conjugación y transducción bacteriana

8/2/2019 conjugacin y transduccin bacteriana

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Bacterial Genetics


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Prokaryote Basics

The largest and most obvious division of livingorganisms is into prokaryotes vs. eukaryotes.

Eukaryotes are defined as having their genetic materialenclosed in a membrane-bound nucleus, separate from

the cytoplasm. In addition, eukaryotes have othermembrane-bound organelles such as mitochondria,lysosomes, and endoplasmic reticulum. almost allmulticellular organisms are eukaryotes.

In contrast, the genome of prokaryotes is not in aseparate compartment: it is located in the cytoplasm(although sometimes confined to a particular regioncalled a nucleoid). Prokaryotes contain no membrane-bound organelles; their only membrane is the membranethat separates the cell form the outside world. Nearly allprokaryotes are unicellular.


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Three Domains of Life


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Prokaryote vs. Eukaryote Genetics

Prokaryotes are haploid, and they contain a singlecircular chromosome. In addition, prokaryotes oftencontain small circular DNA molecules called plasmids,that confer useful properties such as drug resistance.Only circular DNA molecules in prokaryotes can

replicate. In contrast, eukaryotes are often diploid, and eukaryotes

have linear chromosomes, usually more than 1.

In eukaryotes, transcription of genes in RNA occurs inthe nucleus, and translation of that RNA into proteinoccurs in the cytoplasm. The two processes areseparated from each other.

In prokaryotes, translation is coupled to transcription:translation of the new RNA molecule starts before

transcription is finished.


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Bacterial Culture

Surprisingly, many, perhaps even most, of thebacteria on Earth cannot be grown in thelaboratory today.

Bacteria need a set of specific nutrients, the

correct amount of oxygen, and a propertemperature to grow. The common gutbacterium Escherichia coli(E. coli) grows easilyon partially digested extracts made from yeastand animal products, at 37 degrees in a normalatmosphere. These simple growth conditionshave made E. coli a favorite lab organism, whichis used as a model for other bacteria.


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More Culture

Bacteria are generally grown in either of 2ways: on solid media as individual colonies,or in liquid culture.

The nutrient broth for liquid culture allowsrapid growth up to a maximum density.Liquid culture is easy and cheap.

Solid media use the same nutrient broth asliquid culture, solidifying it with agar. Agar apolysaccharide derived from seaweed thatmost bacteria cant digest.

The purpose of growth on solid media is toisolate individual bacterial cells, then groweach cell up into a colony. This is thestandard way to create a pure culture of

bacteria. All cells of a colony are closelyrelated to the original cell that started thecolony, with only a small amount of geneticvariation possible.

Solid media are also used to count thenumber of bacteria that were in a culturetube.


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Bacterial Mutants

Mutants in bacteria are mostly biochemical in nature, because we cant generally seethe cells.

The most important mutants are auxotrophs. An auxotroph needs some nutrient thatthe wild type strain (prototroph) can make for itself. For example, a trp- auxotrophcant make its own tryptophan (an amino acid). To grow trp- bacteria, you need toadd tryptophan to the growth medium. Prototrophs are trp+; they dont need any

tryptophan supplied since they make their own.

Chemoauxotrophs are mutants that cant use some nutrient (usually a sugar) thatprototrophs can use as food. For example, lac- mutants cant grow on lactose (milksugar), but lac+ prototrophs can grow on lactose.

Resistance mutants confer resistance to some environmental toxin: drugs, heavy

metals, bacteriophages, etc. For instance, AmpR

causes bacteria to be resistant toampicillin, a common antibiotic related to penicillin.

Auxotrophs and chemoauxotrophs are usually recessive; drug resistance mutants areusually dominant.


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Replica Plating

A common way to find bacterial mutants is replica plating, whichmeans making two identical copies of the colonies on a petri plateunder different conditions.

For instance, if you were looking for trp- auxotrophs, one plate wouldcontain added tryptophan and the other plate would not have anytryptophan in it.

Bacteria are first spread on the permissive plate, the plate thatallows both mutants and wild type to grow, the plate containingtryptophan in this case. They are allowed to grow fro a while, then acopy of the plate is made by pressing a piece of velvet onto thesurface of the plate, then moving it to a fresh plate with therestrictive condition (no tryptophan). The velvet transfers some cells

from each colony to an identical position on the restrictive plate. Colonies that grow on the permissive plate but not the restrictive

plate are (probably) trp- auxotrophs, because they can only grow iftryptophan is supplied.


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Replica Plating, pt. 2


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Bacterial Sexual Processes

Eukaryotes have the processes of meiosis to reducediploids to haploidy, and fertilization to return the cells tothe diploid state. Bacterial sexual processes are not soregular. However, they serve the same aim: to mix the

genes from two different organisms together. The three bacterial sexual processes:

1. conjugation: direct transfer of DNA from one bacterial cell toanother.

2. transduction: use of a bacteriophage (bacterial virus) to

transfer DNA between cells. 3. transformation: naked DNA is taken up from the environment

by bacterial cells.


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Transformation

We arent going to speak much of this process, except tonote that it is very important for recombinant DNA work.The essence of recombinant DNA technology is toremove DNA from cells, manipulate it in the test tube,

then put it back into living cells. In most cases this isdone by transformation.

In the case of E. coli, cells are made competent to betransformed by treatment with calcium ions and heatshock. E. coli cells in this condition readily pick up DNA

from their surroundings and incorporate it into theirgenomes.


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Conjugation

Conjugation is the closest analogue inbacteria to eukaryotic sex.

The ability to conjugate is conferred bythe F plasmid. A plasmid is a smallcircle of DNA that replicatesindependently of the chromosome.Bacterial cells that contain an F

plasmid are called F+. Bacteria thatdont have an F plasmid are called F-.

F+ cells grow special tubes called sexpilli from their bodies. When an F+cell bumps into an F- cell, the sex pillihold them together, and a copy of theF plasmid is transferred from the F+

to the F-. Now both cells are F+. Why arent all E. coli F+, if it spreadslike that? Because the F plasmid canbe spontaneously lost.


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Hfr Conjugation

When it exists as a freeplasmid, the F plasmid canonly transfer itself. This isntall that useful for genetics.

However, sometimes the Fplasmid can become

incorporated into the bacterialchromosome, by a crossoverbetween the F plasmid and thechromosome. The resultingbacterial cell is called an Hfr,which stands for High

frequency of recombination. Hfr bacteria conjugate just likeF+ do, but they drag a copy ofthe entire chromosome into theF- cell.


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Interrupted Mating

Chromosome transfer from theHfr into the F- is slow: it takesabout 100 minutes to transferthe entire chromosome.

The conjugation process can

be interrupted using a kitchenblender.

By interrupting the mating atvarious times you candetermine the proportion of F-cells that have received agiven marker.

This technique can be used tomake a map of the circular E.coli chromosome.


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Different Hfr Strains

The F plasmid canincorporate into thechromosome in almostany position, and in eitherorientation. Note that the

genes stay in fixedpositions, but the genesenter the F- in differentorders and times, basedon where the F was

incorporated in the Hfr. Data are for initial time ofentry of that gene into theF-.

gene Hfr 1 Hfr 2 Hfr 3

azi 8 29 88

ton 10 27 90

lac 17 20 3

gal 25 12 11


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Intracellular Events in Conjugation

The piece of chromosome that enters the F- form the Hfris linear. It is called the exogenote.

The F- cells own chromosome is circular. It is called theendogenote.

Only circular DNA replicates in bacteria, so genes on theexogenote must be transferred to the endogenote for theF- to propagate them.

This is done by recombination: 2 crossovers betweenhomologous regions of the exogenote and theendogenote. In the absence of recombination,conjugation in ineffective: the exogenote enters the F-,but all the genes on it are lost as the bacterial cellreproduces.


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F-prime (F)

The process of making an Hfr from an F+ involves a crossover between theF plasmid and the chromosome. This process is reversible: an Hfr canrevert to being F+ when the F plasmid DNA incorporated into the Hfrchromosome has a crossover and loops out of the chromosome forming anF plasmid once again.

Sometimes the looping-out and crossing-over process doesnt happen atthe proper place. When this happens, a piece of the bacterial chromosomecan become incorporated into the F plasmid. This is called an F (F-prime)plasmid.

F plasmids can be transferred by conjugation. Conjugation with an F (or aregular F plasmid) is much faster and more efficient than with an Hfr,because only a very small piece of DNA is transferred. Since the F carriesa bacterial gene, this allele can be rapidly moved into a large number ofother strains. This permits its function to be tested rapidly. Also, tests ofdominance can be done.

A cell containing an F is merodiploid: part diploid and part haploid. It isdiploid for the bacterial gene carried by the F (one copy on the F and theother on the chromosome), and haploid for all other genes.


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Transduction

Transduction is the process of moving bacterial DNAfrom one cell to another using a bacteriophage.

Bacteriophage or just phage are bacterial viruses.They consist of a small piece of DNA inside a protein

coat. The protein coat binds to the bacterial surface,then injects the phage DNA. The phage DNA then takesover the cells machinery and replicates many virusparticles.

Two forms of transduction: 1. generalized: any piece of the bacterial genome can be

transferred

2. specialized: only specific pieces of the chromosome can betransferred.


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General Phage Life Cycle

1. Phage attaches to thecell and injects its DNA.

2. Phage DNA replicates,and is transcribed intoRNA, then translated intonew phage proteins.

3. New phage particlesare assembled.

4. Cell is lysed, releasingabout 200 new phageparticles.

Total time = about 15minutes.


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Generalized Transduction

Some phages, such as phage P1, break up the bacterialchromosome into small pieces, and then package it intosome phage particles instead of their own DNA.

These chromosomal pieces are quite small: about 1 1/2minutes of the E. coli chromosome, which has a totallength of 100 minutes.

A phage containing E. coli DNA can infect a fresh host,because the binding to the cell surface and injection ofDNA is caused by the phage proteins.

After infection by such a phage, the cell contains anexogenote (linear DNA injected by the phage) and anendogenote (circular DNA that is the hostschromosome).

A double crossover event puts the exogenotes genesonto the chromosome, allowing them to be propagated.


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Transduction Mapping

Only a small amount of chromosome, a fewgenes, can be transferred by transduction. Thecloser 2 genes are to each other, the more likely

they are to be transduced by the same phage.Thus, co-transduction frequency is the keyparameter used in mapping genes bytransduction.

Transduction mapping is for fine-scale mappingonly. Conjugation mapping is used for mappingthe major features of the entire chromosome.


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Mapping Experiment

Important point: the closer 2 genes are to each other, thehigher the co-transduction frequency.

We are just trying to get the order of the genes here, notput actual distances on the map.

Expt: donor strain is aziR leu+ thr+. Phage P1 is grown onthe donor strain, and then the resulting phage are mixedwith the recipient strain: aziS leu- thr-. The bacteria thatsurvive are then tested for various markers

1. Of the leu+ cells, 50% are aziR, and 2% are thr+. Fromthis we can conclude that azi and leu are near eachother, and that leu and thr are far apart.

But: what is the order: leu--azi--thr, or azi--leu--thr ?


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Mapping Experiment, pt. 2

2. Do a second experiment to determine theorder. Select the thr+ cells, then determine howmany of them have the other 2 markers. 3% arealso leu+ and 0% are also aziR.

By this we can see that thr is closer to leu than itis to azi, because thr and azi are so far apartthat they are never co-transduced.

Thus the order must be thr--leu--azi.

Note that the co-transduction frequency for thrand leu are slightly different for the 2experiments: 2% and 3%. This is attributable toexperimental error.


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Larger Experiment

A few hints:

1. There are 3 experiments shown. In each, 1 geneis selected, and the frequencies of co-transductionwith the other genes is shown.

2. start with 2 genes that are selected and that havea non-zero co-transduction frequency. Put them onthe map.

3. Then locate the other genes relative to the first 2.


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selected

co-transduced

freq selected

co-transduced

freq selected

co-transduced

freq

e a 0 f a 90 c a 74

e b 85 f b 2 c b 32

e c 29 f c 41 c d 0

e d 62 f d 0 c e 21

e f 0 f e 0 c f 39


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Intro to Specialized Transduction

Some phages can transfer only particular genesto other bacteria.

Phage lambda () has this property. To

understand specialized transduction, we need toexamine the phage lambda life cycle.

lambda has 2 distinct phases of its life cycle.The lytic phase is the same as we saw with the

general phage life cycle: the phage infects thecell, makes more copies of itself, then lyses thecell to release the new phage.


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Lysogenic Phase

The lysogenic phase of the lambda life cycle starts the same way:the lambda phage binds to the bacterial cell and injects its DNA.Once inside the cell, the lambda DNA circularizes, then incorporatesinto the bacterial chromosome by a crossover, similar to theconversion of an F plasmid into an Hfr.

Once incorporated into the chromosome, the lambda DNA becomes

quiescent: its genes are not expressed and it remains a passiveelement on the chromosome, being replicated along with the rest ofthe chromosome. The lambda DNA in this condition is called theprophage.

After many generations of the cell, conditions might get harsh. Forlambda, bad conditions are signaled when DNA damage occurs.

When the lambda prophage receives the DNA damage signal, itloops out and has a crossover, removing itself from thechromosome. Then the lambda genes become active and it goesinto the lytic phase, reproducing itself, then lysing the cell.


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More Lysogenic Phase


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Specialized Transduction

Unlike the F plasmid that can incorporate anywhere in the E. coligenome, lambda can only incorporate into a specific site, called att.The gal gene is on one side of att and the bio gene (biotinsynthesis) is on the other side.

Sometimes when lambda come out of the chromosome at the end ofthe lysogenic phase, it crosses over at the wrong point. This is very

similar to the production of an F from an Hfr. When this happens, a piece of the E. coli chromosome is

incorporated into the lambda phage chromosome These phage that carry an E. coli gene in addition to the lambda

genes are called specialized transducing phages. They can carryeither the gal gene or the bio gene to other E. coli.

Thus it is possible to quickly develop merodiploids (partial diploids)for any allele you like of gal or bio. Note that this trick cant be usedwith other genes, but only for genes that flank the attachment site forlambda or another lysogenic phage.

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conjugación y transducción bacteriana