bacterial genetics. bacteria are haploid identify loss-of-function mutations easier recessive...

Bacterial Genetics

Bacterial Genetics Bacteria are haploid

identify loss-of-function mutations easier recessive mutations not masked

6-2Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Bacterial Genetics Bacteria reproduce asexually

Crosses not used genetic transfer

bacterial DNA segments transferred

6-3Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Enhances genetic diversity Types of transfer

Conjugation direct physical contact & exchange

Transduction phage

Transformation uptake from environment

6-4Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Genetic Transfer

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

Genes for conjugation

F-factor Plasmid

Figure 6.4

Conjugation

Figure 6.4

Conjugation

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

1950s, Luca Cavalli-Sforza discovered E. coli strain very efficient at transferring chromosomal genes

designated strain Hfr (high frequency of recombination)

Hfr strains result from integration of F' factor into chromosome

Hfr Strains

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

Figure 6.5b

order of transfer is lac+ – pro+

F– now lac+ pro–

F– now lac+ pro+

Hfr Conjugation

Elie Wollman & François Jacob The rationale

Hfr chromosome transferred linearly interruptions at different times various lengths

transferred order of genes on chromosome deduced by

interrupting transfer at various time

Interrupted Mating Technique

Wollman & Jacob started the experiment with two E. coli strains Hfr strain (donor) genotype

thr+ : Can synthesize threonine leu+ : Can synthesize leucine azis : Killed by azide tons : Can be infected by T1 phage lac+ : Can metabolize lactose gal+ : Can metabolize galactose strs : Killed by streptomycin

F– strain (recipient) genotype thr– leu– azir tonr lac – gal – strr

6-21Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 6.6

Interpreting the Data

6-26Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Minutes that Bacterial

Cells were Allowed to

Mate Before Blender

Treatment

Percent of Surviving Bacterial Colonies with the Following Genotypes

thr+ leu+ azis tons lac+ gal+

5 –– –– –– –– ––

10 100 12 3 0 0

15 100 70 31 0 0

20 100 88 71 12 0

25 100 92 80 28 0.6

30 100 90 75 36 5

40 100 90 75 38 20

50 100 91 78 42 27

60 100 91 78 42 27

After 10 minutes, the thr+ leu+ genotype was obtained

The azis gene is transferred first

It is followed by the tons gene

The lac+ gene enters between 15 & 20 minutes

The gal+ gene enters between 20 & 25 minutes

There were no surviving colonies after 5 minutes of mating

6-27

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

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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

6-28Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The E. coli Chromosome

6-29Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or displayFigure 6.7

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

6-30Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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

Therefore these two genes are approximately 9 minutes apart along the E. coli chromosome

Figure 6.7

Transduction is the transfer of DNA from one bacterium to another via a bacteriophage

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Transduction

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

6-31

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or displayFigure 6.9

Virulent phages only undergo a lytic cycle

Temperate phages can follow both cycles

6-32

Prophage can exist in a dormant

state for a long time

It will undergo the lytic cycle

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

6-54

Plaques

Figure 6.14

Figure 6.10

Any piece of bacterial DNA can be incorporated into the phage

This type of transduction is termed generalized transduction

Transduction

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

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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

Transformation

6-47Figure 6.12

A region of mismatch

By DNA repair enzymes

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

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-48

Transformation

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

Horizontal Gene Transfer

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

6-51Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

6.2 INTRAGENIC MAPPING IN BACTERIOPHAGES

6-52Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 6.13

Contains the genetic material

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:

6-53

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

6-54

Plaques

Figure 6.14

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

6-55Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Benzer studied one category of T4 phage mutant, designated rII (r stands for rapid lysis)

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

6-56Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Benzer collected many rII mutant strains that can form large plaques in E. coli B & none in E. coli K12()

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

To answer this question, he conducted complementation experiments

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-57

Complementation Tests

6-58

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

Figure 6.16Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

6-59

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

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-60

Coinfection

rII mutations

rII mutations

Viruses cannot form plaques in E. coli K12()

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 describes the general strategy for intragenic mapping of rII phage mutations

6-61

6-62

r103

r104

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

Take some of the phage preparation, dilute it somewhat (10-6) & infect E. coli K12()

66 plaques 11 plaques

Total number of phages

Number of wild-type phages produced by intragenic recombination

Both rII mutants & wild-type phages

can infect this strain

rII mutants cannot infect this strain

6-63

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

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The phage preparation used to infect E. coli B was diluted by 108 (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 108 = 6.6 X 109 or 6.6 billion phages per milliliter

The phage preparation used to infect E. coli k12() was diluted by 106 (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 106 or 11 million phages per milliliter

6-64

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

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

2 [wild-type plaques obtained in E. coli

k12()]Frequency of recombinants =Total number of plaques

obtained in E. coli B

2(11 X 106)

6.6 X 109Frequency of recombinants = = 3.3 X 10–3 = 0.0033

In this example, there was approximately 3.3 recombinants per 1,000 phages

6-65

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

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Benzer used deletion mapping to localize many rII mutations to a fairly short region in gene A or gene B

He utilized deletion strains of phage T4 Each is missing a known segment of the rIIA and/or rIIB

genes

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-66

Deletion Mapping

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 formedCopyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-67

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-68Figure 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

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-69

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-70Figure 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

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-71