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BACTERIAL GENETICS
Source: Population Reference Bureau, 2011

Q2A 1ST SEM

Methods of Genetic Transfer

Source: www.hhs.gov

Fig. 4 Nosocomial Infections. Relative frequency by body site. These data are from the National Nosocomial Infections Surveillance, which is conducted by the Centers for Disease Control and Prevention (CDC). (Source: Prescott, L.M., 2002)

Fig. 3 Some Examples of Emerging and Reemerging Infectious Diseases. Although diseases such as HIV are indicated in only one or two significant locations, they are very widespread and a threat in many regions. (Source: Prescott, L.M. 2002)

Recall (from The Prokaryotes)

How do microorganisms acquire traits not innate to them?

Protoplast structure formed when G+ bacteria are treated with penicillin Pili: generally longer than fimbriae; present only on gram-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 & replicate independently of the chromosome or may be integrated with it

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Genetic Transfer in Bacteria

Types of Intermicrobial Exchange

transformation transfer of naked DNA requires the attachment of two related species & formation of a bridge that can transport DNA DNA transfer mediated by bacterial virus

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

conjugation

transduction

TRANSFORMATION

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

the uptake of exogenous DNA (native bacterial chromosome fragments, plasmid, bacteriophage DNA) from a donor to a recipient cell-cell contact is not required

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

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

Griffiths Experiment

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Griffiths Experiment

Griffiths Experiment

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CONJUGATION

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.

Studied by Joshua Lederberg in 1945

Source: Prescott, L.M. 2002

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5. Requires energy in donor cell - primarily ATP.

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CONJUGATION

CONJUGATION
Mechanism (Conjugative System of F Plasmid)
Establishment of Cell Contact F-encoded (sex) pilus:
1.

Conjugative plasmids: Certain large plasmids carry genes that function to promote their own transfer to other cells, including genes that direct the synthesis of the 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 the F pilus) formation. The F plasmid can carry additional genes, such as genes for antibiotic resistance, and these genes are also transferred to recipient cells.
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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.
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CONJUGATION
Mechanism (Conjugative System of F Plasmid)
1.

CONJUGATION
Mechanism (Conjugative System of F Plasmid)
2.

Establishment of Cell Contact

Following contact, the long flexous pilus of F acts as a retractile motor - mating pair formation

F-encoded (sex) pilus:

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.

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).
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Stabilization of the F factor in the recipient cell

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Evidence of Conjugation

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Recap: Transformation VS Conjugation

TRANSDUCTION

1951, Joshua Lederberg and Norton Zinder

Transformation: the ___________ of ______ DNA by _______ cells. Conjugation: transfer of DNA which requires ______ to _______ contact.

transfer of genes from one cell to another via a phage vector without cell-to-cell contact. There are two ways in which this can occur: A. generalized transduction, and B. specialized transduction. In each case, the transducing phage is a temperate phage, so that the recipient cell survives the phage infection.

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BACTERIOPHAGE

BACTERIOPHAGE

(phage) is a virus that replicates inside of a bacterial cell. consists of nothing more than a piece of nucleic acid encapsulated in a protective protein coat. Depending on the phage, the nucleic acid can be DNA or RNA, double-stranded or single-stranded. Can have a size from about 3,000 bases (3 genes) to about 200,000 bases (200 genes). classified as virulent or temperate depending on the nature of their relationship to the host bacterium.

begins with attachment of the phage to receptors on the cell surface, followed by injection of the nucleic acid into the bacterial cell, leaving all or most of the protein outside the cell. [Note: This is in contrast to viral infection of vertebrate cells, where the entire virus is taken up by the cell, and its nucleic acid released intracellularly.

The phage nucleic acid takes over the cell's biosynthetic machinery to replicate its own genetic material, and to synthesize phagespecific proteins (consist of phage-specific enzymes and proteins

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of the phage coat).

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BACTERIOPHAGE

TYPES OF BACTERIOPHAGE
A. Virulent phage

When sufficient coat proteins and new phage DNA have accumulated, these components self-assemble into mature phage particles with the DNA encapsulated by the phage

Infection of a bacterium with a virulent phage inevitably results in the death of the cell by lysis, with release of newly replicated phage particles.

Under optimal conditions, a bacterial cell infected with only one phage particle can produce hundreds of progeny phage in twenty minutes.

coat.

Release of the new phage particles is accomplished by a phage-specific enzyme (a lysozyme) that dissolves the bacterial cell wall.

Examples of extensively studied virulent phage, all of which are able to replicate in E. coli, are T2, T4, T7 (all double-stranded DNA), X174 and MS-13 (both single-stranded DNA), and MS-2 (single-stranded RNA, Figure 10.3B).

Generally, phage that attack one bacterial species do not attack other bacterial species.
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TYPES OF BACTERIOPHAGE
B. Temperate phage

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.

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.

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.

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.

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 mapping bacterial genes at a finer scale than does conjugation).

The temperate bacteriophage lambda is the most thoroughly understood of all complex viruses.

Specialized transduction

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

Source: Prescott, L.M. 2002

the process of becoming a prophage.

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LYSOGENIC BACTERIA

bacteria that carry a prophage. the phenomenon is termed lysogeny, and the bacterial cell is said to be lysogenized. Nonlysogenic bacteria can be made lysogenic by infection with a temperate phage. The association of prophage and bacterial cell is highly stable, but can be destabilized by various treatments, such as exposure to ultraviolet light, that damage the host DNA. When DNA damage occurs, repression of phage genes is lifted, the prophage excises from the host chromosome, replicates autonomously, and produces progeny phage particles. The host cell is lysed just as with a virulent phage.
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Source: Prescott, L.M. 2002

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LYSOGENIC BACTERIA

References:

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 that possesses 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 (Streptococcus pyogenes) produce pyrogenic exotoxins (erythrogenic toxins) The acquisition by bacteria of properties due to the presence of a prophage is called lysogenic conversion.

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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.