Chapter8 Microbial genetics

Chapter8 Microbial genetics

8.4 Plasmids

8.5 Genetic Conjugation,

Transformation, transduction

8.6 Transposons and Insertion Sequences

8.4 Plasmids

• 1-1000 KB in size

• Typical plasmid 1/20 of chromosome

• Most are circular double-stranded DNA, some linear ds DNA

• Transmitted from cell to cell via conjugation process

• Some can integrated into chromosome

• Can carry a variety of genes for production of toxin, resistance to antibiotics and heavy metals et al.

Circular genetic elements that reproduce autonomously and have an extra-chromosomal existence:

Plasmid• Conjugative: plasmids which govern their own transfer by ce

ll-to-cell contact are called conjugative

• Tra region: a set of genes within the plasmid that control the transmissability by conjugation

• Hfr (high frequency of recombination): strains of bacteria that transfer large amounts of chromosomal DNA during conjugation

• Supercoil: plasmids isolated from the cells are in supercoiled configuration

• Plasmid separation: by ultracentrifugation or electrophoresis

• Curing of plasmids: elimination of plasmids from host cells by various treatments.

Plasmids• Replication: Most plasmids of gram-positive

bacteria replicate by a rolling circle mechanism.

• Copy number: The number of plasmids in a cell, can range from only 1-3 copies to 100 copies.

• Incompatibility: Two different types of plasmids can not coexist in a cell.

• Episomes: Plasmids having the ability to integrate into host chromosome

Col plasmids

• Bacteria also harbor plasmids with genes that may give them a competitive advantage in the microbial word.

• Bacteriocins are bacterial protein that destroy other bacteria. Usually act only against closely related strains

F-Plasmid-Fertility Plasmids• 100 KB

• Can be cured with acridine orange

• Incompatibility (inc)

• Origin of replication (oriS)

• Transposable elements (Tn)

• tra region

• phi: phage inhibition

• IS (insertion sequence)

• rep: replication functions

Cell to Cell Transfer of Plasmids• Conjugative: Plasmids that govern their own transfer by

cell-to-cell contact are called conjugative (not all plasmids are conjugative)

• Some conjugative plasmids can transfer genetic information between distintly related organisms (between gram-positive and gram-negative bacteria, between bacteria and plant cells, and between bacteria and fungi), it is important for evolution.

Conjugation (接合作用）

Plasmid Biology

Episomes

Plasmid Biology

Types of Plasmids and Their Biological Significance

• The presence of plasmids in a cell can have a profound influence on the cell’s phenotype:– the ability of conjugation– the ability of Rhizobium to interact with plants– the resistance to antibiotics and heavy metals– the degradation of octane, camphor et al– the production of enterotoxin– the applications in genetic engineering

Resistance Plasmids (R-Plasmids): the most well studied plasmids

• The emergence of bacteria resistant to several antibiotics is medically significant

• Resistance can be transferred via cell-to-cell contact

• This could be one of the reasons for the rapid rise of multiply resistant strains

• Plasmid recombination is one mean by which multiply resistant organisms might have first arisen

• Infectious nature of the R plasmids permits rapid spread of the characteristic through populations

• Typical example: plasmid R100

The presence of multiple antibiotic resistance is due to the fact that a single R plasmid contains a variety of genes coding for different anti

biotic inactiviation enzymes

Biochemical mechanism of resistance mediated by R plasmids

(1) Transformation, which involves donor DNA free in the environment

(2) Transduction, in which the donor DNA transfer is mediated by a virus

(3) Conjugation, in which the transfer involves cell-to-cell contact and a conjugative plasmid in the donor cell

8.5 Three main processes of genetic recombination in prokaryotes fragments of homologous DNA from a donor chromosome are transferred to a recipient cell

DNA Transfer in Bacteria

transformation

transduction

conjugation

8.5.1 Conjugation

• Conjugative plasmids possess genetic information to code for sex pili and for some proteins needed for DNA transfer.

• Rolling circle replication occurs for DNA transfer during conjugation.

F plasmid of E. coli has the special property of being able to mobilize the chromosomeso that it can be transferredduring cell-to-cell contact.

Conjugation and Chromosome Mobilization:F+ and F- strains

• F+ strains: cells possessing an unintegrated F plasmid.• F- strains: cells which can act as recipients for F+ or

Hfr, F- strains lack F plasmid.

• F plasmid provides its host cell with:– ability to synthesize the F pilus– mobilization of DNA for transfer to another cell– alteration of surface receptors so that the cell is no

longer able to behave as a recipient in conjugation

Integration of an F plasmidinto the chromosome with theformation of an Hfr. IS elementsare the sites of insertion.

homology

Hfr strain• Hfr strains ar

ise as a result of the integration of the F plasmid into the chromosome

Important Concept: F’ plasmids

• Integrated F plasmids may be occasionally excised from the chromosome and bring some chromosomal genes with itself into the liberated F plasmid.

• F’-mediated transfer resembles specialized transduction in that only a restricted group of chromosomal genes can be transferred.

Result of selected conjugation

Donor Recipient Molecules transferred Product

F+ F- F plasmid F+ Cell

Hfr F- Initiating segment of F plasmid and variable quantity of chromosomal DNA

F- with variable quantity of chromosomal DNA

F+ F- F+ plasmid and some chromosomal genes it carries with it

F+ Cell with some duplicate gene pairs: one on chromosom, one on plasmid

Transfer of plasmid DNA

by conjugation

• The F plasmid of an F+ cell is being transferred to a F- recipient cell

Details of the replicationand transfer process

Detection of Genetic Conjugation

Manner of formation of different Hfr strains

• The direction in which the F factor is inserted determines which of the chromosomal genes will be inserted first into the recipient

Interrupted Mating

• Mixing Hfr and F- cells.

• Shake the mixture violently at various time.

• Plate the samples on selective medium for recombinant to grow.

Mapping theorder of genes

Conjugation involves a donor cell, which contains a particular type of conjugative plasmid, and a recipient cell, which does not.

The genes that control conjugation are contained in the tra region of the plasmid (see Section 9.8 in your text ). Many genes in the tra region have to do with the synthesis of a surface structure, the sex pilus . Only donor cells have these pili,

The pili make specific contact with a receptor on the recipient and then retract, pulling the two cells together. The contacts between the donor and recipient cells then become stabilized, probably from fusion of the outer membranes, and the DNA is then transferred from one cell to another.

Mechanism of DNA Transfer During Conjugation

A mechanism of DNA synthesis in certain bacteriophages, called rolling circle replication, was presented here to explains DNA transfer during conjugation .

if the DNA of the donor is labeled, some labeled DNA is transferred to the recipient but only a single labeled strand is transferred. Therefore, at the end of the process, both donor and recipient possess completely formed plasmids.

Genetic Recombination• Homologous or General Recombination

– RecA protein participation

– Homologous DNA sequences have the same or nearly the same sequence

– New genotypes only arise when two homologous sequences are genetically distinct

Detection of Recombination

• Requirement:reverse mutation

for the selected characteristic must be low.

This problem can often be overcome by using double mutants.

Complementation Test:cis-tran test

• trans configuration: two mutations are each on separate DNA molecules

• cis configuration: Two mutations were on the same DNA molecule

• Complementation does not involve recombination

• 1928, Fred Griffith• Competent: cells able to take up a molecule of DNA.

Competency is a complex phenomeono and is dependent on several conditions.

1. Bacteria need to be in a certain stage of growth. 2. Secrete a small protein called the competence factor t

hat stimulates the production of 8 to 10 new proteins reauired for transformation.

• Natural transformation has been discovered so far only in certain genera: Streptococcus, Bacillus, Thermoactinomytes, Haemophilus, Neisseria, Moraxella, Acinetobacter, Azotobacter, Pseudoomonas

DNA transformation

The mechanism of Transformationin S. pneumoniae

1. A competent cell binds a ds DNA fragment 2. The DNA is cleaved by endonucleases to 5-15kb.3. One stand is hydrolyzed by an envelop-associated exonuclease, the other strand associate with small proteins and moves through the plasma membrane.4. Integration of transforming DNA

The transformation of Haemophilus influenzae

• Difference: 1. Haemophilus does not produce a competence factor to stimulate the development of competence.

• 2. It takes up DNA from only closed related species.

• 3. Ds DNA, complexed with proteins, is taken in by membrane vesicles.

• 4. DNA must have a special sequence (5’AAGTGCGGTCA3’) to be bound by a competent cell.

Transformation

A number of prokaryotes have been found to be naturally transformable, including certain species of both gram-negative and gram-positive Bacteria and some species of Archaea. However, even within transformable genera, only certain strains or species are transformable

CompetenceA cell that is able to take up a molecule of DNA and be transformed is said to be competent. Competence in most naturally transformable bacteria is regulated, and special proteins play a role in the uptake and processing of DNA. These competence-specific proteins may include a membrane-associated DNA binding protein, a cell wall autolysin, and various nucleases.

Competent cells bind much more DNA than do noncompetent cells as much as 1000 times more

Artificially Induced CompetenceHigh efficiency natural transformation is found only in a few bacteria; Azotobacter, Bacillus, Streptococcus,, for example, are easily transformed. Many prokaryotes are transformed only poorly or not at all under natural conditions. Determination of how to induce competence in such bacteria may involve considerable empirical study, with variation in culture medium, temperature, and other factors

when E. coli is treated with high concentrations of calcium ions and then stored in the cold, the transformation by plasmid DNA is relatively efficient.

(a) Binding of free DNA by a membrane-bound DNA binding protein.

(b) Passage of one of the two strands into the cell while nuclease activity degrades the other strand.

(c) The single strand in the cell is bound by specific proteins, and recombination with homologous regions of the bacterial chromosome mediated by RecA protein occurs.

The introduction of DNA into cells by mixing the DNA and the cell

Transformed cell

DNA Transfer by Electroporationfor artificial induction of competence are being supplanted by a new method termed electroporation.Small pores are produced in the membranes of cells exposed to pulsed electric fields. When DNA molecules arepresent outside the cells during the electric pulse, they can then enter the cells through these pores. This process is called electroporation.

The mechanism of bacterial transformation

Other methods for introducing DNA into bacterial cells

• Transfection: transformed DNA is extracted from a bacterial virus

• Artificially induced competence: e.g treat E. coli with high concentration of Ca ions, and then stored the cells at low T, the E. coli will become competent at low efficiency

• Electroporation: pulsed electrical fields generate pores in the cell membranes, allowing DNA molecules to enter the cells.

• DNA from any source can be introduced into bacteria by splicing it into a plasmid before transformation

Transformation (transfection) of eukaryotic cells

• Transfection: introducing DNA into mammalian cells– phagocytosis in animal cells– Yeast: spheroplasts added with Ca ions plus polyeth

ylene glycol– Electroporation– Particle gun, or gens gun

Agrobacterium and Plant Interactions: Crown gall and Hairy Root

• Crown gall: caused by Agrobacterium tumefaciens which carries a Ti (Tumor induction) plasmid that promotes the crown gall formation

• Hairy Root: caused by Agrobacterium rhizogenes which carries a Ri plasmid that leads to hairy roots formation

Overview of events of crown gall disease following infection of A. tumefaciens

Ti plasmid of Agrobacterium tumefaciens

Mechanism of transferof T-DNA to the plantcell

Wonder of the Genetic Engineering

• Expression of luciferase gene in a plant.

Background of transduction• Lytic cucle: end in lysis of the host• Lysogeny: after adsorption and penetration, viral ge

nome remains within the host cell and is reporduced along with the bacterial chromosome.thia relationship between the phage and its host is called lysogeny.

• Lysogens or lysogenic: bacteria that can produce phage particles under some conditions.

• Temperate phages: phages able to eatablish this relationship.

• Prophage: the latent form of the virus genome that remains within the host without destroy it.

Virus Life Cycle

1. Attachment (adsorption)2. Penetration (injection)3. Early steps in replication4. Replication5. Synthesis of protein subunits6. Assembly and packaging7. Release

Temperate Bacterial Viruses: Lysogeny and Lambda

• Virulent Viruses• Temperate Viruses

– Lysogeny (溶原性）： Viruses can enter a state called lysogeny, where most phage genes are not expressed, and the phage genome is replicated in synchrony with the host chromosome

– Prophage or provirus– Can be induced (lysogenic induction) by UV ra

diation, nitrogen mustards or X-ray.

Temperate Bacterial Viruses:

Lysogeny and Lambda

Transduction

• Generalized transduction: host genes derived from virtually any portion of the host genome become part of the DNA of the mature virus genome.

• Specialized transduction: occurs only in some temperate viruses: a specific group of host genes is integrated directly into virus genome-usually replacing some of the virus genes-and is transferred to the recipient during lysogenization

Generalized transduction

Abortive transduction• About 70 to 90%of transferred DNA is not integrated but oft

en is able to survive and express itself. Abortive transductants are bacteria that contain this nonintrgrated, transduced DNA and are partial diploids.

Specialized Transduction• Under rare conditio

ns, the phage genome is excised incorrectly.

• Lambda dgal (defective galactose) under the assistance of helper, the defective phage can be replicated and can transduce the galactose genes.

Specialized Transduction

• Low-frequency transduction (LFT) lysates: lysates contain only a few transducing particle, the phage genome is excised incorrectly.

• Helper phage: defective lambda phages carring the gal gene can integrate if there is a normal lambda phage in the same cell. This normal phage is termed the help phage.

• High-frequent transduction (HFT) lysate: a lysate containing a fairly equal mixture of defective lambda dgal phage and normal helper phage.

Phage conversion• A prophage is immune to further infection by the same type

of phage.– Change in structure of a polysaccharide on the cell surfac

e of Salmonella anatum upon lysogenization with – Conversion of nontoxin producing strains of Corynebact

erium diphtheriae to toxin producing (pathogenic) strains.• Information for production of these new materials is ap

parently an integral part of the phage genome and is automatically and exclusively transferred upon infection by the phage and lysogenization.

Conjugation

Transformation

Transduction

Transduction involves transfer of host genes from one bacterium to another by viruses. In generalized transduction, defective virus particles randomly incorporate fragments of the cell's chromosomal DNA; virtually any gene of the donor can be transferred, but the efficiency is low. In specialized transduction, the DNA of a temperate virus excises incorrectly and brings adjacent host genes along with it; only genes close to the integration point of the virus are transferred, but the efficiency may be high.

Concept

Transduction

In transduction, DNA is transferred from cell to cell through the agency of viruses. Genetic transfer of host genes by viruses can occur in two ways.


And

Specialized transduction

Specialized transduction: occurs only in some temperate viruses; DNA from a specific region of the host chromosome is integrated directly into the virus genome - usually replacing some of the virus genes.

Generalized transduction: host DNA derived from virtually any portion of the host genome becomes a part of the DNA of the mature virus particle in place of the virus genome.

Transduction has been found to occur in a variety of prokaryotes, including certain species of the Bacteria: Desulfovibrio, Escherichia, Pseudomonas, Rhodococcus, Rhodobacter, Salmonella, Staphylococcus, and Xanthobacter, as well as the archaean Methanobacterium thermoautotrophicum.

Not all phages can be transducer and not all bacteria are transducible



In generalized transduction, virtually any genetic marker can be transferred from donor to recipient

During a lytic infection, the enzymes responsible for packaging viral DNA into the bacteriophage sometimes accidentally package host DNA. This DNA cannot replicate, it can undergo genetic recombination with the DNA of the new host.

the DNA of lambda is inserted into the host DNA at the site adjacent to the galactose genes

On induction, Under rare conditions, the phage

genome is excised incorrectly

A portion of host DNA is exchanged for phage DNA, called lambda dgal ( dgal means "defective galactose“ )

Phage synthesis is completed

Cell lyses and releases defective phage capable of transducing galactose genes

Specialized Transduction

TransfectionBacteria can be transformed with DNA extracted from a bacterial virus rather than from another bacterium, a process known as transfection.

Conjugation

Transformation

Transduction

8.6 Transposons and Insertion Sequences

• Transposition: the process by which gene moves from one place to another in the genome.

• Transposable elements: transposition of genes is linked to the presence of special genetic elements called transposable elements.

• Three types of transposable elements in bacteria:– Insertion sequences (IS)– Transposons (Tn)– Some special viruses (such as Mu)

Three types of transposable elements in bacteria

• Insertion sequences (IS): about 1000 nucleotides, carry only information to move them to new location (IS1, IS2 and IS3).

• Transposons (Tn): larger than IS, carry genes, such as drug resistance markers and other selectable genes.

• Some special viruses (such as Mu)

Both IS and Tn have shortinverted terminal repeats (IR)at the ends of their DNA, IR areinvolved in the transpositionprocess

Insertion of a transposable element generates a duplication

How is the targetedsequence duplicated?

Transposon mutagenesis

• Insertion of transposon within a gene leads to mutation.• Transposon with antibiotic-resistant marker can be used

for selection purposes.• Two tranposons widely used: Tn 5 (neomycin and kana

mycin resistance), Tn10 (tetracycline resistance).

Invertible DNA and the phenomenon of phase variation

When a DNA segment is oriented in one direction, a particular gene is expressed. Whereas when it is oriented in the opposite direction, a different gene is expressed.

Salmonellaflagellasynthesis

Questions for Microbial Genetics• Describe as much as you know about plasmids.• What is the difference between a plasmid and an episome?• What are Hfr strain? F+ or F-, or F’ strain?• Draw the F plasmid and describe functions of various DNA r

egions.• Why is it said that conjugative plasmid contributes to evoluti

on?• How many types of plasmids and their functions you have le

arned?• Schematically describe R100 plasmid and its functions.• How do R plasmids inactivate antibiotics?• What is an engineered plasmid?• What points do F plasmid provide to its host?• How to detect genetic recombination. Please cite one exampl

e.• How is bacterial genome mapped? What are the three types o

f transposable elements?• Explain transposon mutagenesis and its possible application.• Give an example to explain conversible DNA and phase vari

ation.

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Chapter8 Microbial genetics