Chair of Microbiology, Virology, and Immunology GENETICS OF BACTERIA AND VIRUSES BASES OF...
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Chair of Microbiology, Virology, and Immunology
GENETICS OF BACTERIA AND VIRUSES BASES OF BIOTECHNOLOGY
AND GENE ENGENEERING
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Lectures schedule
1. Structure of bacterial genome.
2. Extrachromosomal elements.
3. Mutations.
4. Recombinations.
5. Gene engineering.
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F. Crick i J. Watson – described DNA
structure
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DNA structure
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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
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E. coli DNA
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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.
Plasmid
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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)
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Phenotypic effects
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Structure of R factors
• RTF– Conjugative
plasmid– Transfer genes
Tn 9
Tn
21
Tn 10
Tn 8
RTF
R determinant
• R determinant– Resistance
genes– Transposons
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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
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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
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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
TransposaseABCDEFG GFEDCBA
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.
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Phase Variation in Salmonella H Antigens
ISH1 gene H2 gene
H1 flagella
H2 flagella
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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
IS ISResistance Gene(s)
IS ISResistance Gene(s)
Transposons are not self-replicating genetic elements, however, and they must integrate into other replicons to be maintained stably in bacterial genomes
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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.
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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.
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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.
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Mutation is a stable, heritable change in the genomic nucleotide sequence
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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
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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
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Types of MutationsMissense mutation: a single base
substitution in the DNA that changes a codon from one amino acid to another
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Types of MutationsNonsense mutation: converts a sense
codon to a nonsense or stop codon, results in shortened polypeptide
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Types of Mutations• Frameshift mutation: arise from accidental insertion or deletion
within coding region of gene, results in the synthesis of nonfunctional protein
Insertion
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Frameshift mutation - Deletion
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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)
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• 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
Mutations affect bacterial cell phenotype
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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)
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N. meningitidis genes with high mutation rates include those
involved in:
capsule biosynthesis
LPS biosynthesis
attaching to host cells
taking up iron
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Examples of mutagensCHEMICAL
AGENTACTION
HNO2
Nitrogen mustard NTG
React chemically with one or more bases so that they pair improperly
Intercalating agents (acridine dyes)
Insert into DNA and cause frame-shift mutations by inducing an addition or the subtraction of a base
Base analogs:
5-bromouracil
2-amino purine
Incorporate into DNA and cause mispairing
Analog of T which can pair with C
Analog of A which can pair with C
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Examples of mutagensPHYSICAL
AGENTACTION
UV irradiation Causes formation of adjacent T-T dimers that distorts the DNA backbone, altering the binding properties of bases near the dimer
X-ray Alters bases chemically, causes deletions and induces breaks in DNA chain
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Examples of mutagensBIOLOGICAL
AGENTACTION
Insertion sequences (IS)
Pieces of DNA about a thousand nucleotide bases in length which can insert into a genetic sequence
Transposons genetic elements goverened by IS which can insert into the chromosome within a gene
Viruses Some bacteriophage (e.g. phage µ) can integrate their DNA into random positions in
the bacterial chromosome
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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
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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
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minus lysinecomplete
Lysine auxotrophsdo not grow
All strains grow
Isolation of a Lysine Auxotroph
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Reparation
Light-requiring
Dark
SOS- reactivation
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Light-requiring Reparation
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Dark Reparation
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Exchange of Genetic Information
Recombination
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Transformation
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TransformationDefinition: 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
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Transformation
– Recombination• Legitimate,
homologous or general
• recA, recB and recC genes
• Significance– Phase variation in Neiseseria– Recombinant DNA technology
• Steps
– Uptake of DNA
• Gram +
• Gram -
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Competent cell
S strain
R strain S strain
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Transduction
• Definition: Gene transfer from a donor to a recipient by way of a bacteriophage
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Phage Composition and Structure
• Composition– Nucleic acid
• Genome size
• Modified bases
– Protein• Protection• Infection
• Structure (T4)
– Size– Head or capsid– Tail
Tail
Tail Fibers
Base Plate
Head/Capsid
Contractile Sheath
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Transduction
Types of transduction– Generalized - Transduction in which
potentially any donor bacterial gene can be transferred
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Generalized Transduction
• Release of phage
• Phage replication and degradation of host DNA
• Assembly of phages particles
• Infection of recipient• Legitimate recombination
• Infection of Donor
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Transduction
Types of transduction
–Specialized - Transduction in which only certain donor genes can be transferred
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Specialized TransductionLysogenic Phage
• Excision of the prophage
gal
bio
gal bio
gal bio
gal
bio
bio
gal
• Replication and release of phage
• Infection of the recipient
• Lysogenization of the recipient– Legitimate
recombination also possible
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TransductionTypes 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.
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Transduction
• Significance– Common in Gram+ bacteria– Lysogenic (phage) conversion
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Bacterial ConjugationDefinition: The transfer of genetic
information via direct cell-cell contact
• This process is mediated by fertility factors (F factor) on F plasmids
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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.
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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.
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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
Donor
Recipient
– Recipient• Lacks an F factor
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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 F+
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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
F+ Hfr
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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
Hfr F’
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Mechanism of F+ x F- Crosses
• DNA transfer– Origin of
transfer– Rolling
circle replication
• Pair formation
– Conjugation bridge
F+ F- F+ F-
F+ F+F+ F+
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Mechanism of Hfr x F- Crosses
• DNA transfer– Origin of
transfer
– Rolling circle replication
• Homologous recombination
• Pair formation
– Conjugation bridge
Hfr F- Hfr F-
Hfr F-Hfr F-
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Mechanism of F′ x F- Crosses
• DNA transfer– Origin of
transfer
– Rolling circle replication
• Pair formation
– Conjugation bridge
F’ F’F’ F’
F’ F- F’ F-
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Conjugation
• Significance– Gram - bacteria
• Antibiotic resistance
• Rapid spread
– Gram + bacteria• Production of adhesive material by donor cells
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Map of chromosome
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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.
Recombination DNA and Gene Cloning
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Bacterial plasmids in gene cloning
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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
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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)
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Restriction endonucleases
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Practical DNA Technology Uses
• Diagnosis of disease
• Human gene therapy
• Pharmaceutical products (vaccines)
• Forensics
• Animal husbandry (transgenic organisms)
• Genetic engineering in plants
• Ethical concerns?
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GENES THERAPY
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Biotechnology practical use