Bacteria Genetics &Exchange of Genetic Information

Shibo Jiang (姜世勃)

MOH&MOE Key Lab of Medical Molecular VirologyShanghai Medical College, 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

Di Qu (瞿涤)

MOH&MOE Key Lab of Medical Molecular VirologyShanghai Medical College, Fudan University

复旦大学基础医学院

医学分子病毒学教育部/卫生部重点实验室

Chapter 7

Key WordsChromsomePlasmidTransposable Genetic ElementsPhage

-Lysophage (temperate), virulent phageprophage, lysogen /lysogeny

Horizontal Gene transfer TransformationTransduction

General transductionLysogenic （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 materialAll organisms use the same nucleotidesAll organisms replicate, transcribe and translate DNA

Growth of bacteriaBacterial Multiplication - Binary Division

Chromosome (ds DNA) number doubled

Plasmid (ds DNA) number ?

General characteristics of bacterial genetics

Bacterial DNA can be altered by mutations

Mutations can result in changes in proteins - New traits -diversity-acquisition of (antibiotics) resistance

New traits can be transmitted to -daughter cells -other microbes （Horizontal Gene transfer）

Bacterial Genetic material Chromsome: the prokaryotic genome is circular, haploid

Genome size very in species: Table 7-1

Plasmid： mobileTransposable 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

Chapter 7

Mutations in Bacteria• Mutations arise in bacterial populations

– Point mutation(synonymous/nonsynonymous substitution)

Induced or spontaneous mutations– Genetic Recombination

Why there are so many bacterial mutants?Rare mutations are expressed in bacteria- Bacteria are haploid- Rapid growth rate

(bacteria generation time/doubling time? E.coli)- >1010/ml- Selective advantage enriches for mutants

antibiotics, nutrients…Horizontal gene transfer

Diploid allele mutation –recessiveness

Haploid mutation –dorminance

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

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Table 7–2 Examples of Metabolic Activities Determined by Plasmids

Organism ActivityPseudomonas species Degradation of camphor, toluene, octane,

salicylic acidBacillus stearothermophilus

α-Amylase

Alcaligenes eutrophus Utilization of H2 as oxidizable energy source

Escherichia coli Sucrose uptake and metabolism, citrate uptake

Klebsiella species Nitrogen fixationStreptococcus (group N) Lactose utilization, galactose

phosphotransferase system, citrate metabolism

Rhodospirillum rubrum Synthesis of photosynthetic pigmentFlavobacterium species Nylon degradation

Classification of PlasmidsIntegrations

integrated into chromosome or freeTransfer properties- Conjugative plasmid

containing tra genes, which perform the complex process of conjugation, the transfer of plasmids to another bacterium

- Nonconjugative plasmidincapable of initiating conjugation

Phenotypic effectsFertility (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

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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

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BACTERIOPHAGES

=bacterial virusinfect host cell - bacteria

Phage Composition and Structure

• Composition– Nucleic acid

genomeds, ss DNA

- ProteinCapsid or

headTail 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 MEMBRANECYTOPLASMIC MEMBRANE

EXTERIOR

RECEPTORPROTEIN

INJECTION -PENETRATION

NOBEL (1969)Alfred Hersheydiscovery on the replication of viruses and genetic structure

HEA

DTA

IL

CAPSOMER

CORESHEATH

COLLAR

BASE PLATE

TAIL FIBER (6)

SPIKES

• Adsorption–Tail fibers– Receptor is LPS for T4

• Irreversible attachmentBase 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 Cycletotal time = ~15 mins

attach

Inject DNA

replication

assemble

cell lysisreleasing ~200 phage

Host cell

TEMPERATE PHAGES AND LYSOGENY

Lambda - Infection : Attachment, Penetration, genome integrated into chromosome

Repression of lytic genes, Integration, LysogenyProphage, Lysogen (host cell)

Prophage Induction (a high stress environment)Inducing agent Repression abolished, Lytic gene

expression.ExcisionLytic growth

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

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Lysogen- bacteria

Lysogenic phaseLytic phase

Events Leading to Lysogeny

• Site-specific recombinationPhage 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 - Recombination

Legitimate, 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 that 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 9-2P. 148

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

Specialized TransductionLysogenic 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

Lysogen- bacteria

Lysogenic phaseLytic phase

LyticC

ycle

LysogenicC

ycle

Lysogen- bacteriaprophage

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-

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

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Conjugation

Direct contact between donor and recipient must occur

Sex pilus is encodedby 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+

Non-transmissible plasmid

Transfer mediated by F+ plasmid

Physiological States of F FactorIntegrated into chromosome (Hfr)(High Frequency of Recombination)Characteristics of Hfr x F- crosses:F- rarely becomes Hfr, while Hfr remains HfrHigh 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- crossesF- becomes F’， while F’ remains F’High transfer of donor genes on F’ , low transfer of

other donor chromosomal genes （ Hfr ）

Hfr F’

F plasmid may acquired other genes from chromosome or other plasmid

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 conjugationR 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 questions1. In p121: question 1, 2, 3, 4, 52. Does the phenotype of an organism automatically change when a change in genotype occurs? Why or why not? 3. Can phenotype change without a change in genotype? In both cases, give some examples to support your answer.4. List the biological significances of gene transfer in bacteria.