this ppt gives the comlete nowledge about bacterial genetics

1. Bacterial Genetics

2. GENETICS-Study of genes their structure &
function, heredity & variation
Genomics-Study & analysis of nucleotides of
DNA
Nucleic acid-DNA and RNA

3. Bacterial DNA
 Single Haploid Chromosome
 Super coiled circular dsDNA=1mm
 Exception:
 2 chromosomes : Vibro cholerae

4. Nucleotides-Structural units of Nucleic acids
Nitrogenous bases-Purines(A,G) and
Pyrimidines(T,U,C) Nucleoside
Pentose sugar-Deoxyribose
Phosphate group

6. 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

7.  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

8.  DNA Replication:
Bidirectional replication Rolling circle mechanism

9. RNA
 mRNA
 rRNA
 tRNA

11. 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

12. Extra chromosomal elements
Plasmids
Curing: process of eliminating plasmid from
bacteria
Spontaneous
induced
Acridine
Radiation
Thymine starvation
High temp

13. Classification
On the basis of ability to perform conjugation:
Conjugative/self transmissible plasmid
Non conjugative plasmid
Based on compatibility b/w plasmid:
Compatible
Incompatible

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

15. Variation
 Phenotypic
 Genotypic

16. 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

17. 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

18.  Types:
Forward mutation
 Substitution
 Transversion:
 Transition:

19.  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

20.  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

21.  Types:
 Addition or deletion
 Frame Shift
 Reverse Mutation
 True reversion
AAA (lysine)
Wild type
GAA (glutamine)
mutant
AAA (lysine)
Wild type

22.  Types:
 Reverse Mutation
 Equivalent reversion: 2nd Mutation different codon but same AA
AAA (Serine)
Wild type
GAA (glutamine)
mutant
UCC (serine)
Wild type

23.  Types:
 Reverse Mutation
 Suppressor mutation: 2nd mutation in a different gene that revert the phenotypic
effects of already existing mutation

24. Demonstration of Mutation
 Gene sequencing
 Phenotypic changes
 Fluctuation test
 Replica plating

25. Fluctuation test
 Luria and Delbruck-Mutation is SPONTANEOUS and
RANDOM-
 Growth in presence of selective inhibitory pressure
 Bacteriophage and E. coli

26. Replica plating-Auxotrophic
mutant

27. Ames Test (carcinogenicity
testing)
 Mutational
reversion
assay
Estimate
Mutagenicity
of mutant

28. GENETIC TRANSFER
 Vertical
 Horizontal
 Transformation
 Transduction
 Lysogenic conversion
 Conjugation

29. 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"

30. Transformation
- random uptake of free / naked DNA
incorporation into chromosome


31.  1928: Frederick Griffith (London): First demonstrated
bacterial transformation

32. An "S" or SMOOTH coat strain, which is
lethal to mice.

33. An “R" or rough coat strain, which is
NOT lethal to mice.

34. Griffith found that he could heat inactivate the
smooth strain.

35. 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.

36. GENETIC TRANSFER
 Vertical
 Horizontal
 Transformation
 Transduction
 Lysogenic conversion
 Conjugation

37. Transduction- Transfer of genetic
material through bacteriophage

39. Transduction 2 types:
Generalized:
Packaging error
3 outcome on transduction:
Abortive transduction: 70-90%
Stable gene transfer
Unstable gene transfer


40. Transduction 2 types:


41. Transduction 2 types:
Restricted/specialized
Defect in disintegration of lysogenic phage
2 outcome when transduced to new bacteria
Cross over
Integrated as prophage

42. Transduction 2 types:


43. Importance of transduction
Drug resistance: Pn resistance
in Staphylococci
Treatment: Genetic mapping,
inborn error of metabolism
Phage vectors used in
molecular transformation of
bacteria

44. 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

45.  Lysogenic conversion: Phage DNA itself behave as new genetic element
 Transduction: Phage act as vehicle carrying bacterial gene

46. GENETIC TRANSFER
 Vertical
 Horizontal
 Transformation
 Transduction
 Lysogenic conversion
 Conjugation

47. 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

49.  F+ F- Mating
 HFR conjugation
 F’ Conjugation
Col factor
R factor-RTF + r determinants

51. 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

53. FATE OF DONOR DNA:

54. Bacterial Recombination
 Integration of Donor DNA to recipient chromosome
 General or Homologous
 Site specific

55. 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

56. Site specific
 Integration of bacteriophage DNA into Bacterial DNA is site specific
 Donor DNA not homologous with chromosome it joins

57. TRANSPOSONS

58. 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

60. 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

61.  Target DNA
 Primers
 Polymerase Enzyme- Thermus aquaticus
 Nucleotide
 Thermocycler

62.  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.

64. PCR in Diagnosis
Bacteria
Viruses
Fungi

65. 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.

66. 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

67. BLOTTING TECHNIQUES
 SOUTHERN BLOT
 WESTERN BLOT
 NORTHERN BLOT
 EASTERN BLOT

68. Thank you

69. 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.

70. 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

71.  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

72. 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

75. Sexduction
Hfr F+

76. 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.

77.  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

78.  An antimicrobial is a substance that kills or inhibits the
growth of microbes such as bacteria, fungi, or viruses.
 microbicidal or microbistatic.

79. Chemotherapy
 Refers to treatment of disease by chemicals that kill cells,
specifically those of micro-organisms or cancer

80. Mechanism of action
Penicillin
Cephalosporins
Vancomycin
Bacitracin
Polymyxin
Colistin
Aminoglycosides
Tetracycline
Chloramphenicol
Macrolide
Lincomycin
Sulfonamides
PAS
INNH
Trimethoprim
Rifampin
Quinolones
Antimetabolites

81. PABA Pteridine
Dihydropteroate synthetase
Dihydropteroic acid
Di hydroFolic Acid
Dihydrofolate reductase
Tetrahydrofolate acid
purines
Nucleic Acid synthesis

82. PABA Pteridine
sulfonamides Dihydropteroate synthetase
Dihydropteroic acid
Di hydroFolic Acid
trimethoprim Dihydrofolate reductase
Tetrahydrofolate acid
purines
Nucleic Acid synthesis

83. 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

84. 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

85. 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

86. GENETIC
 Mutation
 Transfer of genes
 Conjugation
 Transduction
 Transposition
 Transformation

87. Drug Resistance
Mutational Transferable
 Decreased permeability to
drug/alt metabolic
path/inactivating enzymes
 Single drug
 Low degree of resistance
 Not transferable
 Metabolically defective
 Virulence maybe lowered
 Combination of drugs useful
 Inactivating enzymes
 Multidrug
 High degree of
resistance
 Transferable
 Metabolically normal
 No decrease in
virulence
 -----------

88. 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