BACTERIAL GENETICS

Dr. R. Someshwaran, MD., Assistant professor,Department of Microbiology, KFMS&R

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Objectives of today’s class

At the end of my class you should be able to Enumerate the principles of Molecular

biology Describe the structure of DNA & plasmid Discuss about phenotypic and genotypic

variation ELUCIDATE THE METHODS OF GENE

TRANSFER EXPLAIN THE GENETIC MECHANISM OF

DRUG RESISTANCE IN BACTERIA Emphasise on Application of Molecular

genetics

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Introduction Bacteria and other microbes Laws of Genetics Pleomorphism (Nageli) Protean capacity for variation Monomorphism (Kohn & Koch) Molecular biology 1940’s

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Basic principles of Molecular biology

Central dogma of Molecular biology – DNA

Essential material of heridity DNA is transcribed to RNA RNA is translated into

polypeptide Polypeptides – Proteins and

Enzymes

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Bacterial cell

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Transcription & Translation

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Bacterial chromosome

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DNA Double Helix

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Double stranded DNA

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DNA structure Double helix (2 chains of

nucleotides) Deoxyribose and phosphate

backbone Nitrogenous bases – Purine &

Pyrimidines Purine – Adenine (A), Thymine (T) Pyrimidine – Guanine (G), Cytosine

(C) Complementary base pair – A:T, G:C Hydrogen bonds between bases of

opposite strand gives stability to DNA double strand

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Complementary base pairs

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DNA structure

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Watson & Crick – Nobel Laureates

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DNA structure Ratio of each pair of bases (A+T) /

(G+C) is constant for each species but varies widely among one species to another

DNA replication begins with unwinding at one end to form a fork

Each strand of the fork acts as template for synthesis of complementary strand – formation of double helix

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RNA Structure Structurally similar to DNA But 2 major difference Ribose instead of Deoxyribose Base Uracil instead of Thymine 3 types: mRNA, tRNA & rRNA DNA acts as template for mRNA

synthesis AGCU (DNA) complementary to TCGA

(mRNA)

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RNA Vs DNA

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Codon (Triplet code) Codon: Genetic information stored in DNA as code Unit of code with sequence of three bases is called

as Triplet code (Codon) Each codon transcribed on mRNA specifies for a

single aminoacid Ex: AGA codes for Arginine But the code is degenrate (ie., >1 codon may exist

for the same aminoacid) and is non-overlapping Ex: AGG, CGU, CGC, CGA and CGG also codes for

Arginine UAA, UGA, UAG – Nonsense or stop codons; Acts

by terminating the message for synthesis of a polypeptide

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RNA to Polypetides & Proteins

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Gene Bacterial chromosome contain double

stranded molecule of DNA arranged in a circular form

DNA contain large number of genes Gene: A segment of DNA carrying codons

specifying a particular polypeptide Genes contains hundreds of thousands of

nucleotides Note: When straightened, the length of DNA

1000µm but usually expressed as kilobases (1000 base pairs)

Bacterial DNA is 4000kb whereas Human genome is 3 million kb long

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Exons & Introns Exons: Stretches of coding genes are

called Exons Introns: Between the coding sequences

of genes there are useless, non-functional, non-coding intrusions called Introns

During transcription both introns and exons are copied in its entirety

But during translation only exons of RNA copy are being translated by ribosomes into proteins where introns are excised

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Extrachromosomal genetic elements

Extra chromosomal genetic elements are not essential for normal life and functioning of host bacterium; but may confer properties such as

Drug resistance & Toxigenicity leading to survival advantage under necessary appropriate conditions

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Plasmids Plasmids are circular DNA molecules

present in cytoplasm of bacteria, capable of autonomous replication (independent replicons).

Important vectors for Genetic engineering

Plasmid DNA may be integrated with chromosoma DNA. Such integrated froms are called Episomes.

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Plasmid

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Episome

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Plasmid typing Plasmid types: Self-transmissible or

Non-transmissible or non-conjugating

Based on property encoded (Sex, Drug Resistance, etc.)

By Restriction Endonuclease Fingerprinting, Incompatibility typing

Based on types of Conjugation tube induced (Which determines the susceptibility of host bacterium to lysis by virulent bacteriophages)

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Genotype and Phenotype

Genome: Sum total of the genes that make up the genetic apparatus of a cell establishes the genotype or heriditary constitution of the cell that is transmitted to the progeny.

Genotype: Complete Genetic potential of the cell, all of which may or may not be expressed in a given environmental situation

Phenotype: (Phaeno – display) Physical expression of the genotype

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Genotypic and Phenotypic variation

1. Example of Phenotypic variation: Salmonella typhi – Flagellated – Normally When grown in Phenol agar – No flagella

synthesised The condition is reversible when subcultured from

phenol agar to broth. Determined by environment2. Escherichia coli - Enzyme Beta-galactosidase –

Lactose fermentation occurs in medium containing lactose

3. When E. coli grown in media containing only glucose, Enzyme will not be produced (Induced enzymes)

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Genotypic and Phenotypic variation

Genotypic variation is stable, heritable and not influenced by environment and results due to alteration in the genome. Causes: Mutation or by one of the mechanisms of genetic transfer or exchange (Transformation, Transduction, Lysogenic conversion & Conjugation)

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Genotypic and Phenotypic variation

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Bacterial DNA Mutation Random, undirected, heritable variation

caused by alteration in nucleotide sequence at some point of DNA of the cell.

Caused spontaneously (mistakes in DNA synthesis) or Induced by Physicochemical forces (UV, X rays, chemical mutagens like alkylating agents, acridine dyes, %-bromo uracil, 2-Aminopurine, etc.)

Since reproduction (Binary fission) is quick e.g. doubling time= Say 20 minutes as in Escherichia coli. # New mutations spread quickly

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Types of Mutation Types: A. Spontaneous or B. Induced. Also mutations may be typed as Addition,

deletion, Substitution (Point mutation), Frame-shift mutation, missense mutation, Non-sense mutation, Transversion, Suppressor mutation (reversal of mutant phenotype),

Lethal mutation, Conditional lethal mutant (Temperature sensitive mutant or ts mutant; permissive temperature 37⁰C, Non-permissive temperature 39⁰C).

Multiple mutation cause extensive chromosomal arrangement

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Spontaneous Mutation Fixed frequency rate for mutation Mutation – can go unrecognised - can affect vital function - can give survival advantage - Can be lethal - Can cause ‘Adaptations’ as in

Streptomycin resistant mutant of Tubercle bacilli, selective multiplication of Drug resistant bacilli followed by replacement of Drug sensitive bacilli when on treatment with that particular drug (Ex: Streptomycin).

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Calculation of incidence of mutations

If doubling time= 20 min, then 23cells/hr Over 12 hr, 236 cells (~1010)produced from a single cellIf spontaneous mutation rate = 1 x 10-7 / gene, then in 12 hr (day) (1010) (10-7)= 103 mutations/gene/dayIf bacteria have ~4000 genes; then (4x103genes(103)= 4 x 106 mutations/day

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Damage to DNA

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DNA Damage – Altered DNA

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Where, What & How to look for?

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In a chromosome

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We have Genes, DNA

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Mutation

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Types of Mutation

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Types of Mutation

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Types of Mutation

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Fluctuation test in Bacteriophage resistant E. coli By Luria and Delbruck

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Simple but elegant ‘Replica plating technique’ By Lederberg

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Bacterial Genetic Diversity:Genetic Recombination (Gene transfer or

Exchange)

Three processes bring bacterial DNA from different individuals together: Transduction Transformation Conjugation

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Transformers!!!

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Transformation Avery, Mc Leod & Mc Carty (1944) Transfer of genetic information through

the agency of free DNA Griffith 1928 – Mice – Experimental

inoculation of Streptococcus pneumoniae – (R) forms Live non-capsular strain type II, (S) forms Heat-Killed capsular type III strain Isolated from Blood culture of Mice. Showed transfer of information of capsule synthesis from heat killed to live strain was demonstrated in vitro also.

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Griffith experiment

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Transduction Most widespread mechanism of gene transfer

among prokaryotes and excellent tool for gene mapping of bacteria and also as a method of Genetic engineering for treatment for some inborn errors of metabolism

Transfer of a portion of the DNA from one bacterium to another by a bacteriophage is known as transduction

Bacteriophages are viruses that parasitise bacteria and consist of a nucleic acid core and a protein coat.

During Bacteriophage assembly, Packaging errors may occur

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Transduction Types of Transformation: A. Generalised,

B. RestrictedRestricted transduction – extensively studied in λ Lambda phage of E.coli

Prophage lambda is inserted into the bacterial chromosome between the genes determining Galactose utilisatio (gal) and Biotin synthesis (bio)

So, it transduces any one of these genes Chromosomal DNA, Plasmid, Episomes can be

transduced,

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Transduction

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Lysogenic conversion Bacteriophages exhibits 2 types of Life cycle. A. Virulent or Lytic, B. Lysogenic Lysogeny is extremely frequent in nature Lysogenic conversion or Phage conversion In transduction --- Phage is a carrier of

genetic material fom one bacteria to another In Lysogenic conversion --- Phage itself is a

new genetic element to the host bacterium

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Lysogenic conversion1. Susceptibility to bacteriophages

(Immunity to superinfection with same or related phages)

2. Antigenic characteristics3. Medical importance: Corynebacterium

diphtheriaere acquire Toxigenicity and therefore virulence by Lysogenisation with phage beta.

4. Toxigenic strain - eliminate betaphage – non-toxigenic

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Lysogenic conversion

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Lysogenic conversion

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Conjugation Conjugation or Sexduction is a process

whereby a male cell or donor bacterium mates or makes physical contact with female cell or recipient bacterium and transfers genetic elements into it.

Plasmids are frequently tranferred by conjugation; and it was in E.coli K12 the role of plasmids in conjugation was first recognised.

“Maleness” Contains F (fertility) genes on plasmid or in genome. Encoded sex pilus (Conjugation tube)- Forms passage way for DNA from donor to recipient

LE 18-17

Sex pilus 5 µm

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Conjugation - Sexduction Plasmids responsible was termed

as Fertility factor (F) or Sex factor. Now Transfer factor is the term used.

F factor (Episome coding For synthesis of Sex pilus), Cells with F factor = F+ Cells; with no F factor = F-Cells; Hfr cells: High frequency cells - Episomes (Integrated state with host chromosome). F+ Cell to Hfr cell conversion is reversible; F factor to F Prime (F’) factor

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Conjugation

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Colicinogenic factor (Col) Coliorm – Colicins (Lethal antibiotic

like substance for other enterobacteria)

Pyocin by Pseudomonas pyocyanea Diphthericin by Corynebacterium

diphtheriae Bacteriocin is the term given to

these factors. Its production is determined by Col

factor.

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Resistance Transfer Factor (RTF)

Spread of drug resistant bacteria Shigella resistant to

Sulphonamides, Streptomycin, Chloramphenicol & Tetracycline

E.coli, Shigella, Other Enterobacteriaceae, Vibrio, Pseudomonas and Pasturella

Resistance determinant (r) + RTF = R factor

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Genetic mechanism of drug resistance in bacteria

Mutational resistance 2 types:1. Stepwise mutation: Penicillin

resistance2. One-step mutation: Streptomycin Clinical significance: MDR-TB Multi drug resistant

Tuberculosis

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Genetic mechanism of drug resistance in bacteria

Mutational drug resistance

Tansferable drug resistance

One drug e at a time Multiple drug resistance

Low degree resistance

High degree resistance

Can be overcome by high drug dose

High dose ineffective

Drug combinations can prevent

Drug combinations cannot

Resistance does not spread

Spreads to same or different species

Mutant may defective

Not defective

Virulence may be low

Virulence not decreased

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Antibiotic resistance in bacteria

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Antibiotic resistance in bacteria

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Transposon

- Transposons or Jumping genes- Barbara Mc Clintock in Plants- Mode of gene transfer- Transposition – a

mechanism for amplifying genetic transfer in nature

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Molecular genetics

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Molecular genetics 1.Genetic engineering

2.Restriction endonucleases3.DNA probes4.Blotting techniques (Southern,

Northern, Western)5.Polymerase chain reaction

(PCR)6.Molecular epidemiology7.Genetic mapping

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Summary & Take home message

DNA – Gene – Codon Plasmid, Episome Mutation Tranformation, Transduction & Conjugation Lysogenic conversion Mutational vs Transferable drug resistance Transposons or jumping genes Molecular genetics - Genetic engineering,

DNA probes, PCR, Western, Southern and Northern blot

Genetic mapping, Molecular epidemiology

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Let a new ‘Revolution’ begin!!!

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To treat any gene mutation, Take these pill !!!

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THANK YOU