10genetica bacteriana

7/31/2019 10Genetica bacteriana

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S

GENETICA

BACTERIANA

INTRODUCCION

S Virus y bacterias son responsables por a 1/3 de las muertesmundiales

S Bacterias son haploides

S Diferencias allicas en cepas diferente

S Es ms fcil identificar las mutaciones loss of function

S Las mutaciones recesivas se expresan

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

GENETICA

S Reproduccin asexual

S Transferencia genticaS Se transfiere material gentico


S Conjungacin

S Transduccin

S Transformacin


TRANSFERENCIA DE MATERIAL

GENETICO


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6-5

Table 6.1

S 1946: Joshua Lederberg and Edward Tatum

S Requerimientos nutricionales

S Auxotrofo: no puede sintetizar nutriente

S Prototrofo: sintetiza nutriente en medio mnimo

S Requiere contacto fsico


Conjugacin

Figure 6.1 6-7

Auxotrofos no crecen en medio mnimo

Se unen ambas cepas y crecen colonias en medio mnimo


Bernard Davis: contacto fsico para que ocurratransferencia

U-tube permite paso DNA, pero no contacto conbacterias

U-tube


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6-10

Figure 6.2

Nutrient agarplates lacking

biotin, methionine,phenylalanine and

threonine

No colonies No colonies

S Thus, without physical contact, the two bacterial strains did nottransfer genetic material to one another


Factor F

S Factor fertilidad

S F+

S F-

S plasmidio

6-12Figure 6.3

These genes play a role in the transfer of DNA

They are thus designated traand trbfollowed by a capital letter

Plasmidio conjugante

6-14Figure 6.4


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Figure: 16-06_a

An F+ x F- mating, demonstrating how the recipient F- cell is converted to F+. During conjugation, theDNA of the F factor i s replicated with one new copy entering the recipient cell, converting it to F+. Toindicate the clockwise rotation during replication, a black bar is shown on the F factor.

CONJUGACION

CONJUGACION

Figure: 16-06_b

An F+ x F- mating, demonstrating how the recipient F- cell isconverted to F+. During conjugation, the DNA of the F factor isreplicated with one new copy entering the recipient cell,converting it to F+. To indicate the clockwise rotation duringreplication, a black bar is shown on the F factor.

S 1950s, Luca Cavalli-Sforza- E. colitransfiere genes cromosomales

S Hfr:for High frequency of recombination

Hfr


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INTEGRACION FACTOR F AL CROMOSOMA

CONVERSION DE F+ F

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-20Figure 6.5b

lac+Ability tometabolize lactose

lac Inability

pro+Ability tosynthesize proline

pro Inability

Therefore, the order oftransfer is lac+ pro+

Transmisin Hfr

S 1950s: Elie Wollman & Franois Jacob

S El tiempo que tardan los genes en transferirse es proporacional alorden en el cromosoma bacteriano

S Hfr se transfiere de forma lineal al F-

6-21

Apareo interrumpido

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


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Caractersticas

S thr+: sintetiza treonina

S leu+: sintetiza leucine

S azis : sensible a azide (txico)

S tons: sensible a infeccin por T1(bacterifago)

S lac+: metaboliza lactosa

S gal+: metaboliza galactosa

S strs : sensible a streptomicina

S thr leu azirtonrlac gal strr

S r = resistant


HfrF-

6-25Figure 6.6

Apareo interrumpido

Resultados


Minutes that

BacterialCells were

Allowed toMate Before

BlenderTreatment

Percent of Surviving Bacterial Colonieswith 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 wasobtained

The azisgene is

transferred first

It is followed bythe tonsgene

The lac+gene

enters between 15

and 20 minutes

The gal+gene

enters between

20 and 25minutes

There were no surviving coloniesafter 5 minutes of mating

6-30Copyright 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 recipientduring a conjugation

experiment


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S Bacterias y levaduras

S Hasta 500,000 bp

S Varias copias por clula


Plasmidios

SFertilidad

S Resistencia

S Degradativo

S Col-plasmids codifican para colicines (para matar bacterias)

S Virulencia


Categoras plasmidios

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

Virulent phages onlyundergo a lytic cycle

Temperate phages canfollow both cycles

6-35

Prophage canexist in a dormant

state for a longtime

It will switch to

the lytic cycle

Transduccin

6-37Figure 6.10

Transduccin

generalizada


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6-44Figure 6.11

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-45

These coloniesmust be met+

To determine whetherthey are also arg+

streak onto plate thatlacks both amino acids

21/50

S 1928: GriffithS Natural

S Artificial

S Clula competente

S Recombinacin no homloga


Transformacin

6-48Figure 6.12

Transformacin


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6-55

Figure 6.13

Contains the

genetic material

Used for attachment to

the bacterial surface

S In the 1950s, Seymour Benzer embarked on a ten-year studyfocusing on the function of the T4 genes

S He conducted a detailed type of genetic mapping known as intragenicor fine structure mapping

S The difference between intragenic and intergenic mapping is:

6-56

S A plaque is a cleararea on an otherwiseopaque bacterial lawnon the agar surface of

a petri dish

S It is caused by thelysis of bacterial cellsas a result of thegrowth andreproduction ofphages

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Plaques

Figure 6.14


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S Some mutations in the phages genetic material can

alter the ability of the phage to produce plaques

S Thus, plaques can be viewed as traits of bacteriophages

S Plaques are visible with the naked eye

S So mutations affecting them lend themselves to easiergenetic analysis

S An example is a rapid-lysis mutant of bacteriophage

T4, which forms unusually large plaques

S Refer to Figure 6.15

S This mutant lyses bacterial cells more rapidly than do thewild-type phages

S Rapid-lysis mutant forms large, clearly defined plaques

S Wild-type phages produce smaller, fuzzy-edged plaques


S Benzer studied one category of T4 phage mutant, designated rII(rstandsfor rapid lysis)

S It behaved differently in three different strains of E. coli

S In E. coli B

S rIIphages produced unusually large plaques that had poor yields of

bacteriophages

S The bacterium lyses so quickly that it does not have time to produce many newphages

S In E. coli K12S

S rIIphages produced normal plaques that gave good yields of phages

S In E. coli K12() (has phage lambda DNA integrated into itschromosome)

S rIIphages were not able to produce plaques at all

S As expected, the wild-type phage could infect all three strains 6-59Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

S Benzer collected many rIImutant strains that canform large plaques in E. coli Band none in E. coliK12()

S But, are the mutations in the same gene or indifferent genes?

S To answer this question, he conductedcomplementation experiments


Complementation Tests

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S Figure 6.16 shows the possible outcomes of

complementation experiments involving coinfection

of plaque formation mutants

Figure 6.16


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6-62

S Benzer carefully considered the pattern of

complementation and noncomplementation

S He determined that the rIImutations occurred in twodifferent genes, which were termed rIIA and rIIB

S Benzer coined the term cistron to refer to thesmallest genetic unit that gives a negativecomplementation test

S So, if two mutations occur in the same cistron, theycannot complement each other

S A cistron is equivalent to a gene

S However, it is not as commonly usedCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

S At an extremely low rate, two noncomplementing strains ofviruses can produce an occasional viral plaque, if intragenicrecombination has occurred

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rIImutations

rIImutations

Viruses cannot

form plaques in

E. coli K12()

Viruses cannot

form plaques inE. 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

S Figure 6.18 describes the general strategy for intragenic mapping ofrIIphage mutations

6-64Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-65Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Total number

of phages

Number of wild-typephages produced byintragenic recombination

r103

r104

Both rIImutants

and wild-typephages can infectthis strain

rIImutants cannot

infect this strain


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6-66

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


The phage preparation used to infect E. coli Bwas diluted

by 108

(1:100,000,000) 1 ml of this dilution was used and 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() wasdiluted by 106 (1:1,000,000)

1 ml of this dilution was used and 11 plaques were produced

Therefore, the total number of wild-type phages is

11 X 106 or 11 million phages per milliliter

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S In this experiment, the intragenic recombination producesan equal number of recombinants

S Wild-type phages and double mutant phages

S However, only the wild-type phages are detected in theinfection of E. coli k12( )

S Therefore, the total number of recombinants is the number of wild-type phages multiplied by two


2 [wild-type plaquesobtained inE. coli k12()]

Frequency of recombinants =Total number of plaques

obtained inE. coli B

2(11 X 106)

6.6 X 109Frequency of recombinants = = 3.3 X 103 = 0.0033

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

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S As in eukaryotic mapping, the frequency of recombinants canprovide a measure of map distance along the bacteriophagechromosome

S In this case the map distance is between two mutations in the samegene

S The frequency of intragenic recombinants is correlated withthe distance between the two mutations

S The farther apart they are the higher the frequency of recombinants

S Homoallelic mutations

S Mutations that happen to be located at exactly the same site in a gene

S They are not able to produce any wild-type recombinants

S So the map distance would be zero


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

S He utilized deletion strains of phage T4

S Each is missing a known segment of the rIIA and/or rIIBgenes


Deletion Mapping

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S Lets suppose that the goal is to know the

approximate location of an rIImutation, such as r103

S E. coli k12() is coinfected with r103and a deletion strain

S If the deleted region includes the same region that contains the r103mutation

S No intragenic wild-type recombinants are produced

S Therefore, plaques will not be formed

S If the deleted region does not overlap with the r103mutation

S Intragenic wild-type recombinants can be produced

S And plaques will be formed

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-70 6-71Figure 6.19

Mutation must be in regioncontained in BP242 butnot in PT1. Thiscorresponds to A4 in therIIA gene

S As described in Figure 6.19, the first step in the deletionmapping strategy localized rIImutations to seven regions

S Six in rIIA and one in rIIB

S Other strains were used to eventually localize each rIImutation to one of 47 regions

S 36 in rIIA and 11 in rIIB

S At this point, pairwise coinfections were made between

mutant strains that had been localized to the same region

S This would precisely map their location relative to each other

S This resulted in a fine structure map with depicting thelocations of hundreds of different rIImutations

S Refer to Figure 6.20Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-72 6-73

Figure 6.20

Contain many mutations

at exactly the same site

within the gene

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S Intragenic mapping studies were a pivotalachievement in our early understanding of genestructure

S Some scientists had envisioned a gene as being a

particle-like entity that could not be furthersubdivided

S However, intragenic mapping revealed convincinglythat this is not the caseS It showed that

S Mutations can occur at different parts within a single gene

S Intragenic crossing over can recombine these mutations, resulting in wild-

type genes


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