gene transfer mechanisms – conjugation (cont.) transformation and transduction
TRANSCRIPT
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