Lecture 9 Bacterial Genetics

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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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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


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


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.

Lecture 9 Bacterial Genetics

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