lecture 9 bacterial genetics
TRANSCRIPT
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BACTERIAL GENETICS
Methods of Genetic TransferQ2A 1ST SEMSource: Population Reference Bureau, 2011 Source: www.hhs.gov
Fig. 3 Some Examples of Emerging and Reemerging Infectious Diseases.Although diseases such as HIV are indicated in only one or two significantlocations, they are very widespread and a threat in many regions. (Source:
Prescott, L.M. 2002)
Fig. 4 Nosocomial
Infections. Relativefrequency by bodysite. These data arefrom the NationalNosocomialInfectionsSurveillance, whichis
conducted by theCenters for DiseaseControl andPrevention (CDC).(Source: Prescott,L.M., 2002)
How do microorganisms acquire
traits not innate to them?
Recall (from The Prokaryotes)
Protoplast structure formed when G+ bacteria are
treated with penicillin
Pili: generally longer than fimbriae; present only ongram-negative bacteria; responsible for conjugation
Capsule - composed of repeating polysaccharide units, of
proteins, or both; responsible for adhesion to host tissues
and evasion of phagocytes
Plasmid: a circular dsDNA that can exist & replicateindependently of the chromosome or may be integrated
with it
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Genetic Transfer in Bacteria
Genetic Transfer - results in Genetic Variation
Genetic Variation - needed for Evolution
3 Ways Genetic Transfer can occur in Bacteria
1. Conjugation
2. Transformation
3. Transduction
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Types of Intermicrobial Exchange
transformation transfer of naked DNA
conjugation
requires the attachment of two
related species & formation of a
bridge that can transport DNA
transductionDNA transfer mediated by bacterial
virus
TRANSFORMATION
the uptake of exogenous DNA (native bacterialchromosome fragments, plasmid, bacteriophageDNA) from a donor to a recipient
cell-cell contact is not required
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TRANSFORMATION
A. Competence State
ability of a bacterium to take up DNA from the
medium
Natural or artificial
its appearance is usually the consequence of a
nutritional shift-down and usually coincides with a
decrease in rate, or a blockage, of DNA synthesis
natural: ~100% of the cells of a population of S.pneumoniae can become synchronously competent;
competence lasts 20 mins; 20% in B. subtilis but lasts
for ~90 mins
TRANSFORMATION
Artificial Competence
Methods
1. the divalent cation method for transforming E. coli
and related Gram (-)
2. the protoplast techniques used to transform a
variety of bacteria, including many which do not
develop natural competence
TRANSFORMATION
TECHNOLOGICAL TRANSFER SYSTEMS
1. Electroporation - application of high-voltage
electrical discharges into a suspension of cells, which
produce localized membrane breaks or pores
through which all manners of macromolecules,
including DNA can enter.
2. Biolistic Transformation - Use of a DNA-coated
tungsten projectiles at a high velocity to penetrate
cells, specifically in the organelle.
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Electroporation Biolistic Transformation
TRANSFORMATION
B. Binding, uptake and fragmentation
of DNA
C. Recombination and integration
of the donor genetic material
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Griffiths Experiment
2 Strains of Streptococcus pneumoniae
1. Virulent strain with a capsule (S) - Pneumonia
2. Avirulent strain without a capsule (R) - no disease
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Griffiths Experiment
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Griffiths Experiment
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Griffiths Experiment
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Griffiths Experiment
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CONJUGATION
Studied by Joshua Lederberg in 1945
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Source: Prescott, L.M. 2002
CONJUGATION
Requirements:
1. Requires cell-cell contact
2. Occurs via a conjugal/membrane pore
3. DNA transfer occurs in one direction - from donor to
recipient not vice versa.
4. Does not require protein synthesis in donor cell.
5. Requires energy in donor cell - primarily ATP.24
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CONJUGATION
Conjugative plasmids: Certain large plasmids carrygenes that function to promote their own transfer to
other cells, including genes that direct the synthesis ofthe sex pili.
The F (fertility) plasmid carries some fourteen genes,including the structural gene for the pilus protein(pilin), that function in sex pilus (in this case called theF pilus) formation.
The F plasmid can carry additional genes, such asgenes for antibiotic resistance, and these genes arealso transferred to recipient cells.
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CONJUGATION
Mechanism (Conjugative System of F Plasmid)1. Establishment of Cell Contact
F-encoded (sex) pilus: A hollow cylinder with an exterior diameter of 8 nm and interior diameter
of 2 nm; up to 20 um in length
traA encodes pilin; 12 other tra genes for maturation and assembly of
pilus
very long (e.g. the pilus produced by F-plasmid) or very short (e.g. the
pilus produced by plasmid RP4)
DNA is not transferred via the sex pilus; for recognition of
recipient cell.26
CONJUGATION
Mechanism (Conjugative System of F Plasmid)1. Establishment of Cell Contact
F-encoded (sex) pilus:
Following contact, the long flexous pilus of F acts as a
retractile motor - mating pair formation
mating bridge is a region of contact between the donor and
recipient cells where the DNA is presumably transferred
via a pore (although the pore has not yet been reliably
visualized).27
CONJUGATION
Mechanism (Conjugative System of F Plasmid)2. DNA Mobilization and Transfer (Donor Conjugal
DNA Synthesis; DCDS)
Transfer of a single strand of F factor DNA to recipient cell;
synthesis of complementary strand.
Stabilization of the F factor in the recipient cell
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Evidence of Conjugation
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TYPES OF BACTERIOPHAGEB. Temperate phage
A bacterium infected with a temperate phage can have the same fate as a
bacterium infected with a virulent phage (lysis rapidly following infection).
However, an alternative outcome is also possible, namely, after entering the
cell, the phage DNA, rather than replicating autonomously, can fuse or
integrate with the chromosome of the host cell.
In this state (the prophage state), the expression of phage genes is repressed
indefinitely by a protein, termed the represser, encoded within the phage
genome. No new phage particles are produced, the host cell survives, and the
phage DNA replicates as part of the host chromosome.
The temperate bacteriophage lambda is the most thoroughly understood of all
complex viruses.
Generalized Transduction a random fragment of bacterial DNA, resulting from phage-induced cleavage of
the bacterial chromosome, is accidentally encapsulated in a phage protein coat in
place of the phage DNA.
When this rare phage particle infects a cell it injects the bacterial DNA fragment
into the cell.
If this fragment becomes integrated into the recipient chromosome by
recombination, the recipient cell will be stably transduced.
Because a random fragment of bacterial DNA is packaged in a phage protein
coat, any bacterial gene can be transduced in this manner.
Because transducing phage package about fifty genes at a time (some one percent
of the bacterial genome), generalized transduction provides a useful means for
ma in bacterial enes at a finer scale than does con u ation .
A
A
Source: Prescott, L.M. 2002
Specialized transduction
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only certain bacterial genes are transduced, namely those that
are located on the bacterial chromosome in close proximity to the
prophage insertion site of the transducing.
The phage acquires the bacterial genes by a rare, abnormal
excision from the bacterial chromosome.
In general, a specialized transducing phage particle contains both
phage and bacterial DNA joined together as a single molecule.
Upon infecting another cell, this joint molecule integrates into the
recipient chromosome just as the phage DNA would normally do in
the process of becoming a prophage.
A
A
Source: Prescott, L.M. 2002
LYSOGENIC BACTERIA
bacteria that carry a prophage.
the phenomenon is termed lysogeny, and the bacterial cell issaid to be lysogenized.
Nonlysogenic bacteria can be made lysogenic by infection witha temperate phage.
The association of prophage and bacterial cell is highly stable,but can be destabilized by various treatments, such asexposure to ultraviolet light, that damage the host DNA.
When DNA damage occurs, repression of phage genes islifted, the prophage excises from the host chromosome,replicates autonomously, and produces progeny phageparticles.
The host cell is lysed just as with a virulent phage.43
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LYSOGENIC BACTERIA The emergence of the virus from its latent prophage state is called
induction.
Lysogenic bacteria sometimes have properties strikingly different from
their nonlysogenic counterparts.
Corynebacterium diphtheriae is pathogenic because it carries a prophage thatpossesses a gene (the fox gene) that encodes the diphtheria toxin
Strains of C. diphtheriae that lack the prophage are non-pathogenic.
Similarly, lysogenic strains of group streptococcus (Streptococcuspyogenes) produce pyrogenic exotoxins (erythrogenic toxins)
The acquisition by bacteria of properties due to the presence of aprophage is called lysogenic conversion.
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References:
Madigan, M.T., J.M. Martinko, J. Parker. 2003. Brock Biology of
Microorganisms. 10th edition. New Jersey, USA: Prentice-Hall,
Inc.
Talaro, K. P. 2009. Foundations in Microbiology. 7th edition.New York, USA: McGraw-Hill Companies, Inc.
Bayer, M. 2005. Microbiology 1: General Microbiology.
Lecture Handouts. UPLB.
__________. 2006. Microbiology 101. Techniques in
Microbiology. Lecture Handouts. UPLB.
Laguardia, M. 2010. Microbiology and Parasitology. Lecture
Presentation. DLSL.
Opulencia, R. 2011. Microbiology 130. Microbial Genetics.
Lecture Handouts. UPLB.