Bacterial GeneticsLearning the Basics

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Dr.T.V.Rao MD

Dr.T.V.Rao MD

Genetics Guide Life

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Genes are EternalRun In Progeny

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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

T.V.Rao MD

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Watson - CrickDiscovery of DNA

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Beginning of Bacterial Genetics

• The principles of Genetics were applied to bacteria and viruses

• Advances in Genetic process also of lead to fundamental advances in Biology and Biochemistry.

A Birth of New Branch of Science

Molecular Biology 6Dr.T.V.Rao MD

DNA A Complex Structure

Makes Life

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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

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Structure of DNA

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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

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Structure of DNA• Attached to each

Deoxyribose and phosphate residues arranged alternatively

• Attached to each Deoxyribose are of four nitrogen bases

• Purines - Adenine, Guanine

• Pyramidine

Thymidine and Cytosine

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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 13Dr.T.V.Rao MD

DNA - RNA

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Knowledge on DNA lead to advances in Molecular Biology

• Central dogma of Life – Deoxyribonucleic acid• DNA carries the Genetic information• DNA is transcribed to RNA – Polypeptides

Cell Function depends upon specific polypeptides – Proteins – Enzymes

DNA is a store house of Protein synthesis

DNA acts a Template for synthesis of mRNA

Virus differs from other as they contains either DNA or RNA

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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 16Dr.T.V.Rao MD

What is a Gene• Gene is a sequence

of DNA carrying codons specifying for particular polypeptide.

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

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Bacterial Chromosome

• Contains a Double stranded molecules of DNA arranged in circular form.

• Length 1,ooo microns.• Bacterial DNA contains about

4,000kilobases• I kb = 1000 base pairs ( A-T ) ( G-C)• Humans have about 3,000 kb pairs.

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How bacterial Genome differs from Higher forms of Life

• Several stretches of DNA don't appear to function as codons, occurs between the coding sequences of Gene. called as INTRONS.

• Coded are called as EXONS• In transcription introns are excised when

form RNA before translated by ribosomal proteins.

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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

Able to produce toxins

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Plasmids

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

• Capable of Autonomous replication

• Can transfer genes from one cell to other

• Act as vectors in Genetic engineering.

• Can also present in Yeasts

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Plasmid ( Blue )

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Plasmids • Plasmid seem to be ubiquitous in bacteria, May

encode genetic information for properties

1 Resistance to Antibiotics 2 Bacteriocins production 3 Enterotoxin production 4 Enhanced pathogen city 5 Reduced Sensitivity to mutagens 6 Degrade complex organic molecules

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R plasmid

R: drug resistance

RTF: transfer of R plasmid

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Plasmids

• Can be integrated with Chromosomal DNA

• Episomes -Integrated form of plasmid with DNA

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Potentials of Plasmids

• Plasmids can be self transmissible and Non transmissible

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

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Classification of Plasmids

• Incompatibility typing• Don't accommodate

others which are similar

• Other methods of Classification

Centrifugation

Electrophoresis

Genetic methods

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Genotypic and 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 in a environment. Change according to environment.

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What is Phenotypic expression

• 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

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Different Enzymes Guided by Genomic configurations

• Inducer enzyme acts in the presence of substrate

• Constitutive enzyme acts irrespective of presence or absence of enzyme.

• Phenotypic variations influenced by environment limited in range by genes

• Genotypic variations are stable not influenced by environment.

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Principles of Genotypic variations

• Mutations• Genotypic by transfer of genes

Transformation

Transduction

(Lysogenic conversion)

Conjugation31Dr.T.V.Rao MD

Replica Plating, pt. 2

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Mutations in Bacteria• Bacteria Multiply by asexual binary fission• Altered Nucleotide sequence in expresses new

or altered characteristics • Selective value to the organism• Evolutionary value• Acquires Antibiotic resistance grows in body

without inhibition• Become a prominent organism• Phenotypic variation occurs when genes

changes in response to the environment but reversible.

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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

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Mutation Type

Frameshift (deletion) (leu) (ser) (arg)

Normal AAT AGT GCC

(leu) (val) (pro)

Mutant AAT AGT GCC A

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Mutation Type

Frameshift (insertion) (leu) (ser) (arg)

Normal AAT AGT GCC

(leu) (glut) (cyst)

Mutant AAT CAGT GCC

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Mutations can occur in any sequence,inveitable, useful for

Survival

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Multiple Mutations• Causes extensive chromosomal rearrangement• Missense mutation -Triplet code is acted 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

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

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Mutations

• Suppressor Mutation is reversal of mutant phenotype by another mutation at a point of DNA distant from that of original mutation.

• All genes are susceptible for mutations, but all mutations are not expressed

• Lethal mutation is harmful destroy the vital functions

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Mutations

• Conditional Lethal mutant may be Live under certain conditions

• Common example is temperature ( its ) mutant• Temp sensitive ( ts) mutant lives at 350c but not

at 390c• Each gene undergoes mutation at a fixed

frequency.

Bacteria undergo mutations at 10-4 - to 10-10

• Tutomerisim T – A is replaced by G - A

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Mutagenic Agents

• U V rays• Alkyl ting

agents• Arcidine Dyes

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GENE TRANSFER Occurs by Complex Mechanisms

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The three bacterial sexual processes

– 1. Conjugation: direct transfer of DNA from one bacterial cell to another.

–2. Transduction: use of a Bacteriophages (bacterial virus) to transfer DNA between cells.

–3. Transformation: naked DNA is taken up from the environment by bacterial cells. 44Dr.T.V.Rao MD

Transformation of Genetic material

( Gene Transfer )

• Different Mechanisms Transformation

Transduction

Conjugation

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Gene Transfer Processes for Bacteria and Their Viruses

1. Conjugation2. Transformation

3. Transduction

4. Infection with bacteriophage

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What is Transformation• Transformation is defined as transfer of Genetic

information through the activity of DNA• 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

Avery, McCleod, Mc Cartny in 1944 identified to be DNA47Dr.T.V.Rao MD

Griffith Phenomenon

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Demonstration of transformation

Avery, MacLeod, and McCarty (1944)

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Transduction

• Generalized involve any segment of DNA

• Restricted when specific Bacteriophages traduces only a particular genetic trait.

• Transduction effects Plasmids ,and Episomes• Plasmid transfer induces Penicillin resistance in

Staphylococcus• Helps Genetic mapping, also in eukaryotic cell• Helps Genetic Engineering T.V.Rao MD

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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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DNA transfer through Bacteriophages

• When the Phage particle infects another bacteria DNA transfer is effected and the recipient cell acquires new characters coded by donor DNA

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Bacteriophages

• Are viruses that parasitize bacteria and consists of Nucleic acid core and a protein coat

• A phage particle may have at its core besides its own nucleic acid and a segment of the Host DNA 53Dr.T.V.Rao MD

Transduction Types

• Two types of Transduction• 1 Lytic and 2 Lysogenic• 1 Virulent or Lytic cycle after

large number of progeny are built up inside the host bacterium ruptures and phages are released 54Dr.T.V.Rao MD

Lysogenicity creates new characters

• Eg - Lysogenic conversion in Diphtheria bacilli which acquires toxigenicity by lysogenization with phage beta

• Elimination of phage for toxigenic strain renders nontoxigenic

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ConjugationLederberg - Tatum

• 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

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Conjugation

• The ability to conjugate is conferred by the F plasmid. A plasmid is a small circle of DNA that replicates independently of the chromosome. Bacterial cells that contain an F plasmid are called “F+”. Bacteria that don’t have an F plasmid are called “ 57Dr.T.V.Rao MD

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

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Mechanism of Transfer I:Conjugation

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Conjugation

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Simple Conjugation

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Conjugation

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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.

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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

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F factor helps transformation

• 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 The process of transfer of host genes

through F’ factor

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DNA transfer through Bacteriophages

• When the Phage particle infects another bacteria DNA transfer is effected and the recipient cell acquires new characters coded by donor DNA

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Types of DNA transfer throughBacteriophages

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Colicinogenic ( Col ) Factor

• Coli form Bacteria produce Colicins

• Colicins are lethal to other Enterobacteriaceae

• Pyocins produce by Pseudomonas

• Diptherocins produced by C.diptheria

• Plasmid transmits col factor leads to self transfer of chromosomal segments

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Resistance Transfer FactorRTF

• Plasmids – helps to spread multiple drug resistance

• Discovered in 1959 Japan• Infections caused due to Shigella spread

resistance to following Antibiotics

Sulphonamides

Streptomycin

Chloramphenicol,

Tetracycline69Dr.T.V.Rao MD

RTF

• Shigella + E.coli excreted in the stool resistant to several drugs in vivo and vitro

• Plasmid mediated –transmitted by Conjugation

• Episomes spread the resistance

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Bacterial Conjugation:High Frequency Transfer (Her) Cells

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Hfr Conjugation

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Sequence of RTF transmission

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Hfr cell conjugating a Normal cell

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Composition of RTF• Plasmid consists of two

components• A transfer factor RT, helps

conjugational transfer and resistant determinants ( r ) to each of the several drugs

• RTF + r determinants are known as R factor

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R factor• R factor can contain

several determinants as many as 8 or > 8 drugs

• Guide the cell for production of Enterotoxins too

• But R factors can be inhibited by

Bile salts

R factors can be transferred to animals

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Genesis of R factors

• In discriminate use of Antibiotics in vet nary Medicine has increased the spread of R factors to Human

• Addition of Antibiotics to Animal feeds to be prohibited.

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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

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Genetic Mechanisms in Bacteria helps to Spread the Infectious diseases

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Mutations• Mutilations can be

1 Stepwise mutation as in Penicillin use

2 One step mutation

Streptomycin use

May show low resistance or High resistance

If tuberculosis is treated with sole drug as of Only Streptomycin some resistant mutants appear and replaces sensitive bacteria in due course

so the occurrence of MDR - TB80Dr.T.V.Rao MD

Other Mechanisms

• Use of Penicillin created resistant Staphylococcus by transduction

• R factors created resistance to several drugs, caused increased virulence

• Spread to several humans and animals

Best option- To restrict use of

Antibiotics81Dr.T.V.Rao MD

Transposable Genetic Elements Structurally / Genetically – Discrete sequence of

DNA – Move around in a cut and paste manner between Chromosomal and Extra chromosomal DNA molecules within cells.

Called as Transposons _ Jumping GenesGenetic transfer due to TranspositionSmall Transposons 1 – 2 KbNot self replicating and depend on Plasmid or

Chromosome for replication.A chunk of DNA is added by Transposons.

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Transposons and R factor

• R forms may have evolved as a collection of Transposons

• Each carrying Genes that confers resistance to one or several Antibiotics

• Seen in Plasmids, Microorganisms AnimalsLaboratory Manipulations are called as Genetic

Engineering

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Molecular Genetics

• Analysis and manipulation of DNA using Biochemical and Microbiological techniques

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Genetic Engineering

• Under standing Molecular genetics in Biochemistry fuels genetic Engineering

• Recombinant DNA (renal) techniques changed the ideals of Medicine

• Genetic Engineering await many surprises?

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Genetic Engineering Was Born from Genetic Recombination•Genetic engineering involves changing the genetic material in an organism to alter its traits or products

•A recombinant DNA molecule contains DNA fragments spliced together from 2 or more organisms

Genetic Engineering

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Modern applications

• Pharmaceutical production– Insulin, interferon, hormones, vaccines

etc.• Genetically engineered plants• Animal gene alterations• Gene probes• DNA fingerprinting• The human genome initiative

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Genetic Engineering

• Isolation of Genes coding for any desired protein from Microorganism or from cell of higher life forms including human beings and their introduction into a suitable microorganism in which genes would function directing the production of specific proteins

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Genetic Engineering changing the Diagnostic and Therapeutic

Protocols in MEDICINE

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Research on Gene transfer shapes the future of Science

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Genetically Engineered Products

• Can prepare desired protein in pure form in economic way

Somatostatin• Commercial

preparations pdf Cloned Human

Insulin Interferons

Hepatitis B vaccine

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Restriction Endonucleases

• A restriction enzyme (or restriction endonuclease) is an enzyme that cuts

double-stranded DNA. The enzyme makes two incisions, one through each of the sugar-phosphate backbones (i.e., each strand) of the double helix without

damaging the nitrogenous bases They work with cutting up foreign DNA, a process called

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Restriction EndonucleasesMade the advances in Genetic

Engineering

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DNA Probes

• There are Radioactive Biotinylated otherwise

labeled copies united single stranded DNA

Contains 20 -25 nucleotides Helps detection of

Homology DNA by Hybridization.

Helps Diagnosis of Infectious Diseases

Minute quantities of DNA can be detected.

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Blotting Techniques

• Drug fragments obtained by restriction enzyme digestion on separation Gel can be transferred to Nitrocellulose or nylon membranes

• Several methods 1 Southern blotting 2 Northern Blotting 3 Western blotting

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western blot• The western blot (alternatively, protein

immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions)

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Western Blotting

• In Western Blot Protein ( Antigen ) mixture is separated by SDS ( Sodium dodecyl sulfate – polyacrylamide gel electrophoresis ) Blotted on to Nitro cellulose strips and identified by radio labeled or enzyme labeled antibodies as probes

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Western Blot

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Western Blot to confirmHIV Infections made land mark

Diagnostic tool• Western Blot testing

is confirmatory test for diagnosis of HIV/AIDS

• Identifies antibodies directed against different antigens in pathogen

Surface,

Core

RT antigen 99Dr.T.V.Rao MD

Polymerase chain reactionKary B Mullis 1983

• Rapid• Automatic

amplification of specific DNA sequences

• Nobel prize winning Technology 1993

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PCR -Sequences

• PCR consists of several cycles of sequential DNA replication where the products of first cycle becomes the template for the Next

• It makes available abundant quantities of specific DNA sequences starting

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Genetic Mapping

• Genetic sequences for Bacteriophages and virus

• Genetic mapping is done most of the Human Genes

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Newer Understanding on GENES

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Human Genome Project

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Human Genome Project

• Completed in 2003, the Human Genome Project (HGP) was a 13-year project coordinated by the U.S. Department of Energy and the National Institutes of Health. During the early years of the HGP, the Welcome Trust (U.K.) became a major partner; additional contributions came from Japan, France, Germany, China, and others

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Genetics are Complex - Leading the birth of BIOINFORMATICS

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Genes Evolved and made us Men

What NEXT ?

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Are We Playing with Genes in the Right Direction ?

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Understanding of human Genome is Changing the Future of Medicine

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• Programmed Created by Dr.T.V.Rao MD for Undergraduate Medical Students in

the Developing World • Email

• doctortvrao@gmail.com

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