Bacterial Genetics

 GENETICS-Study of genes their structure &

function, heredity & variation
 Genomics-Study & analysis of nucleotides of DNA
 Nucleic acid-DNA and RNA
Bacterial DNA

 Single Haploid Chromosome

 Super coiled circular dsDNA=1mm
 Exception:
 2 chromosomes : Vibro cholerae
 Nucleotides-Structural units of Nucleic acids
 Nitrogenous bases-Purines(A,G) and Pyrimidines(T,U,C)
Nucleoside
 Pentose sugar-Deoxyribose
 Phosphate group
 Bacterial DNA

 Ratio of A+T to G+C constant for each species

 Genetic information is stored as a code
 Codon-unit,triplet(3 bases)
 64 codon
 61 sense codon code for 20 AA
 AGA/AGG/CGA-arginine— code is degenerate
 3 codon UAA/UAG/UGA- nonsense codons
 Gene or cistron
 Segment of DNA carrying codons for a particular polypeptide synthesis
 Locus
 a large no of genes
 Genome
 large no of loci

 Letter---------word-----sentence—paragraph---
 Nucleotide—Codon-----Gene--------Locus---
 1000-3000 Gene
 580 -5200 kbp
 length1-1.3mm
 DNA Replication:

Bidirectional replication Rolling circle mechanism

RNA

 mRNA
 rRNA
 tRNA
 Extra chromosomal elements

Plasmids
Free Circular dsDNA-In Cytoplasm for several
generations
Replicate independently
Episome-integrated form
Not essential for life of bacteria
Number: up to 40/cell
contain 50-100 genes
 Extra chromosomal elements
Plasmids
Curing: process of eliminating plasmid from bacteria
 Spontaneous
 induced
 Acridine
 Radiation
 Thymine starvation
 High temp
Classification
On the basis of ability to perform conjugation:
Conjugative/self transmissible plasmid
Non conjugative plasmid

Based on compatibility b/w plasmid:

Compatible
Incompatible
Classification
Based on function:
Fertility/F plasmid: contain tra gene: sex pili expression
Resistance/R plasmid
Col plasmid
Virulence plasmid
Metabolic plasmid
Variation

 Phenotypic

 Genotypic
MUTATION
Random, heritable variation caused by alteration in
nucleotide sequence of DNA

Frequency 10-2 -10-10/bacterium/division

CAUSES
 Spontaneous
 Induced (mutagen) –
 Physical: UV
 Chemical: alkylating agent, 5-FU, acridine dye
Functionally affect:
 Not able to produce Capsule/flagella
 Antigenic structure alteration
 Altered sensitivity to Bacteriophage
 Drug resistance
 Altered pigment production
 Altered Biochemical reactions
 Altered colony morphology
 Types:
Forward mutation
 Substitution
 Transversion:
 Transition:
 Types:
Forward mutation
 Substitution
 Silent: New codon code for same AA
 Neutral: New codon code for functional equivalent AA
 Missense: Different AA
 Non-sense: Stop
 Types:
Forward mutation
 Substitution
 Silent: New codon codes for same
AA
 Neutral: New codon codes for
functionally equivalent AA
 Missense: Different AA
 Non-sense: Stop
 Types:
 Addition or deletion
 Frame Shift
 Reverse Mutation
 True reversion
 Types:
 Reverse Mutation
 Equivalent reversion: 2nd Mutation different codon but same AA
 Types:
 Reverse Mutation
 Suppressor mutation: 2nd mutation in a different gene that revert the phenotypic effects of
already existing mutation
Demonstration of Mutation

 Gene sequencing
 Phenotypic changes

 Fluctuation test
 Replica plating
Fluctuation test

 Luria and Delbruck-Mutation is SPONTANEOUS and

RANDOM-
 Growth in presence of selective inhibitory pressure
 Bacteriophage and E. coli
Replica plating-Auxotrophic mutant
 Ames Test (carcinogenicity
testing)
 Mutational
reversion
assay

Estimate
Mutagenicity
of mutant
GENETIC TRANSFER

 Vertical
 Horizontal
 Transformation
 Transduction
 Lysogenic conversion
 Conjugation
Transformation
- Random uptake of free / naked DNA
incorporation into chromosome
 Natural – S. pneumoniae
 express DNA-binding proteins on cell surface
 natural competent state allows uptake of "naked DNA"
Transformation
- random uptake of free / naked DNA
incorporation into chromosome
                                                                    
 1928: Frederick Griffith (London): First demonstrated bacterial
transformation
An "S" or SMOOTH coat strain, which is
lethal to mice.
                                                                                                                              
An “R" or rough coat strain, which is
NOT lethal to mice.
Griffith found that he could heat inactivate the
smooth strain.

                                                                      
heat-inactivated S strain,
mixed with the R strain, the mouse would die. 
Thus there was some
Material in the heat-killed S strain that was responsible for
"transforming" the R strain into a lethal form.

                                                                                                                          
GENETIC TRANSFER

 Vertical
 Horizontal
 Transformation
 Transduction
 Lysogenic conversion
 Conjugation
Transduction- Transfer of genetic
material through bacteriophage
 Transduction 2 types:
 Generalized:
 Packaging error
 3 outcome on transduction:
 Abortive transduction: 70-90%
 Stable gene transfer
 Unstable gene transfer

 Transduction 2 types:

 Transduction 2 types:
 Restricted/specialized
 Defect in disintegration of lysogenic phage
 2 outcome when transduced to new bacteria
 Cross over
 Integrated as prophage
 Transduction 2 types:

Importance of transduction

 Drug resistance: Pn resistance in

Staphylococci
 Treatment: Genetic mapping,
inborn error of metabolism
 Phage vectors used in molecular
transformation of bacteria
 Lysogenic Conversion

 In Lysogenic bact prophage acts as additional segment of bact chromosome-new

characters-lysogenic conversion eg. C.diphtheriae and its bacteriophage
 Phage coded Toxins:
 Diphtheria toxin
 cholera toxin
 Verocytotoxin of E. coli
 Streptococcus pyrogenic exotoxin A & C
 Botulism toxin C & D
 Lysogenic conversion: Phage DNA itself behave as new genetic element
 Transduction: Phage act as vehicle carrying bacterial gene
GENETIC TRANSFER

 Vertical
 Horizontal
 Transformation
 Transduction
 Lysogenic conversion
 Conjugation
Bacterial Conjugation

 Transfer of genetic information from one bacterium (donor or male) to another

bacterium (recipient or female) bacterium by mating or contact with each other &
forming conjunction tube
 F+ F- Mating
 HFR conjugation
 F’ Conjugation
Col factor
R factor-RTF + r determinants
Colicinogenic (col) factor

 Bacteriocins are the antibiotic like substances produced by one

bacterium that inhibit other bacteria
 Bacteriocins produced by coliform bacteria are called as colicin
 Bacteria other than coliforms also produce similar kind of
substances e.g. pyocin, diphthericin
FATE OF DONOR DNA:
Bacterial Recombination

 Integration of Donor DNA to recipient chromosome

 General or Homologous
 Site specific
General or Homologous

 Recombination b/w similar DNA sequences

 Reciprocal:
 Exchange of pair of Homologous DNA sequence b/w donor & recipient

 Non Reciprocal:
 Bacterial transformation
 Donor ssDNA is inserted into host chromosome & replace piece of host DNA
Site specific

 Integration of bacteriophage DNA into Bacterial DNA is site specific

 Donor DNA not homologous with chromosome it joins
TRANSPOSONS
Genetic engineering

 Deliberate modification of organism genetic information by directly altering its genome

 Done using recombinant DNA technology

 Gene coding for desired property (protein) ---isolated from organism-----inserted to vector----
cloned---desired property express
 POLYMERASE CHAIN REACTION

 Kary Mullis-1983
 PCR is a DNA amplification system that produces a large amount of DNA in vitro
from small amounts of starting material. It amplifies a specific DNA sequence (or
gene) or interest.
 Primer mediated , temp dependant enzymatic amplification of specific target sequence
to detectable levels
 Target DNA
 Primers
 Polymerase Enzyme- Thermus aquaticus
 Nucleotide
 Thermocycler
 Denaturation-940C
 Annealing of primers-50-600C
 Extension of primers

30-40 cycles for 3 hrs- 106 copies

Detection-gel electrophoresis and ethidium bromide staining.
PCR in Diagnosis

Bacteria

Viruses

Fungi
DNA PROBES

 Radiolabelled or chromogenically labelled pieces of ss DNA

which can be used for the detection of homologous DNA by
hybridization.
 Hybridisation is the technique in which two single-strands of
nucleic acid come together to form a stable double-stranded
molecule.
Applications of DNA Probes

 In clinical microbiology :
 Direct detection of microbes in specimens
 To detect microbes which are difficult or impossible to culture
 Identification of culture isolates
 Strain identification
 To identify toxins, virulence factors
 Identification of resistant markers
BLOTTING TECHNIQUES

 SOUTHERN BLOT

 WESTERN BLOT

 NORTHERN BLOT

 EASTERN BLOT
Thank you
Operons-

 An operon is a group of genes that are transcribed at the same

time. Jacob, Monod & Lwoff
 They usually control an important biochemical process.
 They are only found in prokaryotes.
Lac Operon

 The lac operon consists of three genes each involved in

processing the sugar lactose
 One of them is the gene for the enzyme β-galactosidase
(galactoside permease,transacetylase)
 This enzyme hydrolyses lactose into glucose and galactose
 E. coli can use either glucose, which is a monosaccharide, or
lactose, which is a disaccharide
 However, lactose needs to be hydrolysed (digested) first
 So the bacterium prefers to use glucose when it can
1. When glucose is present and lactose is absent the E.
coli does not produce β-galactosidase.

2. When glucose is present and lactose is present the

E. coli does not produce β-galactosidase.

3. When glucose is absent and lactose is absent the E.

coli does not produce β-galactosidase.

4. When glucose is absent and lactose is present the E.

coli does produce β-galactosidase
Sexduction
Hfr F+
 Antibiotic:

A drug used to treat infections caused by bacteria and other microorganisms. Originally,
an antibiotic was a substance produced by one microorganism that selectively inhibits
the growth of another. Synthetic antibiotics, usually chemically related to natural
antibiotics, have since been produced that accomplish comparable tasks.
 In 1926, Alexander Fleming discovered penicillin produced by fungi that inhibited
bacterial growth.
 In 1939, Edward Chain and Howard Florey further studied penicillin and later
carried out trials of penicillin on humans (with what were deemed fatal bacterial
infections).
 Fleming, Florey and Chain shared the Nobel Prize in 1945 for their work which
ushered in the era of antibiotic
 An antimicrobial is a substance that kills or inhibits the growth
of microbes such as bacteria, fungi, or viruses.
 microbicidal or microbistatic.
Chemotherapy

 Refers to treatment of disease by chemicals that kill cells,

specifically those of micro-organisms or cancer
 Mechanism of action

Aminoglycosides Penicillin
Tetracycline Cephalosporins
Chloramphenicol Vancomycin
Macrolide Bacitracin
Lincomycin

Antimetabolites

Sulfonamides Polymyxin
PAS Colistin
INNH Rifampin
Trimethoprim Quinolones
 PABA Pteridine

Dihydropteroate synthetase
Dihydropteroic acid

Di hydroFolic Acid

Dihydrofolate reductase
Tetrahydrofolate acid

purines

Nucleic Acid synthesis

 PABA Pteridine

sulfonamides Dihydropteroate synthetase

Dihydropteroic acid

Di hydroFolic Acid

trimethoprim Dihydrofolate reductase

Tetrahydrofolate acid

purines

Nucleic Acid synthesis

Selective toxicity

 An ideal antimicrobial agent should exhibit ST

 Drug is harmful to parasite without being harmful to host at
the particular dose

1. Receptor specific
2. Biochemical event
Mechanism of Drug Resistance

NON GENETIC AND GENETIC

NON GENETIC-
 Metabolically inactive/non multiplying
 M.orgs lose specific target site
 Drug unable to penetrate the site of infection
Antimicrobial Chemotherapy

 Modern Chemotherapy-Paul Ehrlich(18450-1915)-arsenicals

for syphilis and m.blue for malaria
 Domagk-1935-Prontosil
 Alexander Fleming-Penicillium notatum-
Penicillin
 Antibiotic-
 Antimicrobial
 Chemotherapeutic
GENETIC

 Mutation
 Transfer of genes
 Conjugation
 Transduction
 Transposition
 Transformation
 Drug Resistance
Mutational
 Decreased permeability to drug/alt
Transferable
metabolic path/inactivating enzymes
 Single drug  Inactivating enzymes
 Low degree of resistance
 Not transferable
 Metabolically defective  Multidrug
 Virulence maybe lowered  High degree of resistance
 Combination of drugs useful  Transferable
 Metabolically normal
 No decrease in virulence
 -----------
Biochemical mechanism of Drug
resistance
 Production of enzymes that destroy the active drug
1. Beta lactamases
2. Adenylating/phosphorylating/acetylating
3. Acetyl transferase
 Change of permeability
 Develop altered structural target
 Altered metabolic pathway
 Altered enzyme