GENETICS of BACTERIA

1. GENETIC of BACTERIA
Professor M.Boychenko

2. Bacterial genome
• DNA molecules that replicate as
discrete genetic units in bacteria are
called replicons. In some bacterial
strains, the chromosome is the only
replicon present in the cell. Other
bacterial strains have additional
replicons, such as plasmids and
bacteriophages.

3. Chromosomal DNA
• Bacterial genomes vary in size from
about 0.4 x 109 to 8.6 x 109 daltons
(Da),
• Most bacteria have a haploid
genome, a single chromosome
consisting of a circular, double
stranded DNA molecule.

4. Bacterial genome
• However linear chromosomes have been
found in Gram-positive Borrelia and
Streptomyces spp.,
• and one linear and one circular
chromosome is present in the Gram-
negative bacterium Agrobacterium
tumefaciens.
• V.cholerae possesses 2 circular
chromosomes

5. plasmids
• The term plasmid was first introduced by
the American molecular biologist Joshua
Lederberg in 1952
• Plasmids are considered transferable
genetic elements, or "replicons", capable
of autonomous replication within a suitable
host. Plasmids can be found in all three
major kingdoms, Archea, Bacteria and
Eukarya

6. plasmids
• A plasmid is an
extra-chromosomal
DNA molecule
separate from the
chromosomal
DNA,which is
capable of
replicating
independently of the
chromosomal DNA.
In many cases, it is
circular and double-
stranded. Plasmids
usually occur
naturally in bacteria,
but are sometimes
found in eukaryotic

7. PLASMIDS
• Plasmid size
varies from 1 to
over 200
kilobase pairs
(kbp). The
number of
identical
plasmids within
a single cell can
range anywhere
from one to
even thousands

8. types of plasmids
• There are two
types of plasmid
integration into a
host bacteria: Non-
integrating
plasmids replicate
as with the top
instance; whereas
episome integrate
into the host
chromosome.

9. types of plasmids
• One way of grouping plasmids
is by their ability to transfer to
other bacteria.

10. Types of plasmids
• Conjugative
plasmids contain
so-called tra-
genes, which
mediate process of
conjugation, the
transfer of
plasmids to
another bacterium

11. F + x F-

12. Types of plasmids
• Non-conjugative plasmids are
incapable of initiating conjugation,
hence they can only be
transferred with the assistance of
conjugative plasmids.,

13. Types of plasmids
• It is possible for plasmids of different types
to coexist in a single cell. Seven different
plasmids have been found in E.coli. But
related plasmids are often incompatible, in
the sense that only one of them survives in
the cell line, due to the regulation of vital
plasmid functions. Therefore, plasmids
can be assigned into compatibility groups.

14. Types of plasmids
• .
• Another way to classify plasmids is by
function. There are five main classes:
• Fertility-F-plasmid, which contain tra-
genes. They are capable of conjugation
(transfer of genetic material between
bacteria which are touching).
• Resistance-(R)plasmids, which contain
genes that can build a resistance
against antibiotics or poisons.
Historically known as R-factors, before
the nature of plasmids was understood.

15. Types of plasmids
• Col-plasmids, which contain genes that
code for (determine the production of)
bactericins proteins, that can kill other
bacteria.
• Degradative plasmids, which enable the
digestion of unusual substances, e.g.,
toluene or salicylic acid.
• Virulence plasmids, which turn the
bacterium into a pathogen (one that
causes disease).

16. Using of plasmids
• Another major use of plasmids is to
make large amounts of proteins. In this
case, researchers grow bacteria
containing a plasmid harboring the gene
of interest. Bacteria can be induced to
produce large amounts of proteins from
the inserted gene. This is a cheap and
easy way of mass-producing a gene or
the protein it then codes for, for
example, insulin or even antibiotics.

17. •Plasmids are now being used to
manipulate DNA and may possibly
be a tool for curing many diseases.

18. Using of plasmids
• Major use of
plasmids is to make
large amounts of
proteins. In this
case, researchers
grow bacteria
containing a plasmid
harboring the gene
of interest. Bacteria
can be induced to
produce large
amounts of proteins
from the inserted
gene.

20. Plasmid’s profile
• Comparing plasmid
profiles is a useful
method for assessing
possible relatedness
of individual clinical
isolates of a particular
bacterial species for
epidemiological
studies.

21. Mobile genetics elements
• .Transposons are segments of DNA
that can move from one site in a
DNA molecule to other target sites
in the same or a different DNA
molecule.
• The process is called transposition
and occurs by a mechanism that is
independent of generalized
recombination.

22. Mobile genetics elements
• Transposons are not self-
replicating genetic elements,
however, and they must integrate
into other replicons to be
maintained stably in bacterial
genomes

23. Mobile genetics elements
• Insertion of a transposon often interrupts
the linear sequence of a gene and
inactivates it.
• Transposons have a major role in causing
deletions, duplications, and inversions of
DNA segments as well as fusions between
replicons.

24. Mobile genetics elements
• Most transposons share a number of
common features. Each transposon
encodes the functions necessary for
its transposition, including a
transposase enzyme that interacts
with specific sequences at the ends of
the transposon.

25. Mobile genetics elements
• Complex transposons vary in length
from about 2,000 to more than 40,000
nucleotide pairs and contain insertion
sequences (or closely related
sequences) at each end, usually as
inverted repeats.

26. IS-elements
• Insertion sequences
are simplest in
structure and
encode only the
functions needed for
transposition.
Inverted repeats are
at their endsThe
DNA between the
inverted terminal
repeats contains
transposase genes

27. • During transposition a short sequence
of target DNA is duplicated,
• The duplication is presumed to
involve asymmetric cleavage of
DNA at the target site.
• If the transposon at a donor site is
replicated and a copy is inserted into
the target site, however, the process is
called replicative transposition

28. Mobile genetics elements
• Resolution of the cointegrate into its
component replicons is often accomplished
by a transposon-encoded resolvase that
catalyzes site-specific recombination
between the transposons.
• Transposition differs from site-specific
recombination by duplicating a segment of
the target sequence and by using a variety of
different target sequences for a single donor
sequence.

29. Role of transposone in bacterial evolution
• Some of the multiple antibiotic resistant
plasmids have individual transposons with
several resistance determinants. The
stepwise acquisition of resistance
determinants can lead, in some cases, to
the formation of composite transposons
that encode multiple resistance
determinants.

30. Role of transposone in bacterial evolution
• After a plasmid carrying a transposon is
introduced into a new bacterial host, the
transposon and its determinants can jump
into the chromosome or indigenous
plasmids of the new host. Therefore,
stability of the mobilizing plasmid in a new
bacterial host is not essential for
persistence of genetic determinants
located on a transposon.

31. .The acquisition of resistance gene arrays involves
genetic mobile elements like :
– Plasmids
•Transposons
•Integrons are a system of gene
capture and expression composed of an
intI gene encoding an integrase, a
recombination site attI, and a
promoter.

32. attC1attI
attI
attC2 attC1attI
attC1
attC2
5'conserved
segment
cassette 1
cassette 2
Integrons are a system of gene capture and expression
composed of an intI gene encoding an integrase, a
recombination site attI, and a promoter.
intI
P

33. The integrase is able to integrate or
excise gene cassettes, by a site-
specific system of recombination.
Cassette mobility results in a very
efficient system of dissemination of
resistance genes.

34. Recombination
• Recombination is the
rearrangement of donor and
recipient genomes to form new,
hybrid genomes
• Recombination involves breakage
and joining of parental DNA
molecules to form hybrid,
recombinant molecules.
• .

35. Recombination
• Several distinct kinds of recombination
have been identified that depend on
different features of the participating
genomes and require the activities of
different gene products. Specific
enzymes that act on DNA (for example,
exonucleases, endonucleases,
polymerases, ligases) participate in
recombination

36. Generalized recombination
• Generalized recombination involves
donor and recipient DNA molecules
that have homologous nucleotide
sequences.
• The product of the recA gene is
essential for generalized
recombination, but other gene
products also participate.

37. Site-specific recombination
• Site-specific recombination involves
reciprocal exchanges only between
specific sites in donor and recipient
DNA molecules.
• The recA gene product is not required
for site-specific recombination.
• Integration of the temperate bacteriophage l into
the chromosome of E coli is a well-studied
example of site-specific recombination
• .

38. Site-specific recombination
• In phage l the product of the int gene
(integrase) is required for the site-specific
integration event in lysogenization;
• The products of the int and xis
(excisionase) genes are both needed for
the complementary site-specific excision
event that occurs during induction of lytic
phage development in lysogenic cells.

39. Site-specific recombination
• The specific
attachment (att)
sites on the E coli
chromosome and l
phage DNA have a
common core
sequence of 15
nucleotides, within
which reciprocal
recombination
occurs

40. Exchange of Genetic Information
• Genetic exchanges among bacteria occur by
several mechanisms.
• In transformation, the recipient bacterium
takes up extracellular donor DNA.
• In transduction, donor DNA packaged in a
bacteriophage infects the recipient
bacterium.
• In conjugation, the donor bacterium transfers
DNA to the recipient by mating.

41. Transfer of genetic’s information

42. transformation
• Historically, characterization of
"transforming principle" from S
pneumoniae provided the first
direct evidence DNA is genetic
material.

44. transformation
• In transformation, pieces of DNA released
from donor bacteria are taken up directly
from the extracellular environment by
recipient bacteria. To be active in
transformation, DNA molecules must be at
least 500 nucleotides in length, and
transforming activity is destroyed rapidly
by treating DNA with deoxyribonuclease.

45. transformation
• Molecules of transforming DNA
correspond to very small fragments of the
bacterial chromosome.
• Transformation was discovered in
S.pneumoniae and occurs in other
bacterial genera including Haemophilus,
Neisseria, Bacillus, and Staphylococcus

46. transformation
• The ability of bacteria to take up
extracellular DNA and to become
transformed, called competence,
varies with the physiologic state
of the bacteria.

47. transformation
• Many bacteria that are not usually
competent can be made to take
up DNA by laboratory
manipulations, such as calcium
shock or exposure to a high-
voltage electrical pulse
(electroporation).

48. transformation
• Competent bacteria may also take up
intact bacteriophage DNA (transfection) or
plasmid DNA, which can then replicate as
extrachromosomal genetic elements in the
recipient bacteria. Recombination
occurs between single molecules of
transforming DNA and the
chromosomes of recipient bacteria.

49. transformation

50. Conjugation

51. F+ x F-
• In matings between F+ and F- bacteria,
only the F plasmid is transferred with high
efficiency to recipients.
• In matings between F+ and F- strains, the
F plasmid spreads rapidly throughout the
bacterial population, and most
recombinants are F+.

52. conjugation

53. Conjugation
• Donor strains with integrated F
factors can transfer
chromosomal genes to
recipients with high efficiency,
they are called Hfr (High
frequency recombination)
strains.

54. Hfr x F-
• In matings between Hfr and F-
strains, the segment of the F
plasmid containing the tra region
is transferred last, after the entire
bacterial chromosome has been
transferred.

55. Conjugation
• Formation of recombinant
progeny requires recombination
between the transferred donor
DNA and the genome of the
recipient bacterium.

56. Conjugation
• Most recombinants from matings
between Hfr and F- are
phenotypically F-.
• In matings between F+ and F- strains,
the F plasmid spreads rapidly
throughout the bacterial population,
and most recombinants are F+.

57. conjugation

58. General transduction
• In transduction,
bacteriophages function as
vectors to introduce DNA from
donor bacteria into recipient
bacteria by infection.

59. General transduction
• For some phages, called
generalized transducing phages,
a small fraction of the virions
produced during lytic growth
contain a random fragment of the
bacterial genome instead of
phage DNA.

60. General transduction
• Each individual transducing phage carries
a different set of closely linked genes,
representing a small segment of the
bacterial genome.
• When a generalized transducing phage
infects a recipient cell, expression of the
transferred donor genes occurs

61. General transduction

62. Specialized transduction
• Specialized transduction differs from
generalized transduction in several
ways.
• It is mediated only by specific
temperate phages,
• and only a few specific donor genes
can be transferred to recipient
bacteria.

63. Specialized transduction
• Specialized transducing
phages are formed only when
lysogenic donor bacteria enter
the lytic cycle and release
phage progeny.

64. Specialized transduction
• The specialized transducing
phages lack part of the normal
phage genome and contain part
of the bacterial chromosome
located adjacent to the prophage
attachment site.

65. Specialized transduction
• Specialized transduction
results from lysogenization of
the recipient bacterium by the
specialized transducing phage
and expression of the donor
genes.

67. Specialized transduction
• Phage conversion and
specialized transduction have
many similarities, but the origin of
the converting genes in temperate
converting phages is unknown.

68. 68
Practical genetic

69. Molecular methods in microbial
laboratory
• 1. PCR
• 2. Micro array
technique
• 3.Ribotyping
• 4.Finger printing
• 5.Plasmid’s profile
• 8.PCR-RT
• Identification of
microbes without
isolation of pure
culture
• Intra species
identification

70. 100-200 μm
DNA microarrays are:
glass slides with
hundreds to thousands of
DNA probes bound

71. Community DNA
Specific PCR product
(i.e., 16S rRNA gene)
Fluorescently labelled target
(DNA or RNA)
Oligonucleotide probes
Length: between 15 and 30 nt
Hybridisation
Microarray with the above probes
Data analysis
Methodology

72. Many target molucules
High signal
A few target molecules
Low signal
No target
No signal
Glass slide
DNA probes
Labelled “target” DNA
Attached fluorophore
Emitted light (fluorescence)
The principle of detection of specific DNA sequences

73. Real time pcr

74. Branch-pcr