bio97 lecture 13 2015 to post

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


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


Growing bacteriaGrowing bacteria


Growing coloniesGrowing colonies


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


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)


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


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


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


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


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


http://www.youtube.com/watch?v=9hzUjx_oD8E&NR=1

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

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+


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


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


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


Mapping close genes by cotransductionMapping close genes by cotransduction

bio97 lecture 13 2015 to post

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