Dmp_712 Bact Exam_1 Bacterial Genetics Section 4 Robertson

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Importance of DNA (gene) Transfer in Veterinary Medicine (4)

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1) Development of antibiotic resistance
2) Changes in pathogenicity/virulence
-loss or gain of surface antigens and macromolecules involved in adherence
-loss or gain of toxin genes that damage host
3) Changes in biochemical reactions used to identify pathogens in clinical labs
4) Limit effectiveness of vaccines due to appearance of new antigens

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Properties of Bacterial Chromosome
Length:
# of Genes:
Non growing cells:

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Length: 100X that of cel, supercoiled 19-20loops to fit in cell
# of Genes: 2,000-4,000 (basic unit of heredity)
Non growing cells: haploid (one DNA copy per cell) - 2 copies just before division

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Genotype

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genetic composition of the cell

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Phenotype

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expressed traits

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Mutations in laboratories can change:

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genotype and phenotype

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Mutation types (3)

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1) Spontaneous
2) Induced (chemical and UV light)
3) Host selective pressures - immune response
-change in antigenic types of surface macromolecules
-acquired gene for protein exotoxin

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Mechanisms of mutations (3)

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1) point mutation
2) deletion
3) insertion

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Point mutation
defn:
types (3)

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defn: base pair substutition
examples:
UUG-->UUA - both code for leucine (silent)
UUG-->UAG - stop signal (chain termination)
UUG --> AUG - misennse (may be no problem)

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Frameshifts

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Result from deletions and insertions

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Deletion

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may be single base or 100's of bases

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Insertion, example

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Insertion sequences (transposons)
-transposable genetic elements - "jumping genes"

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Extrachromosomal Genetic Elements (3)

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1) Plasmids
2) Jumping genes
3) Bacteriophage

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Plasmids
defn:
location:
size:
Copy number:
replication:

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defn: double stranded circular DNA that forms supercoiled structure
location: in cell outside of chromosome
size: << than host chromosome (2Kb - 200Kb)
Copy number: per cell varies 1 to >100
replication: mechanism like chromosomal DNA but independent

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episome

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plasmid integrated in host chromosome (eg. E. Coli F Plasmid)

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Plasmid importance (2 examples)

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Do not encode for essential genes, but can provide a selective advantage to host cell
1) antibiotic resistance (R plasmids)
2) transfer virulence factors
- surface proteins that facilitate adherence (adhesins)
- protein exotoxins that cause host damage

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Plasmid, properties encoded by (3)

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1) mating gene that encodes sex pilus
2) chemical resistance (heavy metals like Hg++ and Cd++)
3) Capacity to degrade environmental contaminants

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Plasmids involved in conjugation in two ways

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1) transfer of ssDNA from donor to recipient cell via sex pilus
2) Pilus mediates cell to cell contact (absolute requirement for transfer)

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Insertion Sequences
contain:
encodes:
cause:

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contain: gene that encodes for enzyme called a transposase - recognizes, cuts and ligates DNA
Encodes identical terminal repeats (insertion sequences) at each end (20bp to >1000 bp latter being more common)
-sequences of DNA that become integrated at specific sites on genome
- complementary to sequence in target DNA
Cause mutation by insertion into genes causing a non-functional protein

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Transposons
size:
encode:(3)
designated:
move by:
Mechanism:

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Size: larger than IS
Encode for transposase
Encode for terminal repeat sequences on each end
Encode for one or more additional genes such as antibiotic resistance
Designated by Tn plus a nber
Move by a "cut and paste" process
Some transposons move from one location to another, but some make a new copy and insert at another location

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How Transposons Move (3)

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1) Transposons bind to inverted repeats
2) Target DNA is cut in an offset manner
3) Some transposons require a specific sequence for insertion while others are random

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Bacteriophage
defn:
consist of:
structures:
Nucleic acid:
Types (2):

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defn: viruses that infect bacteria
consist of: nucleic acid and protein
structures: unique head and tail structures for different phages
Nucleic acid: packaged/stuffed into head
Types (2):
- Temperate - phage DNA inserted in bacterial host chromosome by process called lysogeny
- Lytic - phage replicates and causes lysis

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Virion

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complete virus particle (nucleic acid surrounded by protein coat)

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Nucleocapsid

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complex of nucleic acid and proteins

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Termperate bacteriophage

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viral genome replicates as part of host chromosome in state called lysogeny

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Phrophage/provirus -

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bacteriophage genome integrated into host chromosome

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Lysogenic bacteriophage

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Viral genome integrated into host chromosome
- can be inducted to cause lytic infection (usually the same as prophage)

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Lytic bacteriophage

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viral replication after infection causes death and lysis of host cell

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Lytic Bacteriophage Life Cycle (4)

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1) Attachment of virion to susceptible cell
2) injection of viral nucleic acid
3) Replication of viral components
4) Cell lysis and release of viral particles

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Replication of viral components (5)

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1) Replication of virus nucleic acid
2) Synthesis of viral=specific enzymes
3) Modification of host biosynthetic machinery
4) Synthesis of virus coat protein (capsid)
5) Assembly of new virus particles

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Bacteriophage
Components designed to:
Lysogenicphages can be:

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Components designed to: deliver nucleic acid into bacterial host
Lysogenic phages can be: induced and become lytic

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Phage Biotyping of Clinical Isolates

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Bacteriophages can be used to characterize outbreaks due to certain bacteria

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Phage Biotyping steps (4)

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1) three host-specificity groups (I, II, III) observed with standardized set of phages
2) All phages bind to the same surface receptor-differences in later events
3) Certain biotypes are associated with particular disease states
4) Specificity often associated with hospital outbreaks - trace their spread and assess prevalence

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Detection of Bacteriophage
Plaques:
Phage typing:

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Plaques: clear areas in a "lawn" of bacteria due to lysis resulting from growth of the phage.
Phage typing: Plaque formation patterns used to determine the "phage type" of clinical isolates of certain bacteria

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Homologous Recombination
defn:
crossing over:
Mediates:

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Physical exchange of genes from 2 different sources
-genes are similar enough for segments to interact and form base pairs - known as "crossing over" in classical genetics
- mediates the integration of donor DNA into recipient DNA in all three gene transfer mechanisms in bacteria (transformation, conjugation, and transduction)

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Molecular Events in Homologous Recombination (3)

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1) DNA is taken up by competent bacteria
- can be ds or ss - depends on organism
- if ds DNA is internalized - converted to ssDNA by an endonuclease
2) Events after DNA is taken up and converted to ssDNA
- single stranded binding (SSB) protein binds to (and stabilizes ssDNA)
- recA protein binds to SSB protein - DNA complex
- All 3 components bind to recipient ds DNA
= recA facilitates annealing of ss donor DNAto ds recipient DNA and displaces one of the recipient DNA strands - known as strand invasion
= donor and recipient strands exchange DNA (crossover event)
3) Donor and recipient strands are linked by enzymes involved in repair of DNA

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Transformation

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Mechanism of Gene Transfer in bacteria
- by uptake of "naked" DNA fragments

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Conjugation (2)

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Mechanism of Gene Transfer in bacteria
- cell to cell contact required
- donor genes transferred by plasmids

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Transduction

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Mechanism of Gene Transfer in bacteria
- DNA/genes carried by bacteriophage from donor to recipient cell

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Transformation mechanism
DNA binds:
Fragment size:

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"naked"/free DNA is taken up by competent recipient cells
-DNA binds to specific receptors on cell surfaces
-DNA fragments of 10,000 nts can be taken up
-Gram-negative bacteria take up ds DNA, whereas Gram-positive bacteria take up ssDNA
***ssDonor DNA is integrated by homologous recombination

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competence
defn
when?

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-the ability of recipient cells to take up "naked"/free DNA
-observed only at certain times in growth cycle

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Smooth and Rough colonies
based on:

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surface texture, size

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Smooth colonies
description:
due to:
virulence:

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smooth, glistening, moist appearance, "buttery," viscous
-due to polysaccharide (capsule or LPS)
-more virulent( capable of causing disease)

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Rough colonies
description:
due to:
virulence:

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-dry, wrinkled (rugose,) rough appearance, brittle, friable
-due to loss of either capsule or O-antigen
-less virulent or avirulent (non-pathogenic)

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Experiments by F. Griffith in 1920s showed:
What cells, and what results (3)

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First documented example of gene transfer in bacteria
1) Heat-killed S --> mice live
2) Live R --> mice live
3) Mix --> mice die
- only sooth cells isolated from mice
- lysis of smooth cells released DNA

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expansion by Avery, MacLeod and McCarty (1030's) showed (3)

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1) Transformation can be done in test tube
2) Cell free extract of S cells induced transformation
3) Injected a mixture (cell free extract of S cells and live R cells) into mice --> mice died
- isolated bacteria - S type
- active material purified + shown to be DNA

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Griffith's transformation experiment showed:

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Type of capsule not as important as the presence or absence of capsule

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Conjugation
mechanism:
requires:
what is transferred:
efficient?

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- Plasmid-encoded mechanism
- Requires cell to cell contact
- One strand of plasmid DNA is transferred to recipient via pilus
- Plasmid DNA transfer is very efficient

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F plasmid
defn
forms (2)

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Fertility plasmid
-extrachromosomal - self replicating plasmid
-episome - integrated into host cell chromosome

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Changes in cell due to F plasmid (3)

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-capacity to synthesize the F pilus
-when contact is made between donor and recipient cells, donor DNa is mobilized for transfer
-recipient cell can no longer bind pilus - due to changes in surface receptor

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Conjugation can transfer (2)

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Plasmid
Chromosome transfer

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F plasmid placed with cells w/o F
F+ x F- -->

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F+ x F- --> F+
All F+

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High frequency recombination (Hfr)

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F plasmid incorporated into the host chromosome

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Hfr x F- -->
what occurs?
why?

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F- + Donor DNA
F plasmid DNA is transferred last (90 min for complete transfer)

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Transduction 2 types

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Specialized
Generalized

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Specialized Transduction (3)

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1) only a few host genes are transferred
2) same set of genes each time
3) temperate phage integrate into host chromosome at specific site

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Generalized Transduction

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almost any gene can be transferred from donor to recipient

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Transduction significance

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increases virulence of pathogens or converts avirulent cell to virulent cell

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Specialized Transduction
what is it?
what normally happens?
what rarely happens?

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-induction of temperate phage - lambda phage
-usually viral DNA separates from host DNA by reverse of integration
=lambda phage is excised as a complete unit
=result is normal phage and cell lysis
-rare event - phage genome is excised incorrectly - some adjacent bacterial gens are excised along with lambda DNA
=Enzymes involved in utilization of glaactose (lamda dgal)
=amount of DNA i phage head is fixed - if gal genes are included, the phage will be defective (needshelper phage for replication)

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Gnerealized Transduction
what is it?
how does it happen?

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-infection of bacteria with lytic phage
-infection proceeds normally until timethat phage genome is "stuffed" into capsid
=host DNA is packaged by accident into phage capsids - transducing particle
=phage containing host DNA is defective, but can infect new host
=recombination of donor DNA from transducing particle yields a new genotype