Introduction to Microbial Genetics

Recombination

Microorganisms have the ability to acquire genes and thereby undergo the process of recombination.

In recombination, a new chromosome with a genotype different from that of the parent results from the combination of genetic material from two organisms.

This new arrangement of genes is usually accompanied by new chemical or physical properties.

General Recombination

Usually involves a reciprocal exchange of DNA between a pair of DNA sequences.

It occurs anywhere on the microbial chromosome and is typified by the exchanges occurring in bacterial transformation, bacterial recombination, and bacterial transduction.

General Recombination

Site-specific Recombination

involves the integration of a viral genome into the bacterial chromosome.

•also known as conservative site-specific recombination

Site-specific Recombination

Replicative Recombination

which is due to the movement of genetic elements as they switch position from one place on the chromosome to another.

The Bacterial Chromosome and Plasmid

Prokaryotes such as bacteria possess a single chromosome composed of double-stranded DNA in a loop.

The DNA is located in the nucleoid of the cell and is not associated with protein.

Bacterial Cell

Plasmids

Many bacteria (and some yeasts or other fungi) also possess looped bits of DNA known as plasmids, which exist and replicate independently of the chromosome.

Plasmids behave like a mini-chromosomes Plasmids have relatively few genes (fewer

than 30). The genetic information of the plasmid is

usually not essential to survival of the host bacteria.

Plasmid

are circular DNA molecules present in the cytoplasm of the Bacteria

Capable of Autonomous replication Can transfer genes from one cell to other Plasmid Carry genetic information

necessary for conjugation to occur. Only cell that contain such plasmid can

act as donor. the cell lacking a corresponding plasmid act as recipient.

Plasmid

Plasmids contain genes that impart antibiotic resistance. Up to eight genes for resisting eight different antibiotics may be found on a single plasmid. Genes that encode a series of bacteriocins are also found on plasmids. Bacteriocins are bacterial proteins capable of destroying other bacteria. Still other plasmids increase the pathogenicity of their host bacteria because the plasmid contains genes for toxin synthesis.

Plasmid

Curing

Plasmids can be removed from the host cell in the process of curing.

Curing may occur spontaneously or may be induced by treatments such as ultraviolet light.

Chemical agents used for plasmid curing were, Acridine orange, Acriflavine, Ethidium bromide (Intercalating dyes) and Rifampin

Kinds of Plasmid

Certain plasmids, called episomes, may be integrated into the bacterial chromosome.

Conjugative plasmids contain genes for certain types of pili and are able to transfer copies of themselves to other bacteria.

Kinds of Plasmid

Resistance Plasmid (R factor) Carry genes that confer resistance to

certain antibiotics Usually has two types of genes:

1. R-determinant: resistance genes that code for enzymes that inactivate certain drugs

2. RTF (Resistance Transfer Factor): genes for plasmid replication and conjugation.

Fertility (F) Factor

A special plasmid that plays an important role in conjugation.

contains genes that encourage cellular attachment during conjugation and accelerate plasmid transfer between conjugating bacterial cells.

Those cells contributing DNA are called F+ (donor) cells or male, while those receiving DNA are the F- (recipient) cells or female.

The F factor can exist outside the bacterial chromosome or may be integrated into the chromosome.

Hfr Cell

When the F+ plasmid is integrated within the bacterial chromosome, the cell is called an Hfr cell (high frequency of recombination cell).

Transposable elements

also known as transposons or “jumping genes”, are segments of DNA that move about within the chromosome and establish new genetic sequences.

They exhibit the so called “cut and paste mechanism”

First discovered by Barbara McClintock in the 1940s, transposons behave somewhat like lysogenic viruses except that they cannot exist apart from the chromosome or reproduce themselves.

Not self replicating and depend on Plasmid or Chromosome for replication.

they assisted in the transfer of bacterial resistance to antibiotics. Furthermore, it soon became evident that they caused most of the spontaneous mutations occurring in laboratory populations of more sophisticated organisms

Transposable elements

Transposons

Bacterial Recombinations

Three types of bacterial recombination result in a change in the DNA of recipient organisms.

The proteins expressed by the new genes lead to new physiological characteristics in the bacteria.

Bacterial conjugation

Was first postulated in the 1940s by Joshua Lederberg and Edward Tatum.

The essential feature of the process is that two bacterial cells come together and mate such that a gene transfer occurs between them.

One cell, the donor cell (F+), gives up DNA; and another cell, the recipient cell (F−), receives the DNA.

The DNA is directly transferable.

The transfer is nonreciprocal, and a special pilus called the sex pilus joins the donor and recipient during the transfer.

The DNA most often transferred is a copy of the F factor plasmid.

The factor moves to the recipient, and when it enters the recipient, it is copied to produce a double-stranded DNA for integration.

The channel for transfer is usually a special conjugation tube formed during contact between the two cells

Bacterial conjugation

Naked DNA molecule from the environment is taken up by the cell and incorporated into its chromosome in some heritable form

Defined as transfer of Genetic information through the activity of DNA

Bacterial transformation

During transformation, a competent cell takes up DNA and destroys one strand of the double helix.

A single-stranded fragment then replaces a similar but not identical fragment in the recipient organism, and the transformation is complete.

Bacterial transformation

Bacterial transformation

Was discovered by Frederick Griffith in 1928.Griffith Experiment Griffith worked with the pneumococci that

cause bacterial pneumonia. He discovered that if he mixed fragments of

dead pathogenic pneumococci with specimens of live harmless pneumococci, the harmless bacteria took on genes of the bacterial fragments and became pathogenic.

Griffith Experiment

Bacterial transduction

In transduction, bacterial viruses (also known as bacteriophages) transfer DNA fragments from one bacterium (the donor) to another bacterium (the recipient).

The viruses involved contain a strand of DNA enclosed in an outer coat of protein.

After a bacteriophage (or phage, in brief) enters a bacterium, it may encourage the bacterium to make copies of the phage.

At the conclusion of the process, the host bacterium undergoes lysis and releases new phages.

This cycle is called the lytic cycle.

Bacterial transduction

Lytic Cycle

States that the virulent phages are those phages that can multiply only on bacterial cells. At the end of their life cycle, they cause cell lysis that kills the host bacteria.

Lytic Cycle Steps

1. Attachment - Attachment sites on the phage adsorb

to receptor sites on the host bacterium 2. Penetration - phage injects its genome into the

bacterial cytoplasm

3. Replication - The phage replicates its genome and uses the bacterium's metabolic machinery to synthesize phage enzymes and phage structural components

4. Assembly - The phage replicates its genome and uses the bacterium's metabolic machinery to synthesize phage enzymes and phage structural components

5. Release - The cell breaks open and each replicated virus can now infect other cells.

Lysogenic cycle

Under other circumstances, the virus may attach to the bacterial chromosome and integrate its DNA into the bacterial DNA.

It may remain here for a period of time before detaching and continuing its replicative process.

The virus is called a temperate phage, also known as a prophage.

Lysogenic Cycle Steps

1.) Viral genome enters cell2) Viral genome integrates into host cell genome3) Host cell DNA polymerase copies viral chromosomes4) Cell divides, and virus chromosomes are transmitted to cell's daughter cells5) At any moment when the virus is "triggered", the viral genome detaches from the host cell's DNA and enters stage 2 of the lytic cycle.

any part of the bacterial genome can be transferred; occurs during the lytic cycle of virulent and temperate bacteriophages because the host's chromosome is broken down into fragments.

Generalized transduction

Generalized transduction

1. A phage attaches to cell wall of bacterium and injects DNA.

2. The bacterial chromosome is broken down and biosynthesis of phage DNA and protein occurs.

3. The cell lyses, releasing viruses.

4. The phage carrying bacterial DNA infects another cell.

5. Crossing over between donor and recipient DNA can occur producing a recombinant cell.

Specialized Transduction

Transfer of only specific portions of the bacterial genome; carried out only by temperate phages that have integrated their DNA into the host chromosome at a specific site in the chromosome

Specialized Transduction

1. Remember that in the lysogenic cycle, phage DNA can exist as a prophage integrated in the bacterial chromosome)

2. Occasionally when the prophage exits it can take adjacent bacterial genes with it.

3. The phage DNA directs synthesis of new phages(The phage particles carry phage DNA and bacterial DNA.)

4. The cell lyses, releasing the phages. 5. A phage carrying bacterial DNA infects another

cell.

Mutation

A permanent alteration in the sequence of nitrogenous bases of a DNA molecule.

The result of a mutation is generally a change in the end-product specified by that gene.

In some cases, a mutation can be beneficial if a new metabolic activity arises in a microorganism, or it can be detrimental if a metabolic activity is lost.

Mutation

Caused by alteration in the Nucleotide sequence at some point of DNA which can occur due to

Addition

Deletion

Substitution

of one or more bases

Types of mutations

Missense Mutation the new nucleotide alters the codon so as to produce an altered amino acid in

the protein product. (ex. sickle cell anemia)

the new nucleotide changes a codon that specified an amino acid to one of the STOP codons (TAA, TAG, or TGA)

Nonsense Mutation

is Substitution of purine for pyramidine or vice versa in the base pairing

Transversion

Frameshift Mutation

pairs of nucleotides are either added to or deleted from the DNA molecule, with the result that the “reading frame” is shifted.

Chemical mutagens

include nitrous acid, substance converts adenine to hypoxanthine, a molecule that will not pair with thymine, and thus interrupts the genetic code.

base analog is a chemical mutagen that resembles a nitrogenous base and is incorporated by error into a DNA molecule.

Such a DNA molecule cannot function in protein synthesis.

Certain dyes and fungal toxins (for example, aflatoxin) are known to be mutagens.

Physical mutagens

X rays and gamma rays break the covalent bonds in DNA molecules, thereby producing fragments.

Ultraviolet light binds together adjacent thymine bases, forming dimers. These dimers cannot function in protein synthesis, and the genetic code is thereby interrupted.

Radiation damage can be repaired by certain bacterial enzymes, a process known as photo reactivation.

VIRUS, VIROIDS and PRIONS

VIRUS

Viruses contain DNA or RNA And a protein coat Some are enclosed by an envelope Some viruses have spikes Cause infection and disease Obligate intracellular parasites

• Require hosts to multiply

Sizes of Virus

Helical viruses

consist of nucleic acid surrounded by a hollow protein cylinder or capsid and possessing a helical structure

Polyhedral viruses

consist of nucleic acid surrounded by a polyhedral (many-sided) shell or capsid, usually in the form of an icosahedron

Complex Viruses

have neither helical nor polyhedral forms, are pleomorphic (irregular shaped), or have complex structures

Enveloped Viruses

consist of nucleic acid surrounded by either a helical or polyhedral core and covered by an envelope

Viral Structure

Nucleic acid• DNA or

• RNA

• Single or double stranded

• Linear or circular

• Several separate segments• Influenza

• Few thousand to 250,000 nucleotides• Bacteria have 4 million

Viral Structure

Capsid• Protein coat

surrounding nucleic acid

• Most of mass of virus

• Composed of capsomeres• Arrangement

characteristic for a particular virus

• Single protein type

• Several protein types

Viral Structure Envelope

• Covers capsid in some viruses

• Combination of• Lipids

• Proteins

• Carbohydrates

• Can be derived from host cells plasma membrane

• Components can determined by viral nucleic acid

Viral Structure

Spikes• Carbohydrate –

protein complexes

• Project from envelope

• Attachment mechanism

• Means of identification

• Hemagglutination • Clumping of RBC’s

Viral Taxonomy

Family names end in -viridae Genus names end in -virus Viral species: A group of viruses sharing

the same genetic information and ecological niche (host). Common names are used for species

Subspecies are designated by a number

Herpesviridae Herpesvirus Human

herpes virus 1, HHV 2, HHV 3

• Retroviridae

• Lentivirus

• Human Immunodeficiency Virus 1, HIV 2

Viral Taxonomy

Growing Bacteriophages

Bacteriophages• Grow in

• Liquid suspensions of bacteria

• Solid media bacterial cultures

Plaque method• Bacteriophage

mixed with bacteria

• Mixed in melted agar

• Poured in Petri plate over layer of hardened agar

• Mix solidifies into one cell thick layer

Growing Bacteriophages

Growing Animal Viruses

Three methods used

•Living animals

•Embryonated eggs

•Cell cultures

Growing Viruses in Living Animals

Some viruses only grow in living animals (Mice, rats, rabbits, and guinea pigs) Best to study immune responses Used for diagnostic purposes

• Mice inoculation for rabies Some diseases lack adequate animal models

• HIV-1 • Mice have been genetically engineered to act

as animal model

Growing Viruses in Embryonated Eggs

Embryonated eggs• Embryo present

• Hole drilled in shell

• Viral suspension injected into fluid in egg

• Viral growth• Embryo death

• Embryo cell damage

• Typical lesions on membrane

Growing Viruses in Embryonated Eggs

Embryonated eggs

• Four types on inoculation

•Yolk sac

•Allantoic

•Amniotic

•Chorioallantoic

Growing viruses in Cell Cultures Consist of homogenous cells grown in culture media More convenient than living animals or embryonated

eggs Cell cultures can be propagated like bacterial

cultures

Growing Viruses in Cell Cultures

Virus infects cells• Cells in monolayer deteriorate as virus multiplies

• CPE – cytopathic effect or cytopathogenic effect

• Can be detected and counted similar to plaques

Virus Identification

Methods of identification

• Serological

• Western blot method

• Most commonly used

• Is detected and identified by reactions to antibodies

• Cytological changes

• Molecular methods

• Restriction fragment length polymorphisms (RFLPs)

• Polymerase chain reactions (PCR)

Multiplication of Bacteriophages (Lytic Cycle)

Two types of multiplication

• Lytic cycle

•Ends with death and lysis of cell

•T-even bacteriophage

• Lysogenic cycle

•Host cell remains alive

•Bacteriophage λ

Lytic Cycle

States that the virulent phages are those phages that can multiply only on bacterial cells. At the end of their life cycle, they cause cell lysis that kills the host bacteria.

Lytic Cycle Steps

1. Attachment - Attachment sites on the phage adsorb to

receptor sites on the host bacterium

2. Penetration - phage injects its genome into the bacterial cytoplasm

3. Replication - The phage replicates its genome and uses the bacterium's metabolic machinery to synthesize phage enzymes and phage structural components

4. Assembly - The phage replicates its genome and uses the bacterium's metabolic machinery to synthesize phage enzymes and phage structural components

5. Release - The cell breaks open and each replicated virus can now infect other cells.

Lysogenic cycleUnder other circumstances, the virus may attach to the bacterial chromosome and integrate its DNA into the bacterial DNA.

It may remain here for a period of time before detaching and continuing its replicative process.

The virus is called a temperate phage, also known as a prophage.

Lysogenic Cycle Steps

1.) Viral genome enters cell2) Viral genome integrates into host cell genome3) Host cell DNA polymerase copies viral chromosomes4) Cell divides, and virus chromosomes are transmitted to cell's daughter cells5) At any moment when the virus is "triggered", the viral genome detaches from the host cell's DNA and enters stage 2 of the lytic cycle.

One – Step Growth Experiment Burst time

• Time elapse from attachment to release• 20-40 minutes

Burst size• Number synthesized particle released• 50-200

Multiplication of Animal viruses

Attachment Viruses attaches to cell membrane Penetration By endocytosis or fusion Uncoating By viral or host enzymes Biosynthesis Production of nucleic acid and

proteins Maturation Nucleic acid and capsid proteins

assemble Release By budding (enveloped viruses)

or rupture

PrionsPrions are “infectious proteins” They are normal body proteins that get converted into an alternate configuration by contact with other prion proteins They have no DNA or RNAThe main protein involved in human and mammalian prion diseases is called “PrP”

Prion Diseases

scrapie a fatal disease of sheep and goats

Mad cow disease- incurable, fatal brain disease that affects cattle and possibly some other animals, such as goats and sheep. The medical name for mad cow disease is bovine spongiform encephalopathy or BSE for short. It's called mad cow disease because it affects a cow's nervous system, causing a cow to act strangely and lose control of its ability to do normal things, such as walk.

Prion Diseases

Creutzfeldt-Jacob disease is a rare, degenerative brain disorder. Symptoms usually start around age 60. Memory problems, behavior changes, vision problems and poor muscle coordination progress quickly to dementia, coma and death. Most patients die within a year.

Prion Diseases

Kuru is among the fatal neurodegenerative prion protein (PrP) diseases in humans It is now widely accepted that Kuru was transmitted among members of the Fore tribe of Papua New Guinea via cannibalism.

he term "kuru" derives from the Fore word "kuria/guria", 'to shake', a reference to the body tremors that are a classic symptom of the disease; it is also known among the Fore as the laughing sickness due to the pathologic bursts of laughter people would display when afflicted with the disease

Prion Diseases

Viroids

Small, circular RNA molecules without a protein coatInfect plantsPotato famine in Ireland