Genetics of bacteria

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

2. 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
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3. 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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4. 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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5. Bacterial cell
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6. Transcription & Translation
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7. Bacterial chromosome
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8. DNA Double Helix
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9. Double stranded DNA
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10. 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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11. Complementary base pairs
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12. DNA structure
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13. Watson & Crick – Nobel
Laureates
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14. 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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15. 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) 12/15/2015
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16. RNA Vs DNA
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17. 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
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18. RNA to Polypetides & Proteins
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19. 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) 12/15/2015
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20. 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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21. 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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22. 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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23. Plasmid
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24. Episome
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25. 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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26. 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 12/15/2015
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27. 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 environment
2. Escherichia coli - Enzyme Beta-galactosidase
– Lactose fermentation occurs in medium
containing lactose
3. When E. coli grown in media containing only
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28. 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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29. Genotypic and Phenotypic
variation
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31. Bacterial DNA Mutation
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 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

32. Types of Mutation
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 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

33. Spontaneous Mutation
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 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

34. Calculation of incidence of mutations
If doubling time= 20 min, then
23cells/hr
Over 12 hr, 236 cells (~1010)produced from
a single cell
If spontaneous mutation rate = 1 x 10-7 /
gene, then in 12 hr (day) (1010) (10-7)= 103
mutations/gene/day
If bacteria have ~4000 genes; then
(4x103genes(103)= 4 x 106 mutations/day
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36. Damage to DNA
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37. DNA Damage – Altered DNA
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38. Where, What & How to look
for?
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39. In a chromosome
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40. We have Genes, DNA
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41. Mutation
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42. Types of Mutation
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43. Types of Mutation
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44. Types of Mutation
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45. Fluctuation test in
Bacteriophage resistant E. coli
By Luria and Delbruck
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46. Simple but elegant ‘Replica
plating technique’ By Lederberg
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47. 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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48. Transformers!!!
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49. Transformation
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 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.

50. Griffith experiment
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51. 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
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52. Transduction
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 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,

53. Transduction
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54. Lysogenic conversion
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 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

55. Lysogenic conversion
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1. Susceptibility to bacteriophages (Immunity
to superinfection with same or related
phages)
2. Antigenic characteristics
3. Medical importance: Corynebacterium
diphtheriaere acquire Toxigenicity and
therefore virulence by Lysogenisation with
phage beta.
4. Toxigenic strain - eliminate betaphage –
non-toxigenic

56. Lysogenic conversion
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57. Lysogenic conversion
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58. 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
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59. LE 18-17
Sex pilus 5 µm

60. 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 12/15/2015
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61. Conjugation
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62. 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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63. 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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64. Genetic mechanism of drug
resistance in bacteria
 Mutational resistance 2 types:
1. Stepwise mutation: Penicillin resistance
2. One-step mutation: Streptomycin
 Clinical significance:
 MDR-TB Multi drug resistant Tuberculosis
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65. 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
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66. Antibiotic resistance in
bacteria
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67. Antibiotic resistance in
bacteria
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68. Transposon
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- Transposons or Jumping
genes
- Barbara Mc Clintock in
Plants
- Mode of gene transfer
- Transposition – a
mechanism for amplifying
genetic transfer in nature

69. Molecular genetics
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70. Molecular genetics
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1.Genetic engineering
2.Restriction endonucleases
3.DNA probes
4.Blotting techniques (Southern,
Northern, Western)
5.Polymerase chain reaction (PCR)
6.Molecular epidemiology
7.Genetic mapping

71. 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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72. Let a new ‘Revolution’
begin!!!
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73. To treat any gene mutation,
Take these pill !!!
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74. THANK YOU
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