Gene Transfer Mechanisms – Conjugation (cont.)Transformation and Transduction

Insertion sequences and transposons provide regions of homology between plasmids and chromosomes

Brock Biology of Microorganisms, vol. 9, Chapter 9

Brock Biology of Microorganisms, vol. 9, Chapter 9

Plasmids can integrate into the host genome at sites of homology

insertion sequence

Fig. 5.6 Snyder and Champness

An integrated conjugal plasmid can drive the transfer of chromosomal genes – initiates directionally at oriT

Called a High-frequency of recombination (Hfr) donor – results in many recombinant products

Conjugates the a portion of the integrated plasmid and a section of the chromosome

The recipient does not receive an intact plasmid

Fig. 5.7 Snyder and Champness

This transfer process is directional from oriT – genes closer to oriT are transferred relatively efficiently

Transfer of genes becomes less efficient as their location is less close to oriT

Brock Biology of Microorganisms, vol. 9, Chapter 9

The map of the circular E. coli chromosome was initially established using multiple Hfr strains

Fig. 5.8 Snyder and Champness

Integrated plasmids can excise from the chromosome at high frequency

Aberrant excision events can often lead to a plasmid that now stably carries chromosomal genes

This is called a “prime” (eg. F’)

Some plasmids, while not self-conjugal, can piggy-back along with conjugal plasmids – these are called mobilizable.

Requires that they have a functional oriT that is recognized by the conjugal plasmids

Can be engineered to create a mobilizable cloning vector

Conjugative Transposons

These genetic elements share attributes with conjugal plasmids and transposons

A transposition event between two cells

Requires all the functions found in conjugation plus those of transposition

Scott, 1992, J. Bacteriol. 174:6005

Transformation

Tranformation is process of taking up naked DNA in a stably inherited form.

Two major types of transformation

1. Natural transformation (only a subset of microbes do this)

- usually linear DNA

2. Artificially-induced (most, but not all microbes can be induced to take up DNA

- usually plasmid DNA

A cell that is proficient to take up DNA is described as competent

One of the most basic and important techniques in molecular biology is the ability to introduce foreign DNA into a bacterial host.

Often achieved by preparing artificially-competent cells

E. coli will take up and replicate foreign circular DNA

Two types of artificial transformation

Chemical competence

Electroporation

Artificial competence

Chemical competenceIn some bacteria, including E. coli, treatment of cells with divalent cations at low temperature, facilitates the uptake of plasmid DNA into the cell (linear DNA can be taken up, but is shredded by cytoplasmic DNases before it can do anything)Remains unclear how this works

Hanahan and Bloom, 1996, Chapter 132, Escherichia coli and Salmonella, ASM Press

Uptake channels made of polyP, PHB, and Ca

High field strengths result in very transient holes in the cellular envelope

Under the appropriate conditions, DNA leaks in and DNA leaks out.

A high concentration of plasmid outside results in a rapid influx of plasmids into the cell.

Electroporation cuvette

Cells go hereHigh voltageshock

Electroporation

How well has your transformation worked?

Transformation efficiency

Saturating cells (# of transformants/ug of DNA)

106-109/ug of pBR322app. 1011 plasmids/ug pBR322can also be analyzed as % of cells that receive plasmid

Saturating DNA

% of DNA molecules that successfully transform cells

Protocol Sat. cells Sat. DNA

Chemical 1% 12%

Electro 10% 90%

Natural transformation in Gram positives

Examples:

Streptococcus pneumoniaeBacillus subtilis

-no base specificity-limited # of uptake sites (30-75)-nicked internally-complement is degraded during transport-recombines in recipient

Dubnau. 1999. Ann. Rev. Microbiol. 53:217

Natural transformation in Gram negatives

Examples:

Haemophilus influenzaeNeisseriae gonorrhoeae

-sequence specific – uptake sequences-4-8 sites/cell-no cell bound intermediate-import of ds DNA to periplasm-complement is degraded during transport into cytoplasm-recombines in recipient

Dubnau. 1999. Ann. Rev. Microbiol. 53:217

Gram positive uptake machinery

-dedicated machinery for the transport of DNA into the cell

Dubnau. 1999. Ann. Rev. Microbiol. 53:217

the reverse of a conjugal transfer system- some components similar to Tra functions

Gram-negative uptake machinary

-dedicated machinery for the transport of DNA into the cell- must cross periplasm and outer membrane

Dubnau. 1999. Ann. Rev. Microbiol. 53:217

Energy for driving the process?

1. Intracellular ATP hydrolysis2. pH gradient – PMF?3. Complement degradation

Function for natural transformation

1. Nutrition2. DNA repair3. Genetic diversification

Transduction

Genetic exchange mediated by bacterial viruses (bacteriophage)

Two basic types of bacterial viruses

Lytic viruses – infect cells, multiply rapidly,

lyse cells

Lysogenic viruses – infect cells, can integrate into

genome and go dormant (a prophage)

- at some point, can excise, multiply and lyse cells

Bacteriophage have a range of morphologies from simple filaments to large complex structures

May contain either RNA or DNA associated with a protein coat

Almost all bacteria have phage associated with them

Brock Biology of Microorganisms, vol. 9, Chapter 8

Smithsonian (Oct 2000)

Attach to specific receptors on the surface of their host bacteriaTransfer their nucleic acid into the host cell

T4 bacteriophage on the surface of an E. coli cell

Lysogeny of bacteriophage

Integrate into host genome - Enter a semi-dormant state(eg. Lambda phage)

Brock Biology of Microorganisms, vol. 9, Chapter 8

Dormant prophage – integrated bacteriophage – carries genes that alter the phenotype of the microbe

- best examples are pathogens and toxin production

toxin prophage

insertionsite

Corynebacterium diptheriaea

Phage produces diptheria toxin

This is what makes people sick

C.diptheriaea

without phage strain produces no toxin

Does not cause diptheria

Phage conversion