Brooker Chapter 6

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Transcript Brooker Chapter 6

Bacterial Genetics



Bacteria are haploid

 identify loss-of-function mutations easier 

recessive mutations not masked

Bacterial Genetics

 Bacteria reproduce asexually  Crosses not used  genetic transfer  bacterial DNA segments transferred

Genetic Transfer



Enhances genetic diversity



Types of transfer

 Conjugation  direct physical contact & exchange  Transduction  phage  Transformation  uptake from environment

Conjugation

 Many, but not all, species can conjugate  Only certain strains can be donors  Donor strain cells contain plasmid called F factor 

F +

strains  Plasmid circular, extra-chromosomal DNA molecule

F-factor Plasmid

 Genes for conjugation

Figure 6.4

Conjugation

Figure 6.4

Conjugation

 Results of conjugation   recipient cell acquires F factor converted from F – to F + cell  F factor plasmid may carry additional genes  called F’ factors  F’ factor transfer can introduce genes & alter recipients genotype

Hfr

Strains

 1950s, Luca Cavalli-Sforza discovered

E. coli

efficient at transferring chromosomal genes strain very  designated strain

Hfr

(high frequency of recombination)  Hfr strains result from integration of F' factor into chromosome

Figure 6.5a

Hfr Conjugation

 Conjugation of Hfr & F – transfers portion of Hfr chromosome   origin of transfer of integrated F factor  starting point & direction of the transfer takes 1.5-2 hrs for entire Hfr chromosome to be transfered   Only a portion of the Hfr chromosome gets into the F – cell F – cells does not become F + or Hfr  F – cell does acquire donor DNA  recombines with homologous region on recipient chromosome

Hfr Conjugation

F – now

lac

+ pro – order of transfer is

lac

pro

+ –

Figure 6.5b

F – now

lac

+ pro +

Interrupted Mating Technique



Elie Wollman & Fran çois Jacob



The rationale

  Hfr chromosome transferred linearly interruptions at different times  transferred various lengths  order of genes on chromosome deduced by interrupting transfer at various time



Wollman & Jacob started the experiment with two

E. coli

strains

 Hfr strain (donor) genotype 

thr +

: Can synthesize threonine      

leu +

: Can synthesize leucine

azi s

: Killed by azide

ton s

: Can be infected by T1 phage

lac +

: Can metabolize lactose

gal +

: Can metabolize galactose

str

s : Killed by streptomycin  F –  strain (recipient) genotype

thr – leu – azi r ton r lac – gal – str r

Figure 6.6

Interpreting the Data

Minutes that Bacterial Cells were Allowed to Mate Before Blender Treatment

5 10 15 20 25 30 40 50 60

There were no surviving colonies after 5 minutes of mating After 10 minutes, the thr

+ leu +

genotype was obtained Percent of Surviving Bacterial Colonies The azi

gene is with the Following Genotypes transferred first

thr + leu + azi s ton s lac + gal +

–– 100 100 100 100 100 100 100 100 –– 12 70 88 92 90 90 91 91 –– 3 31 71 80 75 75 78 78 –– 0 0 12 28 36 38 42 42 –– 0 0 0 0.6

5 20 27 27

It is followed by the ton The lac enters between 15 & 20 minutes The gal

s + +

 From these data, Wollman & Jacob constructed the following genetic map:  They also identified various Hfr strains in which the origin of transfer had been integrated at different places in the chromosome  Comparison of the order of genes among these strains, demonstrated that the

E. coli

chromosome is circular

The

E. coli

Chromosome

 Conjugation experiments have been used to map genes on the

E. coli

chromosome  The

E. coli

genetic map is 100 minutes long  Approximately the time it takes to transfer the complete chromosome in an Hfr mating

Arbitrarily assigned the starting point Units are minutes Refer to the relative time it takes for genes to first enter an F – recipient during a conjugation experiment

Figure 6.7

 The distance between genes is determined by comparing their times of entry during an interrupted mating experiment  The approximate time of entry is computed by extrapolating the time back to the origin

Figure 6.7

 Therefore these two genes are approximately 9 minutes apart along the

E. coli

chromosome

Transduction

 Transduction is the transfer of DNA from one bacterium to another via a bacteriophage  A bacteriophage is a virus that specifically attacks bacterial cells  It is composed of genetic material surrounded by a protein coat  It can undergo two types of cycles   Lytic Lysogenic  Refer to Figure 6.9

It will undergo the lytic cycle Virulent phages only undergo a lytic cycle

Figure 6.9

Temperate phages can follow both cycles

Prophage can exist in a dormant state for a long time

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Plaques

 A plaque is a clear area on an otherwise opaque bacterial lawn on the agar surface of a petri dish  It is caused by the lysis of bacterial cells as a result of the growth & reproduction of phages

Figure 6.14

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Transduction

Any piece of bacterial DNA can be incorporated into the phage This type of transduction is termed generalized transduction Figure 6.10



Transformation

Bacteria take up extracellular DNA  Discovered by Frederick Griffith,1928, while working with strains of

Streptococcus pneumoniae

   There are two types  Natural transformation DNA uptake occurs without outside help  Artificial transformation DNA uptake occurs with the help of special techniques

Transformation



Natural transformation occurs in a wide variety of bacteria



Bacteria able to take up DNA = competent



carry genes encoding competence factors

 proteins that uptake DNA into bacterium & incorporate it into the chromosome

Figure 6.12

A region of mismatch By DNA repair enzymes

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Transformation

 Sometimes, the DNA that enters the cell is not homologous to any genes on the chromosome  It may be incorporated at a random site on the chromosome  This process is termed nonhomologous recombination  Like cotransduction, transformation mapping is used for genes that are relatively close together

Horizontal Gene Transfer

 Transfer of genes between different species  vs Vertical gene transfer - transfer of genes from mother to daughter cell or from parents to offspring  Sizable fraction of bacterial genes have moved by horizontal gene transfer  Over 100 million years ~ 17% of

E. coli

S. typhimurium

genes have been shared by horizontal transfer

Horizontal Gene Transfer



Genes acquired by horizontal transfer

 Genes that confer the ability to cause disease  Genes that confer antibiotic resistance 

Horizontal transfer has contributed to acquired antibiotic resistance

6.2 INTRAGENIC MAPPING IN BACTERIOPHAGES

 Viruses are not living  However, they have unique biological structures & functions, & therefore have traits  We will focus our attention on bacteriophage T4  Its genetic material contains several dozen genes  These genes encode a variety of proteins needed for the viral cycle  Refer to Figure 6.13 for the T4 structure

Contains the genetic material

Figure 6.13

Used for attachment to the bacterial surface

 In the 1950s, Seymour Benzer embarked on a ten-year study focusing on the function of the T4 genes   He conducted a detailed type of genetic mapping known as intragenic or fine structure mapping The difference between intragenic & intergenic mapping is:

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Plaques

Figure 6.14

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 Some mutations in the phage’s genetic material can alter the ability of the phage to produce plaques  Thus, plaques can be viewed as traits of bacteriophages  Plaques are visible with the naked eye  So mutations affecting them lend themselves to easier genetic analysis  An example is a rapid-lysis mutant of bacteriophage T4, which forms unusually large plaques  Refer to Figure 6.15

 This mutant lyses bacterial cells more rapidly than do the wild-type phages   Rapid-lysis mutant forms large, clearly defined plaques Wild-type phages produce smaller, fuzzy-edged plaques

  Benzer studied one category of T4 phage mutant, designated

rII

(

for rapid lysis) stands It behaved differently in three different strains of

E. coli

 In

E. coli B



rII

phages produced unusually large plaques that had poor yields of bacteriophages  The bacterium lyses so quickly that it does not have time to produce many new phages  In

E. coli K12S



rII

phages produced normal plaques that gave good yields of phages 

In E. coli K12(

) (

has phage lambda DNA integrated into its chromosome) 

rII

phages were not able to produce plaques at all  As expected, the wild-type phage could infect all three strains

Complementation Tests

 Benzer collected many

rII

mutant strains that can form large plaques in

E. coli B K12(

)

& none in

E. coli

 But, are the mutations in the same gene or in different genes?

 To answer this question, he conducted complementation experiments

 Figure 6.16 shows the possible outcomes of complementation experiments involving plaque formation mutants

Figure 6.16

 Benzer carefully considered the pattern of complementation & noncomplementation  He determined that the

rII

mutations occurred in two different genes, which were termed

rIIA

rIIB

 Benzer coined the term cistron to refer to the smallest genetic unit that gives a negative complementation test  So, if two mutations occur in the same cistron, they cannot complement each other  A cistron is equivalent to a gene  However, it is not as commonly used

 At an extremely low rate, two noncomplementing strains of viruses can produce an occasional viral plaque, if intragenic recombination has occurred

rII

mutations Viruses cannot form plaques in

E. coli K12(

)

Coinfection

rII

mutations Viruses cannot form plaques in

E. coli K12(

)

Function of protein A will be restored Therefore new phages can be made in

E. coli K12(

)

Viral plaques will now be formed

Figure 6.17



Figure 6.18

rII

describes the general strategy for intragenic mapping of phage mutations

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r104 r103

Take some of the phage preparation, dilute it greatly (10 -8 ) & infect

E. coli B

Both

rII

mutants & wild-type phages can infect this strain Take some of the phage preparation, dilute it somewhat (10 -6 ) & infect

E. coli K12(

)

Total number of phages

66 plaques

rII

mutants cannot infect this strain Number of wild-type phages produced by intragenic recombination

11 plaques 6-62

 The data from Figure 6.18 can be used to estimate the distance between the two mutations in the same gene  The phage preparation used to infect

E. coli B

was diluted by 10 8 (1:100,000,000)  1 ml of this dilution was used & 66 plaques were produced  Therefore, the total number of phages in the original preparation is 66 X 10 8 = 6.6 X 10 9 or 6.6 billion phages per milliliter  The phage preparation used to infect

E. coli k12(

)

was diluted by 10 6 (1:1,000,000)  1 ml of this dilution was used & 11 plaques were produced  Therefore, the total number of wild-type phages is 11 X 10 6 or 11 million phages per milliliter

  In this experiment, the intragenic recombination produces an equal number of recombinants  Wild-type phages & double mutant phages However, only the wild-type phages are detected in the infection of

E. coli k12(

)

 Therefore, the total number of recombinants is the number of wild type phages multiplied by two Frequency of recombinants = 2 [wild-type plaques obtained in E. coli k12( l

)

] Total number of plaques obtained in E. coli B Frequency of recombinants = 2(11 X 10 6 ) 6.6 X 10 9 = 3.3 X 10 –3 = 0.0033 In this example, there was approximately 3.3 recombinants per 1,000 phages

 As in eukaryotic mapping, the frequency of recombinants can provide a measure of map distance along the bacteriophage chromosome  In this case the map distance is between two mutations in the same gene  The frequency of intragenic recombinants is correlated with the distance between the two mutations  The farther apart they are the higher the frequency of recombinants  Homoallelic mutations   Mutations that happen to be located at exactly the same site in a gene They are not able to produce any wild-type recombinants  So the map distance would be zero

Deletion Mapping

 Benzer used deletion mapping to localize many

rII

mutations to a fairly short region in gene

or gene

 He utilized deletion strains of phage T4  Each is missing a known segment of the

rIIA

and/or

rIIB

genes

 Let’s suppose that the goal is to know the approximate location of an

rII

mutation, such as

r103



E. coli k12(

)

is coinfected with

r103

& a deletion strain  If the deleted region includes the same region that contains the

r103

mutation  No intragenic wild-type recombinants are produced  Therefore, plaques will not be formed  If the deleted region does not overlap with the

r103

mutation  Intragenic wild-type recombinants can be produced  And plaques will be formed

Figure 6.19

 As described in Figure 6.19, the first step in the deletion mapping strategy localized

rII

mutations to seven regions  Six in

rIIA

& one in

rIIB

 Other strains were used to eventually localize each

rII

mutation to one of 47 regions  36 in

rIIA

& 11 in

rIIB

 At this point, pairwise coinfections were made between mutant strains that had been localized to the same region  This would precisely map their location relative to each other  This resulted in a fine structure map with depicting the locations of hundreds of different

rII

mutations  Refer to Figure 6.20

Figure 6.20

Contain many mutations at exactly the same site within the gene

 Intragenic mapping studies were a pivotal achievement in our early understanding of gene structure  Some scientists had envisioned a gene as being a particle-like entity that could not be further subdivided  However, intragenic mapping revealed convincingly that this is not the case  It showed that  Mutations can occur at different parts within a single gene  Intragenic crossing over can recombine these mutations, resulting in wild-type genes