Bacterial Genetics

OverviewTwo general mechanisms of genetic change in bacteria:

Mutation - alteration in existing DNA sequence

DNA transfer - acquisition of DNA from another source

SpontaneousInduced (caused by mutagens)





Why study bacterial genetics?Model system

•Spontaneous mutations occur in all cells at a very low frequency (≈one per billion nucleotides)

•Bacteria quickly grow to high concentrations (109/ml) in culture, making it possible to study rare occurrences

•Test chemicals for potential carcinogens

Understand bacterial adaptation•Resistance to antimicrobial drugs

Agency Urges Change in Antibiotics for Gonorrhea

By LAWRENCE K. ALTMANPublished: NY Times April 13, 2007

The rates of drug-resistant gonorrhea in the United States have increased so greatly in the last five years that doctors should now treat the infection with a different class of antibiotics, the last line of defense for the sexually transmitted disease, officials said yesterday…..

No new antibiotics for gonorrhea are in the pipeline, officials of the centers told reporters by telephone.

“Now we are down to one class of drugs,” said Dr. Gail Bolan, an expert in sexually transmitted diseases at the California Department of Health Services. “That’s a very perilous situation to be in.”





Why study bacterial genetics?Model system

•Spontaneous mutations occur in all cells at a very low frequency (≈one per billion nucleotides)

•Bacteria quickly grow to high concentrations (109/ml) in culture, making it possible to study rare occurrences

•Some mutagens are carcinogens

Understand bacterial adaptation•Resistance to antimicrobial drugs•Acquisition of disease-causing traits

Terms

PhenotypeGenotype

Terms

Phenotype - the observable characteristics of an organism

Genotype - the sequence of nucleotides in the DNA of an organism

Prototroph His- auxotrophdisrupt gene required for histidine synthesis

Wild type - characteristics similar to the organism as it occurs in nature. Prototroph - requires the same nutrients as the wild type.

Auxotroph - a strain that has lost the ability to synthesize a specific compound; as a consequence, that compound must be supplied as a nutrient in the growth medium.

When studying mutations, you only see what you look for

Part I Mutation

•How we can select (and therefore, study) mutants

•How cells can repair errors/damage

•How mutations occur, and their consequences

Spontaneous Mutation

Mistakes during replicationBase substitution

TGGtryptophan

TGCcysteine

TGAStop codon

TGTcysteine

No consequence Consequence varies Truncated protein; generally non-functional

Silent mutation Missense mutation Nonsense mutation


Mistakes during replicationBase substitutionRemoval or addition of nucleotides

TGTTTGACCTAGGT



TGTTTGACCTAGGT

TGTTGACCTAGGT

TGT TTG ACC TAG GT

TGT TGA CCT AGG T

Frameshift mutation•Generates an entirely different set of triplets•Often, a stop codon is generated


Transposons

• Insertional inactivation of the gene in which the transposon lands• A transposon can insert elsewhere in the same DNA molecule, or

into an entirely different DNA molecule • Some transposons simply “hop”; others replicate then hop

“jumping genes”

Mutationsspontaneous

mistakes during replicationbase substitutionaddition/removal of nucleotides

transposable elements

Summary

induced

Induced Mutation

Alter the base-pairing properties

Chemical mutagens (potential carcinogens)Chemicals that modify purines and pyrimidines

Induced Mutation

Example: nitrous acid strips the amino group from nucleotides

:G :A


Chemical mutagensChemicals that modify purines and pyrimidines

Induced Mutation

Resemble nucleotide bases; erroneously incorporated into DNA

Example: nitrous acid strips the amino group from nucleotidesBase analogs



Analog base-pairs with a different nucleotide

T C

Induced Mutation

Intercalating agents Insert between base-pairs, pushing nucleotides apart; extra nucleotide may then be erroneously added during replication

Analog base-pairs with a different nucleotideResemble nucleotide bases; erroneously incorporated into DNA

Example: nitrous acid strips the amino group from nucleotidesBase analogs



Induced MutationTransposons

Intentional use of an agent that naturally creates spontaneous mutations

Ultraviolet irradiationCauses formation of covalent bonds (thymine dimers) between adjacent thymine bases

Distorted DNA can be repaired, but the process (SOS repair) may introduce errors

High doses are used to sterilize surfaces, lower doses to introduce mutations

X raysCauses double- and single-stranded breaks in DNA

Induced Mutation

Radiation


mistakes during replicationbase substitutionremoval or addition of nucleotides

transposable elementsinduced

chemical mutagensradiationtransposons

Summary

Repair of errors in base incorporationDNA polymerase

proofreadingMismatch repair

excision/replacement

DNA Repair

Repair of thymine dimmers

DNA Repair

Repair of thymine dimmers




Light reactivation (photorepair)

DNA RepairRepair of errors in base incorporation

DNA polymeraseproofreading

Mismatch repairexcision/replacement

Repair of thymine dimmersLight reactivation (photorepair)Excision repair (dark repair; light-independent repair)

DNA Repair

Repair of Modified BasesGlycosylase removes oxidized guanine

SOS repair Induction of SOS system

New polymerase (tolerates “slop”)




Repair of thymine dimersLight reactivation (photorepair)Excision repair (dark repair; light-independent repair)

Mutant Selection

Direct selectionObtain resistant mutants (ex. antibiotic resistant)

Obtain prototrophs that have reverted from auxotrophs Auxotrophs

Prototroph (revertant)

Enriched complexmedium

Minimal medium (glucose-salts)

Application of direct selectionAmes Test - screens for mutagens(used to narrow down list of possible carcinogens)

The Ames Test

Also do expt. with liver extract added

Mutant Selection

Indirect selection (replica plating)Obtain auxotrophs 106 prototrophs

1 auxotroph

Indirect selection (replica plating)Obtain auxotrophs

Indirect selection (replica plating)Obtain auxotrophsJoshua and Esther Lederberg


mistakes during replicationtransposons

inducedchemical mutagensradiationtransposons

Repairrepair of errors in base incorporationrepair of thymine dimmersSOS repair

Selecting mutantsdirect - obtain antibiotic resistant mutants, Ames testindirect - obtain prototrophs

Summary

Part II DNA Transfer

Recipient DonorHorizontal (lateral) transfer

DNA Transfer

1920s; Frederick Griffith- strains of Streptococcus pneumoniae that produce capsules kill mice

“transforming principle”(DNA)

DNA Transfer

1/109 =10-9

10-9 x 10-9 = 10-18

DNA Transfer

To be stably maintained, transferred DNA must either be a plasmid (has an origin of replication), or integrate into the host cell’s genome

Recipient Donor

DNA Transfer

Donor Recipient

Integrate into host genome byHomologous recombination (site-specific recombination)

DNA Transfer

Donor Recipient

Integrate into host chromosome byHomologous recombination (site-specific recombination)

DNA Transfer

Donor Recipient


replication

heteroduplex

DNA Transfer

Donor Recipient


DNA Transfer

Donor Recipient

A+, B- A-, B+

A+, B+

A-, B-

A+, B-

B- A-

Horizontal (lateral) gene transfer

DNA Transfer

Recipient Donor

DNA-mediated transformation

Transduction

Conjugation

DNA-Mediated TransformationUptake of naked DNA

Process is sensitive to the addition of DNAse

DNA-Mediated Transformation

Recipient cell must be competent

Uptake of naked DNA

Observed in only certain speciesExample - Streptococcus pneumoniae (GPC)

Example - Haemophilus influenzae (GNR)

Natural competence

Artificial competenceIn the laboratory, treat cells with specific chemicals

Process is sensitive to the addition of DNAse

(plasmids taken up)

•Becomes competent in late log phase•Competent cell binds ds DNA•Enzymes cut DNA into smaller fragments (5 - 15 kb)•Single strand is taken up by cell

•Cell binds DNA only from related species•Takes up ds DNA

Conjugation

Requires cell-to-cell contactInvolves a conjugative plasmid

F plasmid (fertility plasmid) serves as a modelThree types of donors:

F+

HfrF’

Conjugation: F+ donor

“male” “female”

In donor cell, replication replaces strand that’s being transferred

In recipient cell, complement to transferred strand is synthesized


F+ + F- F+ + F+

In donor cell, replication replaces strand that’s being transferred

In recipient cell, complement to transferred strand is synthesized

Note: some R plasmids (encode resistance to one or more antibiotics) are conjugative


ConjugationHfr = High-frequency recombinationFormation of an Hfr cell

Figure 8.25

Conjugation: Hfr donor


•Some F plasmid DNA is transferred first, followed by chromosomal DNA

• In donor cell, replication replaces strand that’s being transferred

• In recipient cell, complement to transferred strand is synthesized

Cells inevitably separate before entire

chromosome is transferred


Hfr + F- Hfr + F-

Significance of Hfr strains:•Chromosomal transfer•Allowed mapping of E. coli chromosome

Recombinant DNA and Biotechnology

DNA RNA protein

Preview

• Fundamental tools of biotechnology

• Molecular Cloning

• PCR

Fundamental Tools Used in Biotechnology

•Restriction Enzymes - used to cut DNA at specific sequences•Gel Electrophoresis - used to separate nucleotide (or protein) fragments•DNA Probes - used to “find” specific nucleotide sequences•Primers - used to initiate DNA synthesis at a specific location

Restriction Enzymes - cut DNA

1

palindromereflects name of org. from which enz. was first isolated

Restriction Enzymes - cut DNA

Gel Electrophoresis - separates fragments

Note: millions of each “player”

Gel Electrophoresis - separates fragments

DNA Probes - “find” sequences

Primers - dictate sites of synthesis initiation

Techniques Used in Genetic Engineering

DNA

vectorinsert

Self-replicating DNA (ex. plasmid)

recombinant molecule

Techniques Used in Genetic Engineering

Cloning Overview

• Cut out the gene of interest from donor

• Put the gene into a vector

• Transfer the vector into a recipient

• Select for the recipient from a mixed population

Cloning Vectors

1) Plasmids

2) Bacteriophage lambda

3) P1 Phage

4) Cosmids

5) Yeast artificial chromosomes (YAC)

Characteristics of cloning vectors

1) Should have it’s own replicon i.e., be capable of autonomous replication in the host cell

2) Should carry one or more selectable markers that permit identification of parent and recombinant vectors

3) Restriction sites in non-essential regions of DNA into which foreign DNA can be inserted

Molecular Cloning

Genetically Engineering Bacterial Cells

Note: millions of each “player”

Applications of molecular cloning

• Medical application– gene therapy– production of drugs (insulin, antibiotics, hormones)

• Agricultural application– Nutrients enriched food– Nitrogen fixing plants

• Scientific research

(PCR) Polymerase chain reaction

ds DNA containing the target

Taq polymerase (Thermus aquaticus)

nucleotides

primers

thermocycler

Amplifies target sequence

PCR

• Medical application: – Genetic diseases– Infectious disease

• Forensic science:– Identify criminals– parental identification

• Research application

DDC: DNA Test Sets Inmate Free After 18 Years

Forensic Resources DNA Diagnostics Center’s Forensics Division provided the DNA testing that eventually resulted in the release of inmate Robert McClendon. McClendon had spent 18 years in prison, convicted of a child rape that he has always maintained he did not commit.McClendon’s is 1 of 30 cases in Ohio that were identified to have “legitimate claims of innocence” in an investigation conducted by The Columbus Dispatch together with the Ohio Innocence Project (OIP). DDC has volunteered to provide DNA testing for the OIP's post-conviction cases free of charge.

http://www.forensicdnacenter.com/resources/

Bacterial Genetics

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