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

1.

2.

Resistance Plasmid (R factor) Carry genes that confer resistance to certain antibiotics Usually has two types of genes: R-determinant: resistance genes that code for enzymes that inactivate certain drugs 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.

Transposable elements
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

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.

Bacterial conjugation
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 transformation
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
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.

Bacterial transduction
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.

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. 2. 3.

Attachment - Attachment sites on the phage adsorb

to receptor sites on the host bacterium

Penetration - phage injects its genome into the

bacterial cytoplasm

Replication - The phage replicates its genome and Assembly - The phage replicates its genome and

uses the bacterium's metabolic machinery to synthesize phage enzymes and phage structural components
4.

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 cell 2) Viral genome integrates into host cell genome 3) Host cell DNA polymerase copies viral chromosomes 4) Cell divides, and virus chromosomes are transmitted to cell's daughter cells 5) 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.

Generalized transduction
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
1.

2.

3. 4.

5.

A phage attaches to cell wall of bacterium and injects DNA. The bacterial chromosome is broken down and biosynthesis of phage DNA and protein occurs. The cell lyses, releasing viruses. The phage carrying bacterial DNA infects another cell. 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.

2. 3.

4. 5.

Remember that in the lysogenic cycle, phage DNA can exist as a prophage integrated in the bacterial chromosome) Occasionally when the prophage exits it can take adjacent bacterial genes with it. The phage DNA directs synthesis of new phages(The phage particles carry phage DNA and bacterial DNA.) The cell lyses, releasing the phages. 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)

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

Transversion

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

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

DNA or RNA Single or double stranded Linear or circular Several separate segments Few thousand to 250,000 nucleotides
Bacteria have 4 million Influenza

acid

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

Can be derived from

host cells plasma membrane Components can determined by viral nucleic acid

Lipids Proteins Carbohydrates

Viral Structure
Spikes

Carbohydrate protein
complexes Project from envelope Attachment mechanism Means of identification Hemagglutination

Clumping of RBCs

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

Viral Taxonomy
Herpesviridae Herpesvirus Human

Retroviridae Lentivirus Human Immunodeficie ncy Virus 1, HIV 2

herpes virus 1, HHV 2, HHV 3

Growing Bacteriophages
Bacteriophages

Grow in

Liquid

suspensions of bacteria Solid media bacterial cultures

Growing Bacteriophages

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 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

Embryo death Embryo cell

injected into fluid in egg Viral growth

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

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. 3.

Penetration - phage injects its genome into the bacterial

cytoplasm

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

bacterium's metabolic machinery to synthesize phage enzymes and phage structural components
5.

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 cell 2) Viral genome integrates into host cell genome 3) Host cell DNA polymerase copies viral chromosomes 4) Cell divides, and virus chromosomes are transmitted to cell's daughter cells 5) 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 Burst size

Time elapse from attachment to release 20-40 minutes Number synthesized particle released 50-200

Multiplication of Animal viruses

Attachment Viruses attaches to cell membrane Penetration By endocytosis or fusion UncoatingBy 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

Prions
Prions

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 RNA The main protein involved in human and mammalian prion diseases is called PrP

Prion Diseases
scrapie

a fatal disease of sheep and

goats

Prion Diseases
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

Viroids
Small,

circular RNA molecules without a protein coat Infect plants Potato famine in Ireland