Bacterial Genetics

Chapter 8

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

Figure 19-15

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

The Discovery of Transformation: Griffith’s experiment with pneumococcal infections in mice

Griffith Experiment

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

Transformation

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

Figure 18-01

Relative size of Phage Bacteria

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+

Conjugation

During conjugation, the plasmid is replicated in the donor cell and is transferred to the recipient After plasmid is

transferred, F- cell becomes F+

Resistance plasmids

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