Bacterial Genetics

1. Bacterial Genetics
By
Asma Firdous
PG Microbiology

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. • Composed of two strands of complementary nucleotides
coiled together in the form of double helix
• 1st discribed by watson and crick.
• Each strand composed of 3 elements
• Deoxyribose sugar
• Phosphate groups
• 4 nitrogenous bases--- 2 purines(A,G) and 2
pyrimidines(T,C)

5. • NUCLEOTIDES –Sugar+nitrogenous bases+phosphates.
• NUCLEOSIDES– sugar +nitogenous bases.

7. 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

8. DNA Replication
Eukaryotes:
2 strands unwind from one another  Each
strand act as template for a new DNA strand

9. •DNA Replication: Prokaryotes
Bidirectional replication Rolling circle mechanism

10. RNA
RNA is structurally similar to DNA except for 2
differences
In sugarribose is present instead of deoxyribose
In Nitrogenous base Uracil replaces thymine
•mRNA
•rRNA
•tRNA

12. 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

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

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

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

16. Variation
Phenotypic
Genotypic

17. MUTATION
Random, heritable variation caused by alteration in
nucleotide sequence of DNA
Frequency 10-2 -10-10/bacterium/division
CAUSES
• Spontaneous – Occurs naturally in any dividing cells without any mutagen
• Induced (mutagen) –
• Physical: UV
• Chemical: alkylating agent, acridine dye

18. 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

19. •Types:
Forward mutation
•Substitution
•At DNA level
•Transversion:
•Transition:

20. Transition:
Point mutation, changes a purine nucleotide to
another purine (A<-->G), a pyrimidine nucleotide
to another pyrimidine(C<-->T)
Transversion:
Substitution of purine for a pyrimidine or vice
versa

21. •Types:
Forward mutation
•Substitution: At codon level
•Silent: New codon code for same AA
•Neutral: New codon code for functional
equivalent AA
•Missense: Different AA
•Non-sense: Stop

22. 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

23. Addition or deletion
Frameshift mutation:
Any addition or deletion of base pairs that is not a multiple of 3
 shift in the normal reading frame of the coded message 
new set of triplet codon  deleterious  synthesis of non
functional proteins
Reverse Mutation:
It is a second mutation that nullifies the effect of first mutation
 gaining back the function of wild phenotype

24. •Reverse Mutation
• True reversion:
Converts the mutant
nucleotides sequence back
to wild type sequence
AAA (lysine)
Wild type
GAA (glutamine)
mutant
AAA (lysine)
Wild type

25. Reverse Mutation
• Equivalent reversion: 2nd Mutation produces
different codon but same Amino Acids
AAA (Serine)
Wild type
GAA (glutamine)
mutant
UCC (serine)
Wild type

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

27. Demonstration of Mutation
• Gene sequencing
• Phenotypic changes
 Fluctuation test
 Replica plating
 Ames test

28. Gene Sequencing:
Method of choice currently used.
Phenotypic methods:
Less commonly used now
Fluctuation test:
Demonstrates spontaneous mutation in bacteria
Replica plating method:
Used to demonstrate auxotrophic mutants(does not grow in the
absence of particular nutrient)
Ames test:
Used to test the carcinogenicity of a mutagen.

29. GENETIC TRANSFER
•Vertical
•Horizontal
•Transformation
•Transduction
•Lysogenic conversion
•Conjugation

30. Transformation
- Random uptake of free/naked DNA and incorporation into
chromosome
•Natural – S. pneumoniae
•express DNA-binding proteins on cell surface
•natural competent state allows uptake of "naked DNA"

31. Transformation
- Random uptake of free/naked DNA incorporation into chromosome

32. •1928: Frederick Griffith (London):
First demonstrated bacterial
transformation

33. He stated that the live non-capsulated strains were transformed into capsulated strains due to the transfer of
capsular genes released from the lysis of killed capsulated strains

34. GENETIC TRANSFER
•Vertical
•Horizontal
•Transformation
•Transduction
•Lysogenic conversion
•Conjugation

35. Transduction- transmission of a portion of DNA from one
bacterium to another by a bacteriophage.

36. Mechanism of transduction
•During transmission  a part of the host DNA 
accidentally incorporated into the bacteriophage
 then gets transferred to the recipient
bacterium  and aquires new characters by the
recipient bacterium coded by the donor DNA

37. Two types of lifecycle inside the host bacteria
1. Lytic or virulent cycle:
Bacteriophage multiples  cytoplasm  large
number of progeny phages  released  death
and lysis of host cell

38. 2. Lysogenic or temperature cycle:
•Host bacterium is unharmed
•Multiplies synchronously with bacterial DNA

40. •Phage DNA disintegrated from host chromosome
 come out into the cytoplasm  lytic phage
•It replicates to produce daughter phages, which
are subsequently released by host cell lysis

41. Types of Transduction
Transduction is of two types.
1. Generalized
2. Restricted

42. Generalized Transduction
Donor bacterial genome
Any part
Into the recipient bacteria

43. •Packaging errors  defective assembly of
the daughter phages
•A part of host DNA may accidentally be
incorporated into the daughter
bacteriophages

44. The donor DNA may have three fates inside
the recipient bacterium
3 outcome on transduction:
1. Abortive transduction: 70-90%
2. Stable gene transfer
3. Unstable gene transfer

45. Abortive transduction
•Not integrated with the recipient bacterial
chromosome
•Able to survive and express itself  called
abortive transductants

46. Stable gene transfer
The donor DNA gets integrated with the
recipient bacterial chromosome

47. Unstable gene transfer
In some cases, the donor DNA gets disintegrated
by the host cell enzymes

49. Restricted or Specialized transduction
•Phage DNA + adjacent part of bacterial
chromosome
•Due to defect in  disintegration of the
lysogenic phage DNA from the bacterial
chromosome
•Studied intensively  the ‘lambda’ phage of
E.coli

50. •Such transducing ohases carrying a part of
bacterial DNA in addition to their DNA, when
infecting another bacterium, the transfer of
donor DNA takes place in two ways
1. The entire transducing genome
2. Crossover

51. The entire transducing genome
• (phage DNA + donor DNA) acts as a prophage and gets
integrated to the recipient’s chromosome. This occurs
if the recipient bacterium is already infected by
another helper bacteriophage

52. Crossover
• Between the donor DNA and a part of recipient DNA
• Leads to an integration of the donor DNA into the
recipient chromosome and a part of recipient DNA
into the phage DNA

54. Importance of transduction
•Drug resistance: Pn increase in staphylococci
•Treatment: Genetic mapping, inborn error of
metabolism
•Phage vectors used I molecular transformation
of bacteria

55. 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

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

57. •Elimination of the phage from a toxigenic
strain renders the bacterium nontoxigenic

58. 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

59. •Lederberg and Tatum
•Donor  contain plasmid  coded for sex pilus-
conjugate tube
•E.coli K12 – role of plasmids in conjugation first
recognized
•Plasmid - ‘sex factor’ or fertility (F) factor

60. F+ x F- Mating
•During conjugation, the plasmid DNA replicates
 the rolling-circle mechanism
•A copy moves to the recipient bacterium
through the conjugation tube
•In the recipient, the entering strand is copied to
produce complete F factor with ds DNA

61. •As a result, the recipient (F-)
becomes (F+) cell and can in turn
conjugate with other (F-) cells

62. •Sometimes  F factor + bact chromosomal
genes transferred
•Donor chromosomal gene  recombination
with  recipient chromosome
•But with a lower frequency

63. HFR Conjugation
• F factor being a plasmid, it may integrate with bacterial
chromosome and behave as episome
• Transfer chromosomal DNA to recipient cells with high
frequency in comparison to F+ cells, therefore, named as
HFR cells
• During conjugation of HFR cells with an F- cell  connection
breaks  whole genome is NOT transferred
• As the entire F factor does not get transferred, hence
following conjugation, F- recipient cells do not become F+
cells

65. F’ Conjugation
• F+ cell to HFR cell is reversible
• F factor integrated with chromosomal DNA
freestate+ some chromosomal DNA from the
adjacent site of its attachment
named as F’ factor (F prime factor)

66. •When F’ cell conjugates with a recipient (F-), it
transfers the host DNA incorporated with it
along with the F factor. The recipient become F’
cell. This is called sexduction

67. F’
F +
F -
Host incorporated DNA
factor F +

69. Colicinogenic (col) factor
•One class of plasmids, colicinogenic (or Col) factors,
determines the production of proteins called colicins
•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

70. Role of Conjugation in Bacterial Drug
Resistance

72. Resistance Transfer Factor (RTF)
•Spread of multiple drug resistance among
bacteria
•Japanese worker reported
•Shigella was resistant to some drugs  same
resistant pattern was seen in E. coli of the same
patient
•Resistance was – plasmid mediated

73. Plasmid: Two components
1. RTF – Resistance Transfer Factor
2. r resistance determinant
R factor = (RTF + r determinant)

74. •R factor + many r determinants (upto 8)
•Sometimes – RTF separated from r  host cell
remain resistant but cannot transfer the
resistance
•Others also sometimes attached  toxin

75. •Transferable drug seen in  Enterobacteriaceae,
vibrio, pseudomonas, pasturella
•Normal gut  anaerobic condition
bile salts
alkaline pH
anaerobic GPC
 minimises contact donor and recipient
 chances of drug resistance is less

76. •Oral antibiotics  disturb normal flora

77. Ttransposons
•First discovered in the 1940s
•Barbara McClintock during her studies on maize
genetics
•She won Nobel prize in 1983

78. Transposons or transposable elements
Intracellular transfer between
a) Chromosome to chromosome
b) Plasmid to plasmid, and
c) Chromosome to plasmid or vice versa
As transposons move around the genome in a cut-and-
paste manner, they are also called jumping genes or
mobile genetic elements

79. Transposition
•Does not require any DNA homology
•Transposons are not self-replicating  but
dependent on chromosomal or plasmid DNA for
replication
•Transposons are also discovered in virus and
eukaryotic genome

80. Types of Transposons
1. Insertion Sequence Transposons
2. Composite Transposons

81. Insertion Sequence Transposons
•Segment of DNA with one or more genes in the
center, the two ends carrying ‘inverted repeat’
sequences of nucleotides – nucleotide sequences
complimentary to each other, but in reverse order
•Each strand of transposon can form a single-
stranded loop carrying the gene and a double
stranded stem formed by hydrogen bonding
between terminal inverted repeat sequences

83. Composite Transposon
•Larger transposons
•Carrying additional genes(genes coding for
antibiotic resistance or toxin production) in the
center and both ends are flanked by insertion
sequences that are identical or very similar in
sequence

86. FATE OF DONOR DNA:

87. Bacterial Recombination
•Integration of Donor DNA to recipient
chromosome
•General or Homologous
•Site specific

88. 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

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

90. TRANSPOSONS

91. 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

93. BLOTTING TECHNIQUES
• SOUTHERN BLOT
• WESTERN BLOT
• NORTHERN BLOT
• EASTERN BLOT

94. Thank You