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Bio 97, Bardwell, Cinquin and Warrior, UCI - Copyright: All rights reserved
Genetics of bacteria and bacteriophages
Genetics of bacteria and bacteriophages
Bio Sci 97Bio Sci 97
Bio 97, Bardwell, Cinquin and Warrior, UCI - Copyright: All rights reserved
Learning goalsLearning goals
• Explain how key characteristics of bacteria make them a powerful model system for genetics
• List possible functions of plasmids
• Of the different ways by which DNA can be transformed into bacteria, identify those that are most suitable for different purposes
• Explain why selection of phenotypes of interest is more advantageous than screening for these phenotypes
• Work out growth medium formulations that select for genotypes of interest
• Predict outcomes of conjugation and transduction experiments
• Build maps from data derived from conjugation and/or co-transduction experiments
• Explain how key characteristics of bacteria make them a powerful model system for genetics
• List possible functions of plasmids
• Of the different ways by which DNA can be transformed into bacteria, identify those that are most suitable for different purposes
• Explain why selection of phenotypes of interest is more advantageous than screening for these phenotypes
• Work out growth medium formulations that select for genotypes of interest
• Predict outcomes of conjugation and transduction experiments
• Build maps from data derived from conjugation and/or co-transduction experiments
Bacteria as model organismsBacteria as model organisms
• What are important characteristics of bacteria?
• small, easy to grow in extremely large numbers; very useful to have lots of individuals
• grow very quickly; typical generation time < 30 min
• small genome: E. coli ~5 Mbp
• many antibiotic resistance and auxotrophy markers
• E. coli workhorse of molecular biology
• What are important characteristics of bacteria?
• small, easy to grow in extremely large numbers; very useful to have lots of individuals
• grow very quickly; typical generation time < 30 min
• small genome: E. coli ~5 Mbp
• many antibiotic resistance and auxotrophy markers
• E. coli workhorse of molecular biology
Bio 97, Bardwell, Cinquin and Warrior, UCI - Copyright: All rights reserved
Growing bacteriaGrowing bacteria
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Growing coloniesGrowing colonies
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E. coli geneticsE. coli genetics
• Haploid; no obscured recessive mutations but no meiosis
• Just 1 chromosome, but can carry plasmids
• Haploid; no obscured recessive mutations but no meiosis
• Just 1 chromosome, but can carry plasmids
Bio 97, Bardwell, Cinquin and Warrior, UCI - Copyright: All rights reserved
Function of plasmids in bacteria
Function of plasmids in bacteria
• No obvious, useful function at all: parasites
• Provide ability to metabolize certain substrates
• Provide antibiotic resistance
• Strong selection if plasmid provides a function necessary for growth or survival (e.g. antibiotic resistance)
• No obvious, useful function at all: parasites
• Provide ability to metabolize certain substrates
• Provide antibiotic resistance
• Strong selection if plasmid provides a function necessary for growth or survival (e.g. antibiotic resistance)
Bio 97, Bardwell, Cinquin and Warrior, UCI - Copyright: All rights reserved
Clicker questionClicker question
• Starting from a large, mixed population of bacteria with different genotypes, which of the following is easiest to select for?
A. A bacterium that is sensitive to the antibiotic ampicillin
B. A bacterium that cannot synthesize amino acids
C. A bacterium that cannot grow in the presence of arabinose
D. A bacterium that can grow using arabinose instead of glucose
E. A bacterium that cannot grow in the absence of arabinose
• Starting from a large, mixed population of bacteria with different genotypes, which of the following is easiest to select for?
A. A bacterium that is sensitive to the antibiotic ampicillin
B. A bacterium that cannot synthesize amino acids
C. A bacterium that cannot grow in the presence of arabinose
D. A bacterium that can grow using arabinose instead of glucose
E. A bacterium that cannot grow in the absence of arabinose
Genes can be transferred between bacteria
Genes can be transferred between bacteria
• A number of mechanisms
• Conjugation
• Transduction
• Transformation
• Why do we care?
• Tools to study bacteria; e.g. chromosome mapping
• Gene transfer very important for antibiotic resistance
• A number of mechanisms
• Conjugation
• Transduction
• Transformation
• Why do we care?
• Tools to study bacteria; e.g. chromosome mapping
• Gene transfer very important for antibiotic resistance
Bio 97, Bardwell, Cinquin and Warrior, UCI - Copyright: All rights reserved
Conjugation: bacteria have “sex” tooConjugation: bacteria have “sex” too
• Unidirectional
• What is the benefit?
• Commonly assumed that the same mixing of “good genes” as for animals
• Unidirectional
• What is the benefit?
• Commonly assumed that the same mixing of “good genes” as for animals
How was conjugation first observed?How was conjugation first observed?
• Lederberg and Tatum (1946)
Clicker questionClicker questionWhat phenotypes will there be in the population grown in complete medium?
•A. met+ leu-
•B. met- leu+
•C. met+ leu+
•D. A and C
•E. A, B, and C
What phenotypes will there be in the population grown in complete medium?
•A. met+ leu-
•B. met- leu+
•C. met+ leu+
•D. A and C
•E. A, B, and C
Bio 97, Bardwell, Cinquin and Warrior, UCI - Copyright: All rights reserved
Digging into the mechanismDigging into the mechanism• Not all strains can transmit their metabolic capacities to others;
sorted between F+ (fertility factor), and F- (the other ones)• Not all strains can transmit their metabolic capacities to others;
sorted between F+ (fertility factor), and F- (the other ones)
What does this tell us?What does this tell us?
Need for physical contactNeed for physical contactbetween bacteriabetween bacteria
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A mechanism that does not require physical contact between cells
A mechanism that does not require physical contact between cells
Zinder and Lederberg (1952)Zinder and Lederberg (1952)SalmonellaSalmonella, but same principle, but same principle
Unlike with the previous experiment, Unlike with the previous experiment, one population forms prototrophs.one population forms prototrophs.
What could be going on?What could be going on?Something much smaller than a cell Something much smaller than a cell
has to be able to go through the filter.has to be able to go through the filter.Viruses are smallViruses are small
Bio 97, Bardwell, Cinquin and Warrior, UCI - Copyright: All rights reserved
Transduction; bacteria have viruses too: phagesTransduction; bacteria have viruses too: phageshttp://www.youtube.com/watch?v=9hzUjx_oD8E&NR=1
QuickTime™ and aMPEG-4 Video decompressor
are needed to see this picture.
• After infection, two possibilities:• the cell dies producing a large number of new phages: lytic cycle• the phage integrates in the genome: lysogenic cycle; what’s the advantage?• If phage kills all the bacteria around it straight away, it will die too
• When is it time to abandon ship?• When the cell is stressed; same as with some human viruses
• After infection, two possibilities:• the cell dies producing a large number of new phages: lytic cycle• the phage integrates in the genome: lysogenic cycle; what’s the advantage?• If phage kills all the bacteria around it straight away, it will die too
• When is it time to abandon ship?• When the cell is stressed; same as with some human viruses
Bio 97, Bardwell, Cinquin and Warrior, UCI - Copyright: All rights reserved
Transformation: some bacteria can pick up DNA from their surroundingsTransformation: some bacteria can
pick up DNA from their surroundings
• Chemically-induced competence: an enormously-useful property. We can put engineered DNA in bacteria; routine technique even for labs that don’t study bacteria
• Chemically-induced competence: an enormously-useful property. We can put engineered DNA in bacteria; routine technique even for labs that don’t study bacteria
QuickTime™ and aMPEG-4 Video decompressor
are needed to see this picture.
http://www.youtube.com/watch?v=MRBdbKFisgI
Different ways of mapping the bacterial genome: (1) conjugation
Different ways of mapping the bacterial genome: (1) conjugation
• F plasmid encodes transfer genes
• Transfer coincides with replication
• Recipient becomes F+
• F plasmid encodes transfer genes
• Transfer coincides with replication
• Recipient becomes F+
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From F+ to Hfr strainFrom F+ to Hfr strain
When an integrated F is transferred, it When an integrated F is transferred, it drags the chromosome along with itdrags the chromosome along with it
Characteristics of the Hfr transferCharacteristics of the Hfr transfer
• It starts from a specific position within the F factor
• It goes in a specific direction
• The last part of the F factor is transferred last (which is why recipients are generally not converted to F+ or Hfr after conjugation with Hfr donor)
• Different strains will start transfer from different positions in the genome, and in different directions
• It starts from a specific position within the F factor
• It goes in a specific direction
• The last part of the F factor is transferred last (which is why recipients are generally not converted to F+ or Hfr after conjugation with Hfr donor)
• Different strains will start transfer from different positions in the genome, and in different directions
Bio 97, Bardwell, Cinquin and Warrior, UCI - Copyright: All rights reserved
Interrupted matingInterrupted mating
http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/plasmids/Hfr-interrupt.gifhttp://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/plasmids/Hfr-interrupt.gif
Need to distinguish betweenNeed to distinguish betweenthe different bacteria; we are the different bacteria; we are
interested in the interested in the exconjugantsexconjugants..
Antibiotic resistanceAntibiotic resistanceand auxotrophy markersand auxotrophy markers
Mating interrupted withMating interrupted witha blendera blender
Assembling maps Assembling maps
Bio 97, Bardwell, Cinquin and Warrior, UCI - Copyright: All rights reserved
Assembling maps: concrete exampleAssembling maps: concrete example
• Note different origins and orientations• Note different origins and orientationsBio 97, Bardwell, Cinquin and Warrior, UCI - Copyright: All rights reserved
Whole genome mapWhole genome map
• Map is in minutes
• Map is in minutes
• Generalized transducing phage: a phage that sometimes packages bacterial DNA instead of itself
• Packaged copy can recombine into host genome
• Generalized transducing phage: a phage that sometimes packages bacterial DNA instead of itself
• Packaged copy can recombine into host genome
Different ways of mapping the bacterial
genome: (2) cotransduction
Different ways of mapping the bacterial
genome: (2) cotransduction
Mapping close genes by cotransduction
Mapping close genes by cotransduction
• Phages can only package a limited amount of DNA
• Genes close together on the chromosome can be packaged together; genes too far away cannot
• The closer the genes, the more likely they are to be packaged together and cotransduced; we can measure the frequency of cotransduction
• Phages can only package a limited amount of DNA
• Genes close together on the chromosome can be packaged together; genes too far away cannot
• The closer the genes, the more likely they are to be packaged together and cotransduced; we can measure the frequency of cotransduction
Bio 97, Bardwell, Cinquin and Warrior, UCI - Copyright: All rights reserved
Mapping close genes by cotransductionMapping close genes by cotransduction