Bacterial Genetics Basics -

7/30/2019 Bacterial Genetics Basics -

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Bacterial GeneticsLearning the Basics

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Dr.T.V.Rao MD

Dr.T.V.Rao MD


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Genetics Guide Life

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Genes are EternalRun In Progeny

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Understanding Genetics

We resemble and differ because ofGenetic configurations

Parents - Son - Daughter, how theyresemble each other.

They breed true from Generation toGeneration

But vary in small proportions inprogeny.

Bacteria too obey the laws of

Genetics 4Dr.T.V.Rao MD


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Watson - CrickDiscovery of DNA

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Beginning of BacterialGenetics

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

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DNAA 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

forkEach 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 theform of a Double

Helix

Each chain has back

bone of Deoxyriboseand Phosphates

residues arranged

alternatively11Dr.T.V.Rao MD


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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 insteadof Deoxyribose

Uracil is present instead of Thymine

Types of RNA

Messenger RNA mRNARibosomal RNA rRNATransfer RNA tRNA

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DNA - RNA

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Knowledge on DNA lead toadvances 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 mRNAVirus 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 threeBases

Code is triplet A-T- C A code can make single Amino acid

More than one code present for making similarsequence of Amino acid

AGA make Arginine AGC, CGU, CGG, also code for similar Amino

acid

Some Codons UAA don't code for any Aminoacid called as Nonsense codon 16Dr.T.V.Rao MD


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What is aGene Gene is a sequence

of DNA carrying

codons specifying for

particular polypeptide. DNA contains many

Genes( A

combinations of

hundreds andthousands 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 differsfrom Higher forms of Life

Several stretches of DNA don't appear tofunction as codons, occurs between the

coding sequences of Gene. called asINTRONS.

Coded are called as EXONS

In transcription introns are excised whenform RNA before translated by ribosomalproteins.

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Extra chromosomalGenetic Elements

Bacteria posses Extra chromosomal

genetic elementsNot 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 circularDNA molecules presentin the cytoplasm of theBacteria

Capable of Autonomousreplication

Can transfer genes fromone cell to other

Act as vectors in Geneticengineering.

Can also present inYeasts

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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 production3 Enterotoxin production

4 Enhanced pathogen city

5 Reduced Sensitivity tomutagens

6 Degrade complex organic molecules

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R plasmid

R: drug resistance

RTF: transfer of Rplasmid

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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

CentrifugationElectrophoresis

Genetic methods

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Genotypic and Phenotypicvariation

Genome Sum total of Gene that makeup the genetic apparatus of cell

established as Genotype. Hereditary constitution of cell this

transmitted to its progeny

Phenotype is the physical expression ina environment. Change according toenvironment.

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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 GalactosidaseWhen lactose present - test is positive

When lactose is absent - test turns negative

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Different Enzymes Guided byGenomic configurations

Inducer enzyme acts in the presenceof substrate

Constitutive enzyme acts irrespectiveof presence or absence of enzyme.

Phenotypic variations influenced by

environment limited in range bygenes

Genotypic variations are stable notinfluenced by environment.

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Principles of Genotypicvariations

Mutations

Genotypic by transfer of genesTransformation

Transduction

(Lysogenic conversion)

Conjugation

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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 bodywithout inhibition

Become a prominent organism

Phenotypic variation occurs when geneschanges in response to the environment butreversible.

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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 canoccur due to

Addition

DeletionSubstitution

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 AGTGCCA

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Mutation Type

Frameshift (insertion)

(leu) (ser) (arg)

Normal AAT AGT GCC

(leu) (glut) (cyst)

Mutant AAT CAGTGCC

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Mutations can occur in any


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Mutations can occur in anysequence,inveitable, useful for

Survival

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Multiple Mutations

Causes extensive chromosomal rearrangement

Missense mutation -Triplet code is acted so asto specify an Aminoacid different from thatnormally located at particular position in theprotein

Nonsense mutation - Deletion of nucleotidewithin a gene may cause premature polypeptidechain termination by nonsense codon

Tran version is Substitution of purine forpyramidine 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 notat 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 tingagents

Arcidine Dyes

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GENE TRANSFEROccurs by Complex Mechanisms

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Th th b t i l l


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The three bacterial sexualprocesses

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


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Transformation of Geneticmaterial

( Gene Transfer )

Different

MechanismsTransformation

Transduction

Conjugation

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G T f P f


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Gene Transfer Processes for

Bacteria and Their Viruses

1. Conjugation

2. Transformation

3. Transduction4. Infection with

bacteriophage

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What is Transformation

Transformation is defined as transfer of Geneticinformation through the activity of DNA

Griffith experiment

Mice injected with Live non capsulated ( R )

Pneumococciwith heat killed capsulated (S) Pneumococci

Lead to death of Mice with isolation of Livecapsulated Pneumococci

It means that some factor from Deadpneumococci transferred to live non pathogenicPneumococci

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


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Griffith Phenomenon

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Demonstration oftransformation

Avery, MacLeod, andMcCarty (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 inStaphylococcus

Helps Genetic mapping, also in eukaryotic cell Helps Genetic Engineering

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Transduction Transduction is

defined astransfer of

portion of DNAfrom onebacteria to

another byBacteriophages,is known as

Transduction51Dr.T.V.Rao MD

DNA t f th h


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DNA transfer throughBacteriophages

When the Phageparticle infects

another bacteriaDNA transfer iseffected and therecipient cell

acquires newcharacters codedby donor DNA

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Bacteriophages

Are viruses that

parasitize bacteria

and consists of

Nucleic acid core anda protein coat

A phage particle may

have at its core

besides its own

nucleic acid and a

segment of the Host

DNA53Dr.T.V.Rao MD


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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 hostbacterium ruptures and

phages are released54Dr.T.V.Rao MD

Lysogenicity creates new


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Lysogenicity creates newcharacters

Eg - Lysogenic conversion in

Diphtheria bacilli which

acquires toxigenicity bylysogenization with phage beta

Elimination of phage fortoxigenic strain renders

nontoxigenic55Dr.T.V.Rao MD

Conjugation


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ConjugationLederberg - Tatum

A process by which a Donor cell or male cellmakes contact with another cell, the recipient orFemale cell.

DNA is directly transferable Plasmid Carry genetic information necessary for

conjugation to occur.

Only cell that contain such plasmids can act asdonor. the cell lacking a corresponding plasmidact as recipient.

Requires direct contact between donor and

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Conjugation

The ability to conjugate is

conferred by the F plasmid. A

plasmid is a small circle of DNAthat replicates independently of

the chromosome. Bacterial cells

that contain an F plasmid arecalled F+. Bacteria that dont

have an F plasmid are called 57Dr.T.V.Rao MD

Conjugation Transferring


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Conjugation - Transferringgenes with plasmids

Plasmids

mediating

conjugation carry

genes coding forproperties, of 1-2

microns long

protein appendagetermed Pilus on the

Donor cell

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M h i f T f I


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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 Pilusare specified by differenttypes of plasmids and

can help in aid of plasmidclassification.

Only one strand ofcircular DNA of theplasmid nicked upon at a

specific site and passedinto 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 identifiablegenetic 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 chromosomeCan transform chromosomal genes to recruitment withhigh frequency are known as Hfr cells

Conversion of F+ cells into Hfr state is reversible.

F factor incorporates some chromosomal genes and iscalled as F

Sexduction The process of transfer of host genesthrough F factor

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DNA transfer through


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DNA transfer throughBacteriophages

When the Phageparticle infects

another bacteriaDNA transfer iseffected and therecipient cell

acquires newcharacters codedby donor DNA

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Types of DNA transfer through


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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

T.V.Rao MD

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Resistance Transfer Factor


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Resistance Transfer FactorRTF

Plasmids helps to spread multiple drug

resistance

Discovered in 1959 Japan

Infections caused due to Shigella spreadresistance to following Antibiotics

Sulphonamides

StreptomycinChloramphenicol,

Tetracycline69Dr.T.V.Rao MD

RTF


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RTF

Shigella + E.coliexcreted in the stoolresistant to several

drugs in vivo and vitro

Plasmid mediatedtransmitted byConjugation

Episomes spread theresistance

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Bacterial Conjugation:


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Bacterial Conjugation:High Frequency Transfer (Her) Cells

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Hf C j i


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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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C i i f RTF


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Composition of RTF

Plasmid consists of twocomponents

A transfer factor RT, helpsconjugational transfer andresistant determinants ( r ) to

each of the several drugs RTF + r determinants are known

as R factor75Dr.T.V.Rao MD

R f t


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R factor R factor can contain

several determinants as

many as 8 or > 8 drugs

Guide the cell for

production ofEnterotoxins too

But R factors can be

inhibited by

Bile saltsR factors can be

transferred to animals

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G i f R f


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Genesis of R factors

In discriminate use of

Antibiotics in vet nary Medicine

has increased the spread of Rfactors to Human

Addition of Antibiotics toAnimal feeds to be prohibited.

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Genetic Mechanisms of Drug


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Genetic Mechanisms of DrugResistance

Bacteria acquire drug resistance through severalMechanisms

Mutations

Genetic transfer

Transformation,Transduction

Conjugation

Several Biochemical Mechanisms

Decreasing permeability of drugs,Attaining alternative pathways

Produce enzymes and inactivate drugs

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Genetic Mechanisms in Bacteria helps to


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Genetic Mechanisms in Bacteria helps toSpread the Infectious diseases

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M t ti


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Mutations

Mutilations can be

1 Stepwise mutation as in Penicillin use

2 One step mutation

Streptomycin useMay show low resistance or High resistance

If tuberculosis is treated with sole drug as of Only

Streptomycin some resistant mutants appearand replaces sensitive bacteria in due course

so the occurrence ofMDR - TB80Dr.T.V.Rao MD

Oth M h i


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Other Mechanisms

Use of Penicillin created resistantStaphylococcus by transduction

R factors created resistance to several drugs,

caused increased virulence Spread to several humans and animals

Best option- To restrict use ofAntibiotics

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T bl G i El


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Transposable Genetic Elements

Structurally / Genetically Discrete sequence ofDNA Move around in a cut and paste mannerbetween Chromosomal and Extra chromosomalDNA molecules within cells.

Called as Transposons _ Jumping GenesGenetic transfer due to Transposition

Small Transposons 1 2 Kb

Not self replicating and depend on Plasmid or

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

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T d R f t


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Transposons and R factor

R forms may have evolved as a collection ofTransposons

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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M l l G ti


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Molecular Genetics

Analysis and

manipulation of

DNA using

Biochemical

andMicrobiological

techniques84Dr.T.V.Rao MD

G ti E i i


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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 Engineeringawait manysurprises?

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Genetic Engineering Was Born from

Genetic Recombination

Genetic engineering involves changingthe 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


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pp

Pharmaceutical production

Insulin, interferon, hormones, vaccines

etc.

Genetically engineered plants

Animal gene alterations

Gene probes DNA fingerprinting

The human genome initiativeDr.T.V.Rao MD 87

Genetic Engineering


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Genetic Engineering

Isolation of Genes coding for anydesired protein from Microorganism

or from cell of higher life formsincluding human beings and theirintroduction into a suitable

microorganism in which genes wouldfunction directing the production ofspecific proteins

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Genetic Engineering changing

the Diagnostic and TherapeuticProtocols inMEDICINE

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Research on Gene transfer


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shapes the future of Science

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Genetically Engineered Products


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Genetically Engineered Products

Can prepare desiredprotein in pure form ineconomic way

Somatostatin

Commercialpreparations pdf

Cloned HumanInsulin

Interferons

Hepatitis Bvaccine

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Restriction Endonucleases


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Restriction Endonucleases

A restriction enzyme (orrestrictionendonuclease) is an enzyme that cuts

double-stranded DNA. The enzymemakes two incisions, one through each ofthe sugar-phosphate backbones (i.e., each

strand) of the double helix without

damaging the nitrogenous bases Theywork with cutting up foreign DNA, aprocess called

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Restriction EndonucleasesMade the advances in Genetic


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Made the advances in GeneticEngineering

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DNA Probes


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DNA Probes

There are Radioactive

Biotinylated otherwiselabeled copies unitedsingle stranded DNA

Contains 20 -25 nucleotidesHelps detection of

Homology DNA byHybridization.

Helps Diagnosis ofInfectious Diseases

Minute quantities of DNAcan be detected.

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Blotting Techniques


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Blotting Techniques

Drug fragments obtained by restrictionenzyme digestion on separation Gel can

be transferred to Nitrocellulose or nylonmembranes

Several methods

1 Southern blotting

2 Northern Blotting

3 Western blotting95Dr.T.V.Rao MD

western blot


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western blot

The western blot (alternatively, proteinimmunoblot) is an analytical techniqueused to detect specific proteins in a givensample of tissue homogenate or extract. It

uses gel electrophoresis to separatenative 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


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Western Blotting

In Western Blot Protein (Antigen ) mixture is

separated by SDS (

Sodium dodecyl sulfate

polyacrylamide gelelectrophoresis ) 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

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Western Blot to confirmHIV Infections made land mark


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HIV Infections made land markDiagnostic tool

Western Blot testingis confirmatory test for

diagnosis of

HIV/AIDS

Identifies antibodies

directed against

different antigens in

pathogenSurface,

Core

RT antigen99Dr.T.V.Rao MD

Polymerase chain reaction


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yKary B Mullis 1983

Rapid

Automatic

amplification ofspecific DNA

sequences

Nobel prize winning

Technology 1993

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PCR Sequences


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PCR -Sequences

PCR consists of several

cycles of sequential DNA

replication where the

products of first cyclebecomes the template for

the Next

It makes available

abundant quantities ofspecific DNA sequences

starting

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Genetic Mapping


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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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Completed in 2003, the Human GenomeProject (HGP) was a 13-year project

coordinated by the U.S. Department of

Energy and the National Institutes ofHealth. During the early years of the HGP,

the Welcome Trust (U.K.) became a major

partner; additional contributions came fromJapan, France, Germany, China, and

others

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Genetics are Complex- Leading


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the birth of BIOINFORMATICS

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Genes Evolved and made usMen


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MenWhat NEXT ?

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Are We Playing with Genes in the


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Right Direction?

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Understanding of human Genome is


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Changing the Future of Medicine

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Programmed Created by Dr.T.V.Rao MDfor Undergraduate Medical Students in

the Developing World Email

[email protected]

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