Bacteria Genetics & Exchange of Genetic...
Transcript of Bacteria Genetics & Exchange of Genetic...
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
8
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
16
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
20
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
28
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
51
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.