Bacterial Genetics and Applications

Dr.T.V.Rao MD

Dr.T.V.Rao MD


Gene Transfer Processes for Bacteria and Their Viruses
1. Conjugation
2. Transformation

3. Transduction 4. Infection with bacteriophage

Dr.T.V.Rao MD


Conjugation Lederberg - 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 Dr.T.V.Rao MD 3 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
Dr.T.V.Rao MD 4

Mechanism of Transfer I: Conjugation

Dr.T.V.Rao MD



Dr.T.V.Rao MD


Simple Conjugation

Dr.T.V.Rao MD



Dr.T.V.Rao MD


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.
Dr.T.V.Rao MD 9

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
Dr.T.V.Rao MD 10

T.V.Rao MD

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 Dr.T.V.Rao MD through F’ factor


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
Dr.T.V.Rao MD 12

Types of DNA transfer through Bacteriophages

Dr.T.V.Rao MD


Colicinogenic ( Col ) Factor
• Coliform 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 Dr.T.V.Rao MD 14

Resistance Transfer Factor RTF
• Plasmids – helps to spread multiple drug resistance • Discovered in 1959 Japan • Infections caused due to Shigella spread resistance to following Antibiotics

Sulphonamides Streptomycin Chloramphenicol, Tetracycline
Dr.T.V.Rao MD 15

• Shigella + E.coli excreted in the stool resistant to several drugs in vivo and vitro • Plasmid mediated – transmitted by Conjugation • Episomes spread the resistance
Dr.T.V.Rao MD 16

Bacterial Conjugation: High Frequency Transfer (Her) Cells

Dr.T.V.Rao MD


Hfr Conjugation

Dr.T.V.Rao MD


Sequence of RTF transmission

Dr.T.V.Rao MD


Hfr cell conjugating a Normal cell

Dr.T.V.Rao MD


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
Dr.T.V.Rao MD 21

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
Dr.T.V.Rao MD 22

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.
Dr.T.V.Rao MD 23

Genetic Mechanisms of Drug Resistance
• Bacteria acquire drug resistance through several Mechanisms • Mutations • Genetic transfer Transformation, Transduction Conjugation Several Biochemical Mechanisms Decreasing permeability of drugs, Attaining alternative pathways Produce enzymes and inactivate drugs
Dr.T.V.Rao MD 24

Genetic Mechanisms in Bacteria helps to Spread the Infectious diseases

Dr.T.V.Rao MD


• 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 - TB
Dr.T.V.Rao MD 26

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 Antibiotics
Dr.T.V.Rao MD 27

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 Genes Genetic 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.
Dr.T.V.Rao MD 28

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 Animals Laboratory Manipulations are called as Genetic Engineering
Dr.T.V.Rao MD 29

Molecular Genetics
• Analysis and manipulation of DNA using Biochemical and Microbiological techniques
Dr.T.V.Rao MD 30

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?
Dr.T.V.Rao MD 31

Genetic Engineering
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
Dr.T.V.Rao MD 32

Modern applications
• Pharmaceutical production
– Insulin, interferon, hormones, vaccines etc.

• Genetically engineered plants • Animal gene alterations • Gene probes • DNA fingerprinting • The human genome initiative
Dr.T.V.Rao MD 33

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
Dr.T.V.Rao MD 34

Genetic Engineering changing the Diagnostic and Therapeutic Protocols in MEDICINE

Dr.T.V.Rao MD


Research on Gene transfer shapes the future of Science

Dr.T.V.Rao MD


Genetically Engineered Products
• Can prepare desired protein in pure form in economic way Somatostatin • Commercial preparations pdf Cloned Human Insulin Interferons

Hepatitis B vaccine

Dr.T.V.Rao MD


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
Dr.T.V.Rao MD 38

Restriction Endonucleases Made the advances in Genetic Engineering

Dr.T.V.Rao MD


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.
Dr.T.V.Rao MD 40

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
Dr.T.V.Rao MD 41

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)
Dr.T.V.Rao MD 42

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
Dr.T.V.Rao MD 43

Western Blot

Dr.T.V.Rao MD


• Western Blot testing is confirmatory test for diagnosis of HIV/AIDS • Identifies antibodies directed against different antigens in pathogen Surface, Core Dr.T.V.Rao MD RT antigen

Western Blot to confirm HIV Infections made land mark Diagnostic tool


Polymerase chain reaction Kary B Mullis 1983
• Rapid • Automatic amplification of specific DNA sequences • Nobel prize winning Technology 1993

Dr.T.V.Rao MD


Polymerase chain reaction (PCR)
• The polymerase chain reaction (PCR) is a biochemical technology in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
Dr.T.V.Rao MD 47

Polymerase chain reaction (PCR)
• Developed in 1983 by Kary Mullis,PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR.
Dr.T.V.Rao MD 48

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
Dr.T.V.Rao MD 49

Genetic Mapping
• Genetic sequences for Bacteriophages and virus • Genetic mapping is done most of the Human Genes

Dr.T.V.Rao MD


Newer Understanding on GENES

Dr.T.V.Rao MD


Human Genome Project

Dr.T.V.Rao MD


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
Dr.T.V.Rao MD 53

Genetics are Complex - Leading the birth of BIOINFORMATICS

Dr.T.V.Rao MD


Genes Evolved and made us Men What NEXT ?

Dr.T.V.Rao MD


Understanding of human Genome is Changing the Future of Medicine

Dr.T.V.Rao MD


• The Programme Created by Dr.T.V.Rao MD for Medical Students in the Developing World

• Email

Dr.T.V.Rao MD


Sign up to vote on this title
UsefulNot useful