Embed Size (px)
DESCRIPTION
microbiology
Citation preview
6/29/2014
1
BIOL 2P98 D3 Spring 2014Principles of MicrobiologyDr. Carolynn E Pietrangeli PhD
Lecture 5 June 30, 2014
CHSC 2P98 Principles of MicrobiologySpring D3 June 16‐July 18 2014
INSTRUCTOR: Dr. Carolynn E. Pietrangeli, PhD
CONTACT : e‐mail: [email protected]: MCF 212
OFFICE HOURS: Monday/Friday 12PM‐1PMAnd by appointment – e‐mail request
FORMAT: Lectures, 3 hours twice weekly Monday/Friday 9AM‐12PM AS216
6/29/2014
2
Where to Now?
• Let’s try on some Genes…
• …See what happens when they mutate … and …
• …Transfer (donate?) them …
Order of Business
• The bacterial genome– The bacterial chromosome and replicationPlasmids– Plasmids
– Transposable elements
• Mutation– Assaying mutation
• Bacterial gene transfer– Recombination– Transformation– Transduction
• Assaying bacterial gene transfer
6/29/2014
3
Before We Visit…Let’s Learn the Language…
• Genome – entire complement of genes present in a cell (or virus), carrying all the heritable traits
• Chromosome – principal genetic element carrying genes essential for life processes, including proliferation
• Additional genetic elementsAdditional genetic elements– Plasmids– Transposable elements
Origin ofreplication
lac operon(lactosedegradation)100/0
90 10
HfrHP804
HfrC
t
Not1 restrictionsites, in kbp
4,639,679 bp
Escherichia coli K‐12
80
70 30
20
KL14
Figure 4.8
trp operon(tryptophanbiosynthesis)
his operon(histidinebiosynthesis)
60
50
40Hfr44
© 2012 Pearson Education, Inc.
E. coli chromosome
6/29/2014
4
Plasmids: Extrachromosomal Prokaryotic DNAReplicationfunctions
mer
sulstr
cat
IS1
94.3/0 kbp
Small circular or linear dsDNAmolecules; often supercoiled
tra
IS1
IS10
IS10
75 kbp 25 kbp
50 kbp
Figure 4.10
Figure 4.9
tetoriT Tn10• Most plasmids are circular dsDNA• Replicate independently of the bacterial chromosome• May integrate into the bacterial chromosome = episome• Several types may co‐exist in a single bacterium; must be from differentIncompatibility (Inc) Groups
• F plasmids (conjugative) may be transferred between bacteria
Figure 4.9
Examples of Bacterial Plasmids
CN = Copy numberhttp://www.gs.washington.edu/academics/courses/manoil/41109/lecture/lecFeb23.pdf
6/29/2014
5
Replication of the Bacterial Chromosome
Replication of the Bacterial Chromosome
Theta (θ) replication
http://www.youtube.com/watch?v=HBwyNrkYnp01. DNA replication in prokaryoteshttp://www.youtube.com/watch?v=w7TjoiubgF42. Claymation DNA replication http://www.youtube.com/watch?v=qSmTtk54JAs3. DNA replication in E coli
replication
6/29/2014
6
Plasmid Replication Strategies
• Theta (θ) replication
• Rolling circle replication• Rolling circle replication
http://www.youtube.com/watch?v=YydvqhNOcVARolling Circle Replication of DNA
Transposable Elements “Jumping genes”
DNA segments that move from one site to another on the same or a different DNA molecule
6/29/2014
7
Conservative
Replicative
Types of Transposable Elements (TEs)
• Insertion sequences• Composite transposons (Tn)• Composite transposons (Tn)• Replicative transposons:
‐ Phage Mu – viral
6/29/2014
8
Insertion SequencesThe Price of Admission
Properties of Insertion Sequences (Is)• Short: 700‐2500 bp• Genes restricted to transposition:
transposase + regulatory sequencestransposase + regulatory sequences
IS911: 1250 bp
orfB is Transposase geneTransposase = enzyme required to achieve transposition
IS carry ONLY the genes required for transposition
Composite transposons (Tn) carry:• Insertion sequences
• Additional genes – antibiotic resistance
6/29/2014
9
Replicative Transposon – Phage Mu [Mutator Phage]
• Random integration into b t i l hbacterial chromosome
• Insertion produces mutation in host DNA
• Replication by transposition –
i htransposon acquires host DNA sequences
Morgan GJ et al J. Mol. Biol. (2002) 317, 337‐359
Functions of Transposable Elements (TE) / Mobile Genetic Elements (MGE)
• Generate mutations
• Increase or decrease the amount of DNA in the genome
• Promote genome rearrangement
• Regulate gene expression
• Induce chromosome breakage and rearrangement• Induce chromosome breakage and rearrangement
6/29/2014
10
The Molecular Toolkit: How We Use Transposons
• Genetic mapping – TE insertions – Behave as point mutations mostly producing null alleles– Behave as point mutations, mostly producing null alleles– Are useful in deletion mapping – determining the gene order – when near but not within a gene of interest
– Can determine the location of genes within or outside of operons (polarity of insertion into operons)
• Mutation transfer selected for by antibiotic resistancey– Track loss of transposon by loss of antibiotic resistance
…Where to Now?... Bacterial Genome Plasticity – Mutation
• Defining mutationsMacrolesions– Macrolesions
– Microlesions
• Nature or nurture – how do mutations occur?– Spontaneous mutations– Induced mutations
l f l d• Genetic analysis of mutants – selection and screening
• How do we exploit bacterial mutations?
6/29/2014
11
…The Ground Rules…
• Mutations are stochastic events• Once a mutation occurs, unless a revertant
b l h f dsubsequently occurs, the progeny of a mutated cell will carry the parent mutation
• Three levels of evaluation of mutations– DNA bp alteration– Amino acid codon changeg– Functional protein/tRNA/rRNA phenotypic change
Basics of Mutation and Mutants
• Wild‐type bacterial strain – strain isolated from nature = parental strain
• Mutation – Heritable change in DNA sequence that canlead to a change in phenotypelead to a change in phenotype
• Mutant bacterial strain – strain that differs from parental strain in any portion of the genome nucleotide sequence
• Revertant bacterial strain – strain in which the parental/wild type phenotype is restored
Wild type Mutant
6/29/2014
12
Defining Mutations and MutantsPerspective: Detection of Mutant Clones
• Selectable mutations – Genetic research tools– Mutant acquires growth advantage under controlled conditions
– Detected by controlled assay
• Nonselectable mutations– Changes in DNA from parent that provide neither an advantage or disadvantage to the organism
– Detected by screening
Macrolesional Mutations
Position with respect to centromerePosition with respect to centromere
6/29/2014
13
DNA
M U T A T I O N Normal DNAreplication
Microlesional Mutations
DNA
mRNAAsparagine
codonStopcodon
Tyrosinecodon
Tyrosinecodon
Transcription
Figure 10.3
Protein
Missensemutation
Nonsensemutation
Silentmutation
Wild type
Faultyprotein
Incompleteprotein
Normalprotein
Normalprotein
Translation
© 2012 Pearson Education, Inc.
Types of Forward MutationsForward mutation: Wild type Mutant
Protein Frame of Reference
Type of Mutation DNA Level Protein LevelType of Mutation DNA Level Protein Level
Silent Change in nucleotide coding sequence
No change in amino acid (aa)
Missense Single base substitution Exchange of aa
Nonsense Single base substitution or more results in STOP codon (CAG UAG)
Chain termination
Frameshift Insertion OR deletion of 1 2 bp Mutant or non functionalFrameshift ‐ Insertion OR deletion of 1‐2 bpinto/out of gene coding region‐ Reading frame shift
Mutant or non‐functional protein
6/29/2014
14
Stop codonsDNA:
‐TAG/TAA/TGARNA:UAG/UAA/UGA
Substitution Point Mutation:THE CAT IS IN THE BAGTHE HAT IS IN THE BAG
How would you classify this mutation?
‐UAG/UAA/UGA
UH OH … STOP
No problem!Same aa; same protein
Wrong protein … Wrong function
CODON – no protein
Missense but close!
6/29/2014
15
Frameshift MutationsCaused by Indels (Insertions and Deletions)
Readingframe
mRNADNA
Normal
CodonsInsertion
Transcriptionoff of lightgreen strands
THE BIG CAT ATE THE FAT RATWild typeproteinDeletion
© 2012 Pearson Education, Inc.
THE BIG CAT ATE THE FAT RATTHE BIG CAT −TET HEF ATR AT. … …
Wild typeMutant
Reversion of Mutation
• Same‐site reversion• Second‐site reversion/Suppressor mutation
6/29/2014
16
Mutation Origins
• Spontaneous – arising without exernalintervention
• Induced – resulting from external intervention
– Exposure to toxins, radiation
– Laboratory tool to identify genetically engineered organisms
– Site‐directed mutagenesis
Induced and Conditional Mutations• Induced mutations – Mutagen – external pressure that causes a heritable
alteration in DNA‐ Chemicals‐ Radiation – ioninzing (X‐ray) or non‐ionizing (UV)‐ Viral incorporation
• Conditional mutation – expressed only under fixed environmental conditions– Permissive vs. Restrictive conditions– Biochemical mutants ‐ Auxotrophs
6/29/2014
17
Mutation Rates
• DNA replication errors: 106 to107/kb
• DNA virus error rates: 103 to104/kb• DNA virus error rates: 10 to10 /kb
• RNA genome mutation rate is 1000‐fold higher than DNA genome mutation rate
DNA Repair• Direct reversal – base can be corrected without reference tocomplementary strand
• Single‐strand repair – utilizes complementary strand • Double‐strand repair – highly error‐prone• SOS regulatory repair – large‐scale damage; highly error‐prone; responsible for acquisition of antibiotic resistance in some bacteria
Clancy, S. (2008) DNA damage & repair: mechanisms for maintaining DNA integrity. Nature Education 1
6/29/2014
18
Induced and Conditional Mutagenesis
• Induced mutations – Mutagen – external pressure that causes a heritable alteration in DNA
‐ Chemicals
‐ Radiation – ioninzing (X‐ray) or non‐ionizing (UV)
‐ Viral incorporation
• Conditional mutation – expressed only under fixed environmental conditions
– Permissive vs. Restrictive conditions
– Biochemical mutants ‐ Auxotrophs
Examples of Chemical Mutagens
• Nucleotide base analogs: resemble nucleotides• Alkylating agents – e.g. nitrosoguanidine• Hydroxylating agents• Intercalating agents – induce frameshiftmutations – e.g. acridines
6/29/2014
19
Prescott Table 14.1
Do Mutations Occur RANDOMLY or in RESPONSE to Environmental Pressure?
6/29/2014
20
Utilizing Viruses to Detect BACTERIALMutants
Bacteriophage (Phage) – virus that infects bacteria
Lytic phage T4 bacteria lysed following viralLytic phage – T4 – bacteria lysed following viral replication
Temperate phage – ʎ Phage – viral genome integrates into host DNA and replicates with it
Prophage – phage genome inserted and integrated into the circular bacterial DNA chromosome
Replication• Lytic cycle• Lysogenic cycle
How do We Detect Mutants in a Bacterial Population?
• Use a selectable marker – antibiotic resistance; phage resistance; nutritional alteration ‐ auxotroph
• Screen for changes in colony morphology
• Perform replica plating
T1 phage
6/29/2014
21
Nature or Nurture: How do Mutations Occur?
Spontaneous mutation The Luria and Delbruck Fluctuation Test 1943
Nobel Prize 1969
Observation:• Phage T1 kills most bacteria (Tons)
• Rare bacteria survive (Tonr)
Possible Explanations for Tonr phenotype
• Random mutation: Tons Tonr
• Induced adaptation: Mutation induced by physiologic response to phage
Replica Plating
‐E. coli Tonr ‐
A t hAuxotroph‐Temperature sensisitive
Griffths et al., Fig. 15‐22, 7th ed.
6/29/2014
22
Master plate; growth
Detecting Nutritional Auxotrophsthrough Replica Plating
Master plate; growthon complete medium
Velveteen;sterilized
Plastichoop
Woodenblock
Velveteen;with imprintof allcolonies
Complete medium All colonies grow
Minimal medium Mutants do not grow
Press plate ontovelveteen
Transfer imprintof colonies tofresh media
Incubate
Figure 10.2© 2012 Pearson Education, Inc.
Auxotroph – bacteria that carry a mutation resulting in inability to synthesize an essential compound
The Luria and Delbruck Fluctuation Test
System: grow bacteria over several generations in presence of phage
Readout: number of Tonr bacterial colonies
Hypothesis testing:
Source of mutation Expected resultNumber of Tonr colonies
Spontaneous Highly variableMutation occurs before contact with phage
PhysiologicMutation is induced by contact with phage
Uniform
6/29/2014
23
20 x 0.2 mL
1 x 10 mL
Earlyculture
Latecultures
Fluctuation Test
Large Early Culture ResultsFluctuation Test
Small Late Culture ResultsIf mutation occurred in RESPONSE to stimulus, the variance would
be very low
6/29/2014
24
Mutagenesis at Work for Us:The Ames Test
Interpreting the Result of the Ames Test
Figure 10.8© 2012 Pearson Education, Inc.
6/29/2014
25
Mutation: If it’s WRONG, why do it?
• Mutation generates diversity
• Mutation is a major means of achieving• Mutation is a major means of achieving …
Laboratories
• Schedule: As per posting on Sakai
• Laboratory Demonstrators
– Christene Carpenter‐Cleland MC F210
Extension 5788 [email protected]
– Mark Lukewich MCF 213
Extension 3398 [email protected]
6/29/2014
26
Evaluation
• Laboratory 35% See Laboratory Manual
• Midterm Examination 20% Friday July 4 9:00AM‐10:30AM
Discussion Forum will followDiscussion Forum will follow
• Final Examination 20% 2 hour duration
• Discussion Forum 25% ‐ 5 exercises/5 marks each
See Sakai for Instructions
20% content
5% participationp p
There are NO make‐up or supplemental examinations in BIOL 2P98 This applies to Lecture Examinations
(Midterm AND Final Examination) AND to theLaboratory Examination
BIOL 2P98 SP Laboratory ScheduleLaboratory Schedule
Week of Laboratory Exercise Title
Jun 16 Lab 1 Lab Safety Techniques & Enumeration
Labs are in IH 309.
Jun‐16 Lab 1‐ Lab Safety, Techniques & Enumeration
Jun‐23 Lab 2‐ Bacterial Growth & Identification
Jun‐30 Canada Day week NO LABS Assignment 2 on Sakai
Jul‐07 Lab 3‐ The Control of Microbial Growth
Jul‐14 Lab Exam Based on all lab material
Lab sections IH309
Section 5 Monday 2:00pm ‐ 5:00pmSection 3 Tuesday 10:00am ‐ 1:00pmSection 1 Tuesday 2:00pm ‐ 5:00pmSection 2 Wednesday 2:00pm ‐ 5:00pm
‐ Read all Appendices & Lab 1 on Sakai‐ Create a flowchart for Lab 1‐ Bring a lab coat & safety glasses/goggles, Lab 1, flowchart and Photographic Atlas
6/29/2014
27
Discussion Forums
• Occur each Friday during last lecture hour (11AM‐12PM)
• Each week, we evaluate a case relevant to lecture/laboratory
content in Microbiology in assigned Small Groupscontent in Microbiology in assigned Small Groups
• Each case will be accompanied by sets of questions to be discussed
and worked on by each Group
• Each Group submits a SINGLE electronic Report (no paper
submission) responding to the case questions set for that Group
• ELECTRONIC Report (no paper submission) due by 5PM on the
T d f th f ll i k ( C O tli )Tuesday of the following week (see Course Outline)
• The Group will report the specific contribution of each member
• Each member of the Group earns the SAME GRADE for the Report
• Discussion Forum Instructions posted on Sakai – includes Group
listings
Discussion Forum Participation
• 20% of the total grade earned for the Discussion Forums (5% of the final grade inDiscussion Forums (5% of the final grade in BIOL 2P98) is derived from participation in Discussion Forum exercises
• Attendance will be recorded by sign‐in sheet at the beginning of each Discussion Forum
Di i G ill b k d t d ib• Discussion Groups will be asked to describe the specific contribution of each member to each Group Report
6/29/2014
28
Resources
Required Textbook
Brock Biology of Microorganisms. Fourteenth Edition. Madigan MT, MartinkoJM, Bender KS, Buckley DH, Stahl DA. Pearson Boston 2012
• Provides a basic review of concepts to be discussed during the course
• Does NOT substitute for course lectures or Discussion Forum content
• Textbook chapters will not be assigned; students will match the lecture content to relevant chapters in the textbook
• Textbook content will be supplemented by readings made available on Sakai. Postings of additional readings will be announced during lectures and on Sakai
Recommended Textbook for Laboratory
A Photographic Atlas for the Microbiology Laboratory. Leboffe MJ, Pierce BE 4th Edition. Morton Publishing 2011
General Background Microbiology
http://www.mhhe.com/biosci/cellmicro/talaro/links.mhtml
Academic Integrity A Reflection of Personal Integrity
• Many things may be stolen from us by other people over the course of our lives
• Integrity is one thing we can steal only from ourselves
Integrity
• Respecting it Respecting yourself• Respecting it = Respecting yourself
• Preserve it
• Be proud of upholding it
