Second MBBS undergraduate students - Microbiology - Bacterial Genetics

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 BACTERIA12/15/2015
2

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. 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. Bacterial cell
12/15/2015
5

6. Transcription & Translation
12/15/2015
6

7. Bacterial chromosome
12/15/2015
7

8. DNA Double Helix
12/15/2015
8

9. Double stranded DNA
12/15/2015
9

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. Complementary base pairs
12/15/2015
11

12. DNA structure
12/15/2015
12

13. Watson & Crick – Nobel
Laureates
12/15/2015
13

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. 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. RNA Vs DNA
12/15/2015
16

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. RNA to Polypetides & Proteins
12/15/2015
18

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. 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. 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. 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. Plasmid
12/15/2015
23

24. Episome
12/15/2015
24

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. 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. 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. 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. Genotypic and Phenotypic
variation
12/15/2015
29

30. 12/15/2015
30

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. 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. 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. 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. 12/15/2015
35

36. Damage to DNA
12/15/2015
36

37. DNA Damage – Altered DNA
12/15/2015
37

38. Where, What & How to look
for?
12/15/2015
38

39. In a chromosome
12/15/2015
39

40. We have Genes, DNA
12/15/2015
40

41. Mutation
12/15/2015
41

42. Types of Mutation
12/15/2015
42

43. Types of Mutation
12/15/2015
43

44. Types of Mutation
12/15/2015
44

45. Fluctuation test in
Bacteriophage resistant E. coli
By Luria and Delbruck
12/15/2015
45

46. Simple but elegant ‘Replica
plating technique’ By Lederberg
12/15/2015
46

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. Transformers!!!
12/15/2015
48

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. Griffith experiment
12/15/2015
50

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. 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. Transduction
12/15/2015
53

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. 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. Lysogenic conversion
12/15/2015
56

57. Lysogenic conversion
12/15/2015
57

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. 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
60

61. Conjugation
12/15/2015
61

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. 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. 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. 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. Antibiotic resistance in
bacteria
12/15/2015
66

67. Antibiotic resistance in
bacteria
12/15/2015
67

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. Molecular genetics
12/15/2015
69

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. 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. Let a new ‘Revolution’
begin!!!
12/15/2015
72

73. To treat any gene mutation,
Take these pill !!!
12/15/2015
73

74. THANK YOU
12/15/2015
74