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BACTERIAL
GENETICS
Dr. R. Someshwaran, MD., Assistant professor,
Department of Microbiology, KFMS&R
Objectives of today’s class
At the end of my class you should be able to
 Enumerate the principles of Molecular
biology
...
Introduction
 Bacteria and other microbes
 Laws of Genetics
 Pleomorphism (Nageli)
 Protean capacity for variation
 M...
Basic principles of Molecular
biology
 Central dogma of Molecular biology –
DNA
 Essential material of heridity
 DNA is...
Bacterial cell
12/15/2015
5
Transcription & Translation
12/15/2015
6
Bacterial chromosome
12/15/2015
7
DNA Double Helix
12/15/2015
8
Double stranded DNA
12/15/2015
9
DNA structure
 Double helix (2 chains of nucleotides)
 Deoxyribose and phosphate backbone
 Nitrogenous bases – Purine &...
Complementary base pairs
12/15/2015
11
DNA structure
12/15/2015
12
Watson & Crick – Nobel
Laureates
12/15/2015
13
DNA structure
 Ratio of each pair of bases (A+T) / (G+C) is
constant for each species but varies widely
among one species...
RNA Structure
 Structurally similar to DNA
 But 2 major difference
 Ribose instead of Deoxyribose
 Base Uracil instead...
RNA Vs DNA
12/15/2015
16
Codon (Triplet code)
 Codon: Genetic information stored in DNA as
code
 Unit of code with sequence of three bases is
cal...
RNA to Polypetides & Proteins
12/15/2015
18
Gene
 Bacterial chromosome contain double
stranded molecule of DNA arranged in a
circular form
 DNA contain large number...
Exons & Introns
 Exons: Stretches of coding genes are
called Exons
 Introns: Between the coding sequences of
genes there...
Extrachromosomal genetic
elements
 Extra chromosomal genetic elements are
not essential for normal life and
functioning o...
Plasmids
 Plasmids are circular DNA molecules
present in cytoplasm of bacteria, capable
of autonomous replication (indepe...
Plasmid
12/15/2015
23
Episome
12/15/2015
24
Plasmid typing
 Plasmid types: Self-transmissible or Non-
transmissible or non-conjugating
 Based on property encoded (S...
Genotype and Phenotype
 Genome: Sum total of the genes that make
up the genetic apparatus of a cell
establishes the genot...
Genotypic and Phenotypic
variation
1. Example of Phenotypic variation:
 Salmonella typhi – Flagellated – Normally
 When ...
Genotypic and Phenotypic
variation
Genotypic variation is stable, heritable and
not influenced by environment and results
...
Genotypic and Phenotypic
variation
12/15/2015
29
12/15/2015
30
Bacterial DNA Mutation
12/15/2015
31
 Random, undirected, heritable variation caused
by alteration in nucleotide sequence...
Types of Mutation
12/15/2015
32
 Types: A. Spontaneous or B. Induced.
 Also mutations may be typed as Addition,
deletion...
Spontaneous Mutation
12/15/2015
33
 Fixed frequency rate for mutation
 Mutation – can go unrecognised
 - can affect vit...
Calculation of incidence of mutations
If doubling time= 20 min, then
23cells/hr
Over 12 hr, 236 cells (~1010)produced from...
12/15/2015
35
Damage to DNA
12/15/2015
36
DNA Damage – Altered DNA
12/15/2015
37
Where, What & How to look
for?
12/15/2015
38
In a chromosome
12/15/2015
39
We have Genes, DNA
12/15/2015
40
Mutation
12/15/2015
41
Types of Mutation
12/15/2015
42
Types of Mutation
12/15/2015
43
Types of Mutation
12/15/2015
44
Fluctuation test in
Bacteriophage resistant E. coli
By Luria and Delbruck
12/15/2015
45
Simple but elegant ‘Replica
plating technique’ By Lederberg
12/15/2015
46
Bacterial Genetic Diversity:
Genetic Recombination (Gene transfer or
Exchange)
 Three processes bring bacterial DNA
from ...
Transformers!!!
12/15/2015
48
Transformation
12/15/2015
49
 Avery, Mc Leod & Mc Carty (1944)
 Transfer of genetic information through the
agency of fr...
Griffith experiment
12/15/2015
50
Transduction
 Most widespread mechanism of gene transfer
among prokaryotes and excellent tool for gene
mapping of bacteri...
Transduction
12/15/2015
52
 Types of Transformation: A. Generalised, B.
RestrictedRestricted transduction –
extensively s...
Transduction
12/15/2015
53
Lysogenic conversion
12/15/2015
54
 Bacteriophages exhibits 2 types of Life cycle.
 A. Virulent or Lytic, B. Lysogenic
...
Lysogenic conversion
12/15/2015
55
1. Susceptibility to bacteriophages (Immunity
to superinfection with same or related
ph...
Lysogenic conversion
12/15/2015
56
Lysogenic conversion
12/15/2015
57
Conjugation
 Conjugation or Sexduction is a process
whereby a male cell or donor bacterium mates
or makes physical contac...
LE 18-17
Sex pilus 5 µm
Conjugation - Sexduction
 Plasmids responsible was termed as
Fertility factor (F) or Sex factor. Now
Transfer factor is t...
Conjugation
12/15/2015
61
Colicinogenic factor (Col)
 Coliorm – Colicins (Lethal antibiotic like
substance for other enterobacteria)
 Pyocin by Ps...
Resistance Transfer Factor
(RTF)
 Spread of drug resistant bacteria
 Shigella resistant to Sulphonamides,
Streptomycin, ...
Genetic mechanism of drug
resistance in bacteria
 Mutational resistance 2 types:
1. Stepwise mutation: Penicillin resista...
Genetic mechanism of drug
resistance in bacteria
Mutational drug
resistance
Tansferable drug
resistance
One drug e at a ti...
Antibiotic resistance in
bacteria
12/15/2015
66
Antibiotic resistance in
bacteria
12/15/2015
67
Transposon
12/15/2015
68
- Transposons or Jumping
genes
- Barbara Mc Clintock in
Plants
- Mode of gene transfer
- Transpos...
Molecular genetics
12/15/2015
69
Molecular genetics
12/15/2015
70
1.Genetic engineering
2.Restriction endonucleases
3.DNA probes
4.Blotting techniques (Sou...
Summary & Take home
message
 DNA – Gene – Codon
 Plasmid, Episome
 Mutation
 Tranformation, Transduction & Conjugation...
Let a new ‘Revolution’
begin!!!
12/15/2015
72
To treat any gene mutation,
Take these pill !!!
12/15/2015
73
THANK YOU
12/15/2015
74
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Bacterial genetics 2015 December Second MBBS students Microbiology

Second MBBS undergraduate students - Microbiology - Bacterial Genetics

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Bacterial genetics 2015 December Second MBBS students Microbiology

  1. 1. BACTERIAL GENETICS Dr. R. Someshwaran, MD., Assistant professor, Department of Microbiology, KFMS&R
  2. 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 BACTERIA12/15/2015 2
  3. 3. Introduction  Bacteria and other microbes  Laws of Genetics  Pleomorphism (Nageli)  Protean capacity for variation  Monomorphism (Kohn & Koch)  Molecular biology 1940’s 12/15/2015 3
  4. 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 12/15/2015 4
  5. 5. Bacterial cell 12/15/2015 5
  6. 6. Transcription & Translation 12/15/2015 6
  7. 7. Bacterial chromosome 12/15/2015 7
  8. 8. DNA Double Helix 12/15/2015 8
  9. 9. Double stranded DNA 12/15/2015 9
  10. 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 12/15/2015 10
  11. 11. Complementary base pairs 12/15/2015 11
  12. 12. DNA structure 12/15/2015 12
  13. 13. Watson & Crick – Nobel Laureates 12/15/2015 13
  14. 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 12/15/2015 14
  15. 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 15
  16. 16. RNA Vs DNA 12/15/2015 16
  17. 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 12/15/2015 17
  18. 18. RNA to Polypetides & Proteins 12/15/2015 18
  19. 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 19
  20. 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 12/15/2015 20
  21. 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 12/15/2015 21
  22. 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. 12/15/2015 22
  23. 23. Plasmid 12/15/2015 23
  24. 24. Episome 12/15/2015 24
  25. 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) 12/15/2015 25
  26. 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 26
  27. 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 only12/15/2015 27
  28. 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) 12/15/2015 28
  29. 29. Genotypic and Phenotypic variation 12/15/2015 29
  30. 30. 12/15/2015 30
  31. 31. Bacterial DNA Mutation 12/15/2015 31  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. 32. Types of Mutation 12/15/2015 32  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. 33. Spontaneous Mutation 12/15/2015 33  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. 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/day12/15/2015 34
  35. 35. 12/15/2015 35
  36. 36. Damage to DNA 12/15/2015 36
  37. 37. DNA Damage – Altered DNA 12/15/2015 37
  38. 38. Where, What & How to look for? 12/15/2015 38
  39. 39. In a chromosome 12/15/2015 39
  40. 40. We have Genes, DNA 12/15/2015 40
  41. 41. Mutation 12/15/2015 41
  42. 42. Types of Mutation 12/15/2015 42
  43. 43. Types of Mutation 12/15/2015 43
  44. 44. Types of Mutation 12/15/2015 44
  45. 45. Fluctuation test in Bacteriophage resistant E. coli By Luria and Delbruck 12/15/2015 45
  46. 46. Simple but elegant ‘Replica plating technique’ By Lederberg 12/15/2015 46
  47. 47. Bacterial Genetic Diversity: Genetic Recombination (Gene transfer or Exchange)  Three processes bring bacterial DNA from different individuals together: Transduction Transformation Conjugation 12/15/2015 47
  48. 48. Transformers!!! 12/15/2015 48
  49. 49. Transformation 12/15/2015 49  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. 50. Griffith experiment 12/15/2015 50
  51. 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 12/15/2015 51
  52. 52. Transduction 12/15/2015 52  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. 53. Transduction 12/15/2015 53
  54. 54. Lysogenic conversion 12/15/2015 54  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. 55. Lysogenic conversion 12/15/2015 55 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. 56. Lysogenic conversion 12/15/2015 56
  57. 57. Lysogenic conversion 12/15/2015 57
  58. 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 pilus12/15/2015 58
  59. 59. LE 18-17 Sex pilus 5 µm
  60. 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 60
  61. 61. Conjugation 12/15/2015 61
  62. 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. 12/15/2015 62
  63. 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 12/15/2015 63
  64. 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 12/15/2015 64
  65. 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 defective12/15/2015 65
  66. 66. Antibiotic resistance in bacteria 12/15/2015 66
  67. 67. Antibiotic resistance in bacteria 12/15/2015 67
  68. 68. Transposon 12/15/2015 68 - Transposons or Jumping genes - Barbara Mc Clintock in Plants - Mode of gene transfer - Transposition – a mechanism for amplifying genetic transfer in nature
  69. 69. Molecular genetics 12/15/2015 69
  70. 70. Molecular genetics 12/15/2015 70 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. 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 epidemiology12/15/2015 71
  72. 72. Let a new ‘Revolution’ begin!!! 12/15/2015 72
  73. 73. To treat any gene mutation, Take these pill !!! 12/15/2015 73
  74. 74. THANK YOU 12/15/2015 74

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