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Diversity in Bacteria
Bacteria use three different mechanism to adapt to changing environments Mutation Gene transfer Regulation of gene expression
Spontaneous Mutation
Spontaneous mutations occur in natural environment Occur infrequently and randomly
Rate of mutation probability that a mutation will be observed in
a given gene each time the cell divides Rate is generally between 1 in 10,000 and 1 in
a trillion Low rate is due to cellular repair mechanisms
Mutations are stable heritable changes in the base sequence of DNA
Can occur from Base substitutions Removal or addition of nucleotides Transposable elements
Spontaneous Mutation
Spontaneous Mutation
Base substitutions Most common type of mutation Results from mistakes during DNA
synthesis Incorrect base is incorporated into
DNA Point mutations
Occur when one base pair is changes
Missense mutation Mutation resulting from amino
acid substitution is called Nonsense mutation
Mutation that changes a amino acid codon to a stop codon
Spontaneous Mutation
Removal and addition of nucleotides Shifts the translational
reading frame Shifts the codons
Mutation called frameshift mutation
Affects all amino acids downstream from addition or deletion
Mutations frequently result in premature stop codons
Effect of Deletion, Addition, Sub.
THEREDFOXATETHETANRAT (Normal) THE RED FOX ATE THE TAN RAT
THEEDFOXATETHETANRAT (Deletion) THE EDF OXA TET HET ANR AT
THERREDFOXATETHETANRAT (Addition) THE RRE DFO XAT ETH ETA NRA T
THEYEDFOXATETHETANRAT (Substitution) THE YED FOX ATE THE TAN RAT
Transposable elements Special segments of DNA that move
spontaneously from gene to gene Elements called transposons
Transposons disrupt proper function of gene Gene or gene product generally non functional
Spontaneous Mutation
DNA of genomeTransposon
is copied
Mobile transposon
Transposon
Insertion
New copy oftransposon
Transposon movement (“copy-and-paste” mechanism)
Retrotransposon movement
DNA of genome
Insertion
RNA
Reversetranscriptase
RetrotransposonNew copy of
retrotransposon
Induced Mutations
Mutations are essential for understanding genetics Mutations can be intentionally produced to
demonstrate function of particular gene or set of genes These mutations are termed induced
Mutations can be induced via Chemical mutagens Transposition Radiation
Chemical mutagens Nitrous acid
Converts amino group to a keto group Changes cytosine to uracil
Uracil binds to adenine while cytosine binds to guanine
Alkylating agents Largest group of chemical mutagens
Alters hydrogen bonding of bases Nitrosoguanine is common alkylating agent
Induced Mutations
Induced Mutations Chemical mutagens
Base analogs Chemicals that are
structurally similar to the nitrogenous bases but have slightly altered base pairing properties
Base analogs include: 2-aminopurine which
incorporates in the place of adenine but binds with cytosine
5-bromouracil which incorporates in the place of thymine but binds with guanine
Induced Mutations
Chemical mutagens Intercalating agents
Molecules that insert themselves between adjacent bases
Creates space between bases Extra base is often added to fill space
Ethidium bromide is common intercalating agent Potential carcinogen
Transposition Common procedure used to induce mutation
in laboratory Gene that receives transposon will undergo a
knockout mutation Termed insertion mutation
Induced Mutations
Induced Mutations
Radiation Two types
Ultraviolet light Causes covalent bonding between
adjacent thymine bases Forms thymine dimers which
distorts DNA X rays
Cause breaks and alterations in DNA
Breaks that occur on both strand are often lethal
Repair of Damaged DNA
Repair of base substitution Cells develop two methods of
repair Proofreading
DNA polymerase has proofreading function
Able to excise incorrect base and replace with correct one
Mismatch repair Endonuclease enzyme
removes short stretch of nucleotide
DNA polymerase fills gap DNA ligase joins ends
Mismatch repair
Repair of Damaged DNA Repair of thymine dimers
Two mechanisms Light repair
A.k.a photoreactivation Enzyme uses visible light to break covalent bonds between bases
Dark repair A.k.a excision repair Endonuclease excises damages section New section replicated and joined to original strand
Thymine dimer repair
Repair of Damaged DNA
Repair of modified bases Enzyme cuts DNA backbone and removes
base DNA polymerase incorporates new base
SOS repair Last ditch effort to bypass damage Damage induces SOS system
Produces new DNA polymerase Highly error prone
Mutations can arise from synthesis with new enzyme
Mutations and Their Consequences Mutation provide organism way to respond to
changing environments Environment selects for cells suited to survive
Environment does not cause mutation
Mutation Selection
Major problem in induced mutation is identifying bacteria with desired mutation
Techniques used include Direct selection
Involves inoculating population of bacteria on medium on which only mutants will grow
Used to select antimicrobial resistant organisms
Indirect selection Required to isolate organisms that
require growth factor that parent strain does not
Replica plating
Replica Plating
Mutation Selection
Testing for cancer causing chemicals Many mutagens are also
carcinogens Cancer causing agents
Microbes used to test potential carcinogenic activity
Tests are based on effect chemical has on microbial DNA
Ames test common chemical carcinogen test
Test rate of reversion of Salmonella auxotroph
Also test potential lethality
Mechanisms of Gene Transfer
Genes are naturally transferred between bacteria using three mechanisms DNA-mediated transformation Transduction Conjugation
Movie
Gene exchange in bacteria Two event occur in gene exchange
1. Donor DNA is transferred and accepted by the recipient cell
Donor DNA is transferred one of three ways Transformation Transduction Conjugation
2. Donor DNA is integrated onto the recipient cell’s chromosome
Mechanisms of Gene Transfer
DNA-mediated transformation Definition: the transfer of naked DNA from
one bacterium to another Discovered by Fredrick Griffith in 1928 while
working with Streptococcus pneumoniae
Mechanisms of Gene Transfer
Griffith realized S. pneumonic existed in two forms Encapsulated, virulent form
Smooth in appearance Nonencapsulated, avirulent form
Rough in appearance Griffith wondered if injections with the smooth strain
could be used as a vaccine against pneumonia He injected mice with the different strains and recorded
his results
Mechanisms of Gene Transfer
DNA-mediated transformation Involves the transfer of naked DNA from the
environment to the recipient cell Cells rupture during the stationary and death phase
The chromosome breaks into small pieces and explode through the ruptured cell wall
Recipient cell picks up piece of the naked DNA The naked DNA is integrated onto the recipient
chromosome Replaces the homologous gene on the chromosome
of the recipient cell
Mechanisms of Gene Transfer
DNA-mediated transformation Natural transformation occurs when bacterial
cells are “competent” Competence is a condition in which bacterial cells
are capable of taking up and integrating larger fragments of DNA
Competence occurs during the late log, early stationary phase
Mechanisms of Gene Transfer
Natural transformation occurs in four stages Entry of the DNA
Only single strands enter, double strands are degraded
Integration of the donor DNA Donor DNA is integrated by hydrogen
bonding Enzymes cleave recipient DNA Donor DNA is put in place
Mismatch repair Repair mechanism remove either donor
or recipient DNA that doesn’t match Repairs with correct nucleotides
Cell multiplication Transformed cells multiply under
selective conditions in which non-transformed cell will not grow
Transduction Bacterial DNA that is transferred from donor to
recipient via a bacterial virus (bacteriophage) Two types of transduction
Generalized Any gene of donor can be transferred
Specialized Only specific genes can be transferred
Mechanisms of Gene Transfer
Transduction is a mis-packaging of DNA during viral replication The mis-packaged phage
infects a new bacterial cell and insert the donor DNA into the recipient cell
The donor DNA is integrated and mismatched pairs are repaired
Conjugation – only form of gene exchange in which donor survives Conjugation is mediated by a plasmid
Plasmid is self replicating extrachromosomal piece of DNA
Can code for traits that give bacteria advantage Conjugation requires direct contact between cells Cells must be of opposite mating types
Donor cells carry a plasmid that codes for fertility factor or “F factor”
This cell is designated F+
Recipient cell does not carry a plasmid This cell is designated F-
Mechanisms of Gene Transfer
LE 18-15
Mutantstrainarg+ trp–
Mutantstrainarg+ trp–
Mixture
Mixture
Nocolonies(control)
Nocolonies(control)
Coloniesgrew
Mutantstrainarg– trp+
Mutantstrainarg– trp+
During conjugation, the plasmid is replicated in the donor cell and is transferred to the recipient After plasmid is
transferred, F- cell becomes F+
In some F+ cells, the F factor integrates onto the host chromosome Converts F+ to Hfr
Hfr= High frequency of recombination
Conjugation between Hfr and F- cell results in only a portion of the F factor being replicated and transferred
F- remain F-
F- has new information but may not have the F factor gene
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