bacteria

prophage

Integration

Conjugation

• Definition: Gene transfer from a donor

to a recipient by direct physical

contact between cells with F pili

• Mating types in bacteria

– Donor

• F factor (Fertility factor)

– F (sex) pilus

– Encoded by a plasmid

– F+

Donor

Recipient

– Recipient

• Lacks an F factor

-F-

F+

F-

52

F factor and Conjugation

• F (fertility) factor is a conjugative plasmid transferred from cell to cell by conjugation

• F factor is an episome , genetic element that can insert

into chromosome or replicate as circular plasmid

• ~100 kb in length

• A low-copy-number plasmid, 1–2 copies per cell

• Replicates once per cell cycle and segregates to both

daughter cells in cell division

F-Pilus for Conjugation

Conjugation

Direct contact between donor and recipient must occur

Sex pilus is encoded

by fertility (F) plasmid

Physiological States of F Factor

Characteristics of F+ x F- crosses:

F- becomes F+, F+ remains F+

Low transfer of donor chromosomal genes

F+

Mechanism of F+ x F- Crosses

• DNA transfer

– Origin of transfer

– Rolling circle

replication

• Pair formation

– Conjugation bridge

F+ F- F+ F-

F+ F+F+ F+

Physiological States of F Factor

Integrated into chromosome (Hfr)

(High Frequency of Recombination)

Characteristics of Hfr x F- crosses:

F- rarely becomes Hfr, while Hfr remains Hfr

High transfer of certain donor chromosomal genes

F+ Hfr

Mechanism of Hfr x F- Crosses

• DNA transfer

– Origin of transfer

– Rolling circle replication

• Homologous

recombination

• Pair formation

– Conjugation bridge

Hfr F- Hfr F-

Hfr F-Hfr F-

Physiological States of F Factor

• Autonomous with donor genes (F’)

Characteristics of F’ x F- crosses

F- becomes F’， while F’ remains F’

High transfer of donor genes on F’ , low transfer of

other donor chromosomal genes （ Hfr ）

Hfr F’

Mechanism of F’ x F- Crosses

• DNA transfer

– Origin of transfer

– Rolling circle replication

• Pair formation

– Conjugation

bridge

F’ F’F’ F’

F’ F- F’ F-

Structure of R Factors

• RTF(resistance transfer factor)

– Conjugative plasmid

– Transfer genes

RTF

R determinant

• R determinant

– Resistance genes

– Transposons

R plasmid

R: drug resistance

RTF: transfer of R

plasmid

Bacteria do not reproduce sexually but can acquire new

DNA through transformation, transduction or conjugation

R plasmids

- resistance to antibiotics, metals

-Virulence factors (that make bacteria pathogenic)

Transposons can insert themselves into genome or

plasmid (and out of it)

Thus bacteria have many ways of obtaining new genes

horzontally to enhance survival

-These natural processes have been modified so that

DNA can be deliberately incorporated into host microbes-

even genes that would normally never be transferred this

way

Review questions

1. In p118: question 1, 2, 5

2. Question 3, 4, and give the explains, if possible.

3. Does the phenotype of an organism automatically

change when a change in genotype occurs? Why or why

not?

4.Can phenotype change without a change in genotype? In

both cases, give some examples to support your answer.

5. List the biological significances of gene transfer in

bacteria.

Ribosome

• Protein synthesis；• Targets of antibiotics

70S :

30S (16S rRNA)

50S (5S & 23S rRNA)

Erythromycin

Streptomycin

68

Procaryotic ribosome