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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
48
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
49
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
www.2dix.com
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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
www.2dix.com
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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.
www.2dix.com
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 .
www.2dix.com
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steps in Generalised Transduction (contd)
7. The donor bacterium's DNA is exchanged for some of the recipient's DNA.
www.2dix.com
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.
www.2dix.com
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.
www.2dix.com
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