Chair of Microbiology, Virology, and Immunology

1
Chair of Microbiology, Virology, and Immunology
GENETICS OF BACTERIA AND VIRUSES
BASES OF BIOTECHNOLOGY AND GENE
ENGENEERING
2
Lectures schedule

• 1. Structure of bacterial genome.
• 2. Extrachromosomal elements.
• 3. Mutations.
• 4. Recombinations.
• 5. Gene engineering.

3
F. Crick i J. Watson described DNA structure
4
DNA structure
5
E. coli DNA
The chromosome of E. coli
has a contour length of approximately 1.35 mm,
several hundred times longer than the bacterial
cell, but the DNA is supercoiled and tightly
packaged in the bacterial nucleoid. The time
required for replication of the entire chromosome
is about 40 minutes
6
E. coli DNA
7
Plasmid
Definition Extrachromosomal genetic elements
that are capable of autonomous replication
(replicon) Episome - a plasmid that can integrate
into the chromosome They are usually much
smaller than the bacterial chromosome, varying
from less than 5 to more than several hundred
kbp. Most plasmids are supercoiled, circular,
double-stranded DNA molecules, but linear
plasmids have also been demonstrated in Borrelia
and Streptomyces.
8
Classification of Plasmids

• Transfer properties
• Conjugative (This plasmids code for functions
  that promote transfer of the plasmid from the
  donor bacterium to other recipient bacteria)
• Nonconjugative (do not)
• Phenotypic effects
• Fertility
• Bacteriocinogenic plasmid
• Resistance plasmid (R factors)

9
Phenotypic effects
10
Structure of R factors

• RTF
• Conjugative plasmid
• Transfer genes

• R determinant
• Resistance genes
• Transposons

11
The average number of molecules of a given
plasmid per bacterial chromosome is called its
copy number. Large plasmids (40 kilobase pairs)
are often conjugative, have small copy numbers (1
to several per chromosome). Plasmids smaller than
7.5 kilobase pairs usually are nonconjugative,
have high copy numbers (typically 10-20 per
chromosome), rely on their bacterial host to
provide some functions required for replication,
and are distributed randomly between daughter
cells at division. Some plasmids are cryptic
and have no recognizable effects on the bacterial
cells that harbor them
12
(No Transcript)
13
Transposable Genetic Elements

• Definition Segments of DNA that are able to move
  from one location to another
• Properties
• Random movement
• Not capable of self replication
• Transposition mediated by site-specific
  recombination
• Transposase
• Transposition may be accompanied by duplication

14
Types of Transposable Genetic Elements

• Insertion sequences (IS)
• Definition Elements that carry no other genes
  except those involved in transposition
• Nomenclature - IS1
• Structure

• Importance
• Mutation
• Plasmid insertion
• Phase variation

The known insertion sequences vary in length from
approximately 780 to 1500 nucleotide pairs, have
short (15-25 base pair) inverted repeats at their
ends, and are not closely related to each other.
15
Phase Variation in Salmonella H Antigens
IS
16
Types of Transposable Genetic Elements

• Transposons (Tn)
• Definition Elements that carry other genes
  except those involved in transposition
• Nomenclature - Tn10
• Transposons can move from one site in a DNA
  molecule to other target sites in the same or a
  different DNA molecule.
• Structure

Transposons are not self-replicating genetic
elements, however, and they must integrate into
other replicons to be maintained stably in
bacterial genomes
17
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. The entire complex element can transpose
as a unit.
18

• Importance
• they cause mutations,
• mediate genomic rearrangements,
• function as portable regions of genetic
  homology, and acquire new genes,
• contribute to their dissemination within
  bacterial populations.
• 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.

19
In medically important bacteria, genes that
determine production of adherence antigens,
toxins, or other virulence factors, or specify
resistance to one or more antibiotics, are often
located in complex transposons. Well-known
examples of complex transposons are Tn5 and Tn10,
which determine resistance to kanamycin and
tetracycline, respectively.
20
Mutation is a stable, heritable change in the
genomic nucleotide sequence
21
How do mutations occur?

• Spontaneous mutations - Arise occasionally in all
  cells are often the result of errors in DNA
  replication (random changes)
• Frequency of naturally occurring (spontaneous)
  mutation varies from 10-6 to 10-9 (avg 10-8)
• This means that if a bacterial population
  increases from 108 to 2 x 108, on the average,
  one mutant will be produced for the gene in
  question.
• Induced mutations - Arise under an
  influence of some factors
• Errors in replication which cause point
  mutations
• other errors can lead to frameshifts
• Point mutation - mismatch substitution of one
  nucleotide base pair for another
• Frameshift mutation - arise from accidental
  insertion or deletion within coding region of
  gene, results in the synthesis of nonfunctional
  protein

22
Types of Mutations

• Point mutation affects only 1 bp at a single
  location
• Silent mutation a point mutation that has no
  visible effect because of code degeneracy

23
Types of Mutations

• Missense mutation a single base substitution in
  the DNA that changes a codon from one amino acid
  to another

24
Types of Mutations

• Nonsense mutation converts a sense codon to a
  nonsense or stop codon, results in shortened
  polypeptide

25
Types of Mutations

• Frameshift mutation arise from accidental
  insertion or deletion within coding region of
  gene, results in the synthesis of nonfunctional
  protein

Insertion
26
Frameshift mutation - Deletion
27
Other Types of Mutations

• Forward mutation a mutation that alters
  phenotype from wild type
• Reverse mutation a second mutation which may
  reverse wild phenotype and genotype (in same
  gene)
• Suppressor mutation a mutation that alters
  forward mutation, reverse wild phenotype (in same
  gene - intragenic, in another gene - extragenic)

28
Mutations affect bacterial cell phenotype

• Morphological mutations-result in changes in
  colony or cell morphology
• Lethal mutations - result in death of the
  organism
• Conditional mutations - are expressed only under
  certain environmental conditions
• Biochemical mutations - result in changes in the
  metabolic capabilities of a cell
• 1) Auxotrophs - cannot grow on minimal media
  because they have lost a biosynthetic capability
  require supplements
• 2) Prototrophs - wild type growth characteristics
• Resistance mutations-result in acquired
  resistance to some pathogen, chemical, or
  antibiotic

29

• Induced mutations-caused by mutagens
• Mutagens Molecules or chemicals that damage DNA
  or alter its chemistry and pairing
  characteristics
• Base analogs are incorporated into DNA during
  replication, cause mispairing
• Modification of base structure (e.g., alkylating
  agents)
• Intercalating agents insert into and distort the
  DNA, induce insertions/deletions that can lead to
  frameshifts
• DNA damage so that it cannot act as a replication
  template (e.g., UV radiation, ionizing radiation,
  some carcinogens)

30
N. meningitidis genes with high mutation rates
include those involved in
capsule biosynthesis LPS biosynthesis attaching
to host cells taking up iron
31
(No Transcript)
32
(No Transcript)
33
(No Transcript)
34
Mutant Detection

• In order to study microbial mutants, one must be
  able to detect them and isolate them from the
  wild-type organisms
• Visual observation of changes in colony
  characteristics
• Mutant selection - achieved by finding the
  environmental condition in which the mutant will
  grow but the wild type will not (useful for
  isolating rare mutations)
• Screen for auxotrophic mutants A lysine
  auxotroph will only grow on media that is
  supplemented with lysine

35
Mutant Detection
Mutants are generated by treating a culture of E.
coli with a mutagen such as nitrosoguanidine The
culture will contain a mixture of wild-type and
auxotrophic bacteria
Out of this population we want to select for a
Lysine auxotrophic mutant
36
Isolation of a Lysine Auxotroph
37
Reparation Light-requiring Dark SOS-
reactivation
38
Light-requiring Reparation
39
Dark Reparation
40
Exchange of Genetic Information Recombination
41
Transformation
42
Transformation

• Definition Gene transfer resulting from the
  uptake of DNA from a donor.
• Factors affecting transformation
• DNA size and state (DNA molecules must be at
  least 500 nucleotides in length)
• Sensitive to nucleases (deoxyribonuclease)
• Competence of the recipient (Bacillus,
  Haemophilus, Neisseria, Streptococcus)
• Competence factor
• Induced competence

43
Transformation

• Steps
• Uptake of DNA
• Gram
• Gram -

• Recombination
• Legitimate, homologous or general
• recA, recB and recC genes

• Significance
• Phase variation in Neiseseria
• Recombinant DNA technology

44
S strain
R strain
Competent cell
S strain
45
Transduction

• Definition Gene transfer from a donor to a
  recipient by way of a bacteriophage

46
Phage Composition and Structure

• Composition
• Nucleic acid
• Genome size
• Modified bases
• Protein
• Protection
• Infection

• Structure (T4)
• Size
• Head or capsid
• Tail

47
Transduction

• Types of transduction
• Generalized - Transduction in which potentially
  any donor bacterial gene can be transferred

48
Generalized Transduction

• Infection of Donor

• Phage replication and degradation of host DNA

• Assembly of phages particles

• Release of phage

• Infection of recipient

• Legitimate recombination

49
Transduction

• Types of transduction
• Specialized - Transduction in which only certain
  donor genes can be transferred

50
Specialized TransductionLysogenic Phage

• Excision of the prophage

• Replication and release of phage
• Infection of the recipient
• Lysogenization of the recipient
• Legitimate recombination also possible

51
Transduction

• Types of transduction
• Abortive transduction refers to the transient
  expression of one or more donor genes without
  formation of recombinant progeny, whereas
  complete transduction is characterized by
  production of stable recombinants that inherit
  donor genes and retain the ability to express
  them.
• In abortive transduction the donor DNA fragment
  does not replicate, and among the progeny of the
  original transductant only one bacterium contains
  the donor DNA fragment. In all other progeny the
  donor gene products become progressively diluted
  after each generation of bacterial growth until
  the donor phenotype can no longer be expressed.

52
Transduction

• Significance
• Common in Gram bacteria
• Lysogenic (phage) conversion

53
Bacterial Conjugation

• Definition The transfer of genetic information
  via direct cell-cell contact
• This process is mediated by fertility factors (F
  factor) on F plasmids

54
In conjugation, direct contact between the donor
and recipient bacteria leads to establishment of
a cytoplasmic bridge between them and transfer of
part or all of the donor genome to the recipient.
Donor ability is determined by specific
conjugative plasmids called fertility plasmids or
sex plasmids.
55
The F plasmid (also called F factor) of E coli is
the prototype for fertility plasmids in
Gram-negative bacteria. Strains of E coli with an
extrachromosomal F plasmid are called F and
function as donors, whereas strains that lack the
F plasmid are F- and behave as recipients.
56
Conjugation

• Gene transfer from a donor to a recipient by
  direct physical contact between cells
• Mating types in bacteria
• Donor
• F factor (Fertility factor)
• F (sex) pilus

• Recipient
• Lacks an F factor

57
Physiological States of F Factor

• Autonomous (F)
• Characteristics of F x F- crosses
• F- becomes F while F remains F
• Low transfer of donor chromosomal genes

58
Physiological States of F Factor

• Integrated (Hfr)
• Characteristics of Hfr x F- crosses
• F- rarely becomes Hfr while Hfr remains Hfr
• High transfer of certain donor chromosomal genes

59
Physiological States of F Factor

• Autonomous with donor genes (F')
• Characteristics of F x F- crosses
• F- becomes F while F remains F
• High transfer of donor genes on F and low
  transfer of other donor chromosomal genes

60
Mechanism of F x F- Crosses

• Pair formation
• Conjugation bridge

• DNA transfer
• Origin of transfer
• Rolling circle replication

61
Mechanism of Hfr x F- Crosses

• Pair formation
• Conjugation bridge

• DNA transfer
• Origin of transfer
• Rolling circle replication
• Homologous recombination

62
Mechanism of F' x F- Crosses

• Pair formation
• Conjugation bridge

• DNA transfer
• Origin of transfer
• Rolling circle replication

63
Conjugation

• Significance
• Gram - bacteria
• Antibiotic resistance
• Rapid spread
• Gram bacteria
• Production of adhesive material by donor cells

64
Map of chromosome
65
Recombination DNA and Gene Cloning
Gene cloning is the process of incorporating
foreign genes into hybrid DNA replicons. Cloned
genes can be expressed in appropriate host cells,
and the phenotypes that they determine can be
analyzed. Some key concepts underlying
representative methods are summarized here.
66
Bacterial plasmids in gene cloning
67
Steps for eukaryotic gene cloning

• Isolation of cloning vector (bacterial plasmid)
  gene-source DNA (gene of interest)
• Insertion of gene-source DNA into the cloning
  vector using the same restriction enzyme bind
  the fragmented DNA with DNA ligase
• Introduction of cloning vector into cells
  (transformation by bacterial cells)
• Cloning of cells (and foreign genes)
• Identification of cell clones carrying the gene
  of interest

68
DNA Cloning

• Restriction enzymes (endonucleases) in nature,
  these enzymes protect bacteria from intruding
  DNA they cut up the DNA (restriction) very
  specific
• Restriction site recognition sequence for a
  particular restriction enzyme
• Restriction fragments segments of DNA cut by
  restriction enzymes in a reproducable way
• Sticky end short extensions of restriction
  fragments
• DNA ligase enzyme that can join the sticky
  ends of DNA fragments
• Cloning vector DNA molecule that can carry
  foreign DNA into a cell and replicate there
  (usually bacterial plasmids)

69
Restriction endonucleases
70
Practical DNA Technology Uses

• Diagnosis of disease
• Human gene therapy
• Pharmaceutical products (vaccines)
• Forensics
• Animal husbandry (transgenic organisms)
• Genetic engineering in plants
• Ethical concerns?

71
GENES THERAPY
72
Biotechnology practical use