Bacterial Genetics

Prabin Shah BScMLT,

MSc(Biochemistry)

Understanding Genetics

We resemble and differ because of Genetic configurations

Parents - Son - Daughter, how they resemble each other.

They breed true from Generation to Generation

But vary in small proportions in progeny. Bacteria too obey the laws of

Genetics

Watson - CrickDiscovery of DNA

DNA A Complex Structure Makes Life

Prokaryotes Vs Eukaryotes Genetics

Prokaryotes Eukaryotes

Prokaryotes are haploid eukaryotes are often diploid

contain a single circular chromosome.

eukaryotes have linear chromosomes, usually more than 1

Prokaryotes often contain “plasmids”.

Doesnot contain plasmids

In prokaryotes, translation is coupled to transcription: translation of the new RNA molecule starts before transcription is finished

In eukaryotes, transcription of genes in RNA occurs in the nucleus, and translation of that RNA into protein occurs in the cytoplasm. The two processes are separated from each other.

DNA( Deoxyribonucleic Acid )

DNA is composed of Many Units of

Adenine – Thymine A - T

Guanine – Cytosine G - C

A+ T

G+C proportion differ for each species DNA replicates first unwinding at one end to

form a fork Each strand of fork acting as template for the

synthesis of complementary strand

DNA

A DNA molecule is composed of two chains of Nucleotides wound together in the form of a Double Helix

Each chain has back bone of Deoxyribose and Phosphates residues arranged alternatively

Structure of DNA

Attached to each Deoxyribose and phosphate residues arranged alternatively

Attached to each Deoxyribose are of four nitrogen bases

Purines - Adenine, Guanine

Pyrimidines

Thymidine and Cytosine

How RNA differs from DNA

RNA contains - Sugar Ribose instead of Deoxyribose

Uracil is present instead of Thymine Types of RNA Messenger RNA mRNA Ribosomal RNA rRNA Transfer RNA tRNA

What is a Code in Genetics?

Code is a unit consists of sequence of three Bases Code is triplet A-T- C A code can make single Amino acid More than one code present for making similar

sequence of Amino acid AGA make Arginine AGC, CGU, CGG, also code for similar Amino acid Some Codons UAA don't code for any Amino acid

called as Nonsense codon

What is a Gene?

Gene is a sequence of DNA carrying codons specifying for particular polypeptide.

DNA contains many Genes(combinations of hundreds and thousands of Nucleotides )

Bacterial Chromosome Contains a Double stranded molecules

of DNA arranged in circular form. Length 1,000 microns. Bacterial DNA contains about

4,000kilobases 1 kb = 1000 base pairs (A-T) (G-C)

Extra chromosomal Genetic Elements

Bacteria posses Extra chromosomal genetic elements

Not Essential for survival of Bacteria But makes the Bacteria Resistant to

antibiotics, and makes them survive

& also able to produce toxins

Plasmids

Plasmids are circular DNA molecules present in the cytoplasm of the Bacteria

Their size varies from 1 kbp to over 400 kilobase pairs (kbp).

Capable of Autonomous replication

Can transfer genes from one cell to other

Act as vectors in Genetic engineering.

Plasmids Plasmid seem to be ubiquitous in bacteria, May

encode genetic information for properties 1 Resitance to Antibiotics 2 Bacteriocins production 3 Enterotoxin production 4 Enhanced pathogenicity 5 Reduced Sensitivity to mutagens 6 Degrade complex organic molecules

Plasmids are classified

1. by their ability to be transferred to other bacteria

Conjugative The sexual transfer of plasmids to another bacterium through a pilus.

Non-conjugative Non-conjugative plasmids don’t initiate conjugaison.They can only be transferred with the help of conjugative plasmids.

Mobilisable An intermediate class of plasmids are mobilisable, and

carry only a subset of the genes These plasmids can 'parasitise' another plasmid,

transferring at high frequency presence of a conjugative plasmid

Incompatibility groups: Several types of plasmids could coexist in a single cell. On the other hand, related plasmids are often

'incompatible', resulting in the loss of one of them from the cell line.

2. Function

1. Fertility-(F) plasmids, They are capable of conjugation (they contains the genes for

the pili).

2. Resistance-(R) plasmids,

Contain gene (s) that can build resistance against one or several antibiotics or poisons.

3. Col-plasmids,

Contain genes coding for colicines, proteins that can kill other bacteria. .

4. Degradative plasmids, able to digest unusual substances, e.g., toluene or

salicylic acid 5. Virulence plasmids turn a bacterium into a pathogen.

6. Addiction system. These plasmids produce both a long-lived poison and a

short-lived antidote. Daughter cells that retain a copy of the plasmid survive while a daughter cell that fails to inherit the plasmid dies

or suffers a reduced growth-rate because of the lingering poison from the parent cell.

Plasmids

Can be integrated with Chromosomal DNA

Episomes -Integrated form of plasmid with DNA

Potentials of Plasmids

Plasmids can be self transmissible and Non transmissible

Transfers the Sex and Drug resistance with the help of restriction end nucleases

Genotypic & Phenotypic variation

Genome – Sum total of Gene that make up the genetic apparatus of cell established as Genotype.

Hereditary constitution of cell this transmitted to its progeny

Phenotype – is the physical expression of genotype in a environment & can change according to environment.

Phenotypic variation

Exhibit – different phenotypes Appearance differs in different situations. Eg: Typhoid bacilli flagellated normally But grown in Phenol agar don't grow flagella so

flagella are lost physical variation Lactose fermentation in E.coli dependent on Beta

Galactosidase

When lactose present - test is positive

When lactose is absent - test turns negative

Genotypic variations

Stable, heritable and not influenced by environment

May occur by Mutations Genotypic by transfer of genes

Transformation

Transduction(Lysogenic conversion)

Conjugation

Mutations Mutation is a Random, Undirected,

Heritable variation 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

Mutation Rate Different types of mutations can occur at

different frequencies. For a typical bacterium, mutation rates of 10–7 to 10–11 per base pair are generally seen

Although RNA and DNA polymerases make errors at about the same rate, RNA genomes typically accumulate mutations at much higher frequencies than DNA genomes.

Mutations can occur in any sequence, inveitable, useful for Survival

Multiple Mutations Causes extensive chromosomal rearrangement Missense mutation Triplet code is altered so as to specify an

aminoacid different from that normally located at particular position in the protein.

Nonsense mutation Deletion of nucleotide within a gene may

cause premature polypeptide chain termination by nonsense codon

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

Possible effects of bp substitution

Replica Plating Method A common way to find bacterial mutants is

replica plating, which means making two identical copies of the colonies on a petri plate under different conditions.

For instance, if you were looking for trp- auxotrophs, one plate would contain added tryptophan and the other plate would not have any tryptophan in it.

Bacteria are first spread on the permissive plate, the plate that allows both mutants and wild type to grow, the plate containing tryptophan in this case..

They are allowed to grow for a while, then a copy of the plate is made by pressing a piece of velvet onto the surface of the plate

then moving it to a fresh plate with the restrictive condition (no tryptophan).

The velvet transfers some cells from each colony to an identical position on the restrictive plate.

Colonies that grow on the permissive plate but not the restrictive plate are (probably) trp- auxotrophs, because they can only grow if tryptophan is supplied

Replica Plating

Transmission of Genetic material ( Gene Transfer )

Different Mechanisms

Transformation

Transduction Conjugation

Transformation Transformation is defined as transfer of Genetic

information through the activity of free DNA first experiment that showed DNA was the

genetic information Griffith experiment Mice injected with Live non capsulated ( R ) Pneumococci & with heat killed capsulated (S) Pneumococci Lead to death of Mice with isolation of Live capsulated

Pneumococci It means that some factor from dead pneumococci

transferred to live non pathogenic Pneumococci

Griffith Phenomenon

Certain prokaryotes exhibit competence, a state in which cells are able to take up free DNA released by other bacteria.

Incorporation of donor DNA into a recipient cell requires the activity of single-stranded binding protein, RecA protein, and several other enzymes.

Only competent cells are transformable

Conjugation A process by which a Donor cell or male cell makes

contact with another cell, the recipient or Female cell.

DNA is directly transferable Plasmid Carry genetic information necessary for

conjugation to occur. Only cell that contain such plasmids can act as donor.

the cell lacking a corresponding plasmid act as recipient.

Requires direct contact between donor and recipient

Conjugation - Transferring genes with plasmids

Plasmids mediating conjugation carry genes coding for properties, of 1-2 microns long protein appendage termed Pilus on the Donor cell

Conjugation

Pilus helps Conjugation

Different types of Pilus are specified by different types of plasmids and can help in aid of plasmid classification.

Only one strand of circular DNA of the plasmid nicked upon at a specific site and passed into a recipient.

Spread to all other cells.

F factor

Transfer factor that contains the genetic information necessary for synthesis of Sex Pilus and for self transfer without any other identifiable genetic materials such as drug resistance

F+ called as Donor bacteria can transform F- into F+ cell

Can be Episomes able to exist in some cells in the integrated state in the donor cell chromosome

Can transform chromosomal genes to recruitment with high frequency are known as Hfr cells

Conversion of F+ cells into Hfr state is reversible. F factor incorporates some chromosomal genes

and is called as F’ Sexduction : process of transfer of host genes

through F’ factor

Transduction

Transduction is defined as transfer of portion of DNA from one bacteria to another by Bacteriophages, is known as Transduction

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Historical Lederberg & Zinder

Transduction was first discovered in 1952 by Joshua Lederberg and Norton Zinder

Joshua Lederberg Norton Zinder

Alcamo's and Prescott , google images

How transduction was discovered

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Studied in Salmonella typhimurium Plated two auxotrophic strains (LA-2 and LA-22)

individually on minimal medium, no cells grew. Plated a mixture of the two auxotrophic strains on

minimal medium, cells grew into colonies. Thus, genetic exchange was taking place between

the two cell types.

www.bio.ilstu.edu

U-tube Experiment

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TYPES OF TRANSDUCTION

GENERALIZED TRANSDUCTION

SPECIALIZED TRANSDUCTION

GENERALIZED TRANSDUCTION

process by which any bacterial gene may be transferred to another bacterium via a bacteriophage.

typically carries only bacterial DNA and no viral DNA

Generalised Transduction

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1. A lytic bacteriophage adsorbs to a susceptible bacterium.

2. The bacteriophage genome enters the bacterium. The genome directs the bacterium's metabolic machinery to manufacture bacteriophage components and enzymes

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3. Occasionally, a bacteriophage head or capsid assembles around a fragment of donor bacterium's nucleoid instead of a phage genome by mistake.

steps in Generalised Transduction (cont’d)

4. The bacteriophages are released.

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steps in Generalised Transduction (cont’d)

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5. The bacteriophage carrying the donor bacterium's DNA adsorbs to a recipient bacterium

6. The bacteriophage inserts the donor bacterium's DNA it is carrying into the recipient bacterium .

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steps in Generalised Transduction (contd)

7. The donor bacterium's DNA is exchanged for some of the recipient's DNA.

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SPECIALIZED TRANSDUCTION

Discovered by Morse et al Process by which a restricted set of bacterial genes are transferred to another bacterium Partially coded prophage DNA is called a "heterogenote".

Specialised Transduction

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1. A temperate bacteriophage adsorbs to a susceptible bacterium and injects its genome .

2. The bacteriophage inserts its genome into the bacterium's nucleoid to become a prophage.

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steps in Specialised Transduction (cont’d)

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3. Occasionally during spontaneous induction, a small piece of the donor bacterium's DNA is picked up as part of the phage's genome in place of some of the phage DNA which remains in the bacterium's nucleoid.

4. As the bacteriophage replicates, the segment of bacterial DNA replicates as part of the phage's genome. Every phage now carries that segment of bacterial DNA. www.2dix.com

steps in Specialised Transduction (cont’d)

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5. The bacteriophage adsorbs to a recipient bacterium and injects its genome.

6. The bacteriophage genome carrying the donor bacterial DNA inserts into the recipient bacterium's nucleoid.

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Genetic Mechanisms of Drug Resistance

Bacteria acquire drug resistance through several Mechanisms

Mutations Genetic transfer Transformation, Transduction ConjugationSeveral Biochemical Mechanisms Decreasing permeability of drugs, Attaining alternative pathways Produce enzymes and inactivate drugs

Genetic Mechanisms in Bacteria helps to spread the Infectious diseases

Transposons and Insertion Sequences

Transposons and insertion sequences are

genetic elements that can move from one location on a chromosome to another by a process called transposition.

a type of site-specific recombination.

Transposition is linked to the presence of special genetic elements called transposable elements. Transposition can be either replicative or conservative

Summary A DNA molecule is composed of two chains of

Nucleotides wound together in the form of a Double Helix

Gene is a sequence of DNA carrying codons specifying for particular polypeptide

Plasmids are circular DNA molecules present in the cytoplasm of the Bacteria

Phenotype variation – is the physical expression of genotype in a environment & can change according to environment

Genotype variation – Stable, heritable and not influenced by environment

Mutation – alteration in the Nucleotide sequence at some point of DNA which can occur due to Addition, Deletion, Substitution of one or more bases

Replica plating Transformation – is defined as transfer of Genetic

information through the activity of free DNA Conjugation – process by which a Donor cell or

male cell makes contact with another cell, the recipient or Female cell.

Transduction – is defined as transfer of portion of DNA from one bacteria to another by Bacteriophages

Generalised transduction – process by which any bacterial gene may be transferred to another bacterium via a bacteriophage

Specialised transduction – Process by which a restricted set of bacterial genes are transferred to another bacterium

Reference Ananthnarayan and Paniker`s Text

book of Microbiology 8th Edition C.P.Baveja Textbook of

Microbiology