Coniugazione: un metodo per trasferire la resistenza all'ampicillina dal donatore al ricevente E. coli
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Overview
Fonte: Alexander S. Gold1, Tonya M. Colpitts1
1 Dipartimento di Microbiologia, Boston University School of Medicine, National Emerging Infections Diseases Laboratories, Boston, MA
Scoperta per la prima volta da Lederberg e Tatum nel 1946, la coniugazione è una forma di trasferimento genico orizzontale tra batteri che si basa sul contatto fisico diretto tra due cellule batteriche (1). A differenza di altre forme di trasferimento genico, come la trasformazione o la trasduzione, la coniugazione è un processo naturale in cui il DNA viene secreto da una cellula donatrice a una cellula ricevente in modo unidirezionale. Questa direzionalità e la capacità di questo processo di aumentare la diversità genetica dei batteri ha dato alla coniugazione la reputazione di una forma di “accoppiamento” batterico, che si ritiene abbia contribuito notevolmente al recente aumento dei batteri resistenti agli antibiotici (2, 3). Utilizzando pressioni selettive, ad esempio l’uso di antibiotici, la coniugazione è stata manipolata per l’uso in laboratorio, rendendola un potente strumento per il trasferimento genico orizzontale tra batteri e, in alcuni casi, da batteri a lieviti, cellule vegetali e animali (4). Oltre alle applicazioni in laboratorio, il trasferimento genico batterio-eucariota mediante coniugazione è un’entusiasmante via di trasferimento del DNA con una moltitudine di possibili applicazioni biotecniche e implicazioni naturali (5).
Si pensa che la coniugazione funzioni con un “meccanismo a due fasi” (6). In primo luogo, prima che qualsiasi DNA possa essere trasferito, la cellula donatrice deve entrare in contatto diretto cellula a cellula con il ricevente. Questo processo è stato caratterizzato meglio nei batteri gram-negativi, il più studiato dei quali è Escherichia coli. Il contatto cellula-cellula è stabilito dalla presenza di una complessa rete di filamenti extracellulari sul donatore nota come sex pilus, un elemento coniugativo codificato dal gene trasferibile noto come fattore F (fertilità) (7, 8). Oltre a stabilire un contatto tra donatore e ricevente, diverse proteine vengono trasportate attraverso il pilus sessuale al citoplasma ricevente, formando un condotto del sistema di secrezione di tipo IV (T4SS) tra le due cellule, una struttura necessaria per la seconda fase di coniugazione, il trasferimento del DNA (6). Combinando questa funzione del pilus sessuale con la replicazione del cerchio rotolante del DNA, la cellula donatrice è in grado di trasferire il DNA sotto forma di un elemento trasponibile, come un plasmide o un trasposone, al ricevente mediante un modello “shoot and pump” (6). In questo caso, lo “shooting” è il trasporto della proteina pilota, con DNA collegato, da parte del T4SS nella cellula ricevente, e il “pumping” è il trasporto attivo del DNA al ricevente, un processo dipendente dal T4SS e catalizzato dall’accoppiamento delle proteine (6). Il macchinario utilizzato in questo processo è composto da un’origine di sequenza di trasferimento (oriT), che deve essere fornita dal DNA nei geni cis e trans, che codificano una relaxasi, un complesso di formazione di coppie di accoppiamenti e una proteina di accoppiamento di tipo IV, e possono essere presenti in cis o trans (9). Questa relaxasi fende il sito nic all’interno della sequenza oriT e si attacca covalentemente all’estremità 5′ del filamento trasferito per produrre il relaxosoma, un complesso DNA-relaxasi a singolo filamento con altre proteine ausiliarie (9). Una volta formato, il relaxosoma si collega al complesso di formazione della coppia di accoppiamento, tramite la proteina di accoppiamento di tipo IV, che consente il trasferimento del complesso ssDNA-relaxasi nelle cellule riceventi da parte del T4SS (10). Una volta nel citoplasma del ricevente, il DNA può integrarsi nel genoma del ricevente o esistere separatamente sotto forma di plasmide, entrambi i quali consentono l’espressione dei suoi geni.
In questo esperimento, il ceppo di donatore di coniugazione ampiamente utilizzato E. coli WM3064 è stato utilizzato per trasferire il gene che codifica per la resistenza all’ampicillina al ceppo ricevente E. coli J53. Mentre entrambi i ceppi dei batteri gram-negativi erano resistenti alla tetraciclina, solo il ceppo donatore WM3064 aveva il gene per la resistenza all’ampicillina, codificato per nel vettore navetta pWD2-oriT, ed era auxotrofico all’acido diaminopimetico (DAP) (11-13). Questo esperimento consisteva in due fasi principali, la preparazione di ceppi donatore e ricevente, seguita dal trasferimento del gene di resistenza all’ampicillina dal donatore al ricevente mediante coniugazione (Figura 1).
Figura 1: Schema di coniugazione. Questo schema mostra il successo del trasferimento di un plasmide, solo un esempio di elemento di DNA trasponibile, da una cellula donatrice a una cellula ricevente usando la coniugazione. Al contatto con la cellula ricevente da parte della cellula donatrice attraverso il pilus sessuale, il plasmide si replica mediante replicazione del cerchio rotolante, si muove attraverso il complesso multiproteico unendo le due cellule e forma un nuovo plasmide a lunghezza intera nella cellula ricevente.
Incubando una miscela di cellule donatrici e riceventi, quindi placcando successivamente queste cellule in presenza di tetraciclina e DAP, ciò ha permesso il trasferimento riuscito del gene di resistenza all’ampicillina. Successivamente, le cellule placcate cresciute da questa miscela in presenza di tetraciclina e ampicillina, hanno rimosso tutte le cellule donatrici a causa della mancanza di DAP e di eventuali cellule riceventi che potrebbero non aver acquisito il gene di resistenza all’ampicillina, producendo batteri del ceppo J53 strettamente riceventi che hanno acquisito resistenza all’ampicillina (Figura 2). Una volta effettuato, il successo del trasferimento del gene di resistenza all’ampicillina è stato confermato dalla PCR. Poiché la coniugazione ha avuto successo, il ceppo J53 di E. coli conteneva pWD2-oriT ed era resistente all’ampicillina, e il gene che codifica per questa resistenza è rilevabile dalla PCR. Tuttavia, in caso di insuccestamento non ci sarebbe stato alcun rilevamento del gene di resistenza all’ampicillina e l’ampicillina avrebbe comunque funzionato come un antibiotico efficace contro il ceppo J53.
Figura 2: Schema del protocollo. Questo schema mostra una panoramica del protocollo presentato.
Figura 3A: La conferma del successo della coniugazione mediante PCR. A) Le scorte congelatrici dei campioni di controllo coniugati e negativi sono state strisciate su piastre di agar e una colonia è stata selezionata (rossa) per l’isolamento del DNA.
Procedure
Results
If conjugation was successful, a 500 base-pair sized band PCR product will be observed in the well in which PCR reaction 1 was loaded (Well #2 in Figure 3B), while no bands will be observed in the well in which PCR reaction 3 was loaded (Well #4 in Figure 3B). The presence of this band confirms the successful transfer of the ampicillin resistance gene, thereby conferring ampicillin resistance to the J53 strain of E. coli.
Figure 3B: The confirmation of successful conjugation by PCR. B) PCR analysis was done using DNA isolated from the select colony. The contents of each well are as follows: 1) DNA ladder, 2) Conjugation DNA and ampicillin primers, 3) Conjugation DNA and housekeeping primers, 4) Negative control DNA and ampicillin primers, 5) Negative control DNA and housekeeping primers, 6) No DNA and ampicillin primers, and 7) No DNA and negative control primers. The presence of a ~ 500 base-pair band PCR product from PCR reaction 1 (well 2), and the lack of this product from PCR reaction 3 (well 4), confirms successful conjugation.
Applications and Summary
Conjugation is a naturally occurring process of horizontal gene transfer that relies on the direct cell-to-cell contact of a donor cell and a recipient cell. This process is shared among all kinds of bacteria and has been instrumental in bacterial evolution, most notably antibiotic resistance. In the lab, conjugation can be used as an effective method of gene transfer that is much less disruptive when compared to other techniques. Outside of the laboratory, the ability to transfer DNA from bacteria to eukaryotes via conjugation offers an exciting new avenue of gene therapy and understanding the implications of these naturally occurring gene transfers, for example the relationship between bacterial infection and cancer, is a rapidly emerging area of research.
References
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- Lacroix B, Citovsky, V. Transfer of DNA from Bacteria to Eukaryotes. mBio. 2016;7(4):1-9.
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Transcript
Bacterial cells, such as E. coli, are able to transfer genetic information from cell-to-cell. Conjugation differs from other mechanisms of DNA transfer, such as transduction or transformation, in that it requires physical contact between the cells.
To proceed, conjugation requires a donor cell that expresses the fertility, or F, factor and a recipient cell without it, an F minus cell. The process requires two steps. The first is the establishment of direct cell-to-cell contact. To do this, the donor cell generates an extracellular filamentous structure called a sex pilus. It is named this since conjugation is a form of mating for asexually reproducing bacteria, but it should be noted that it is not true sexual reproduction as no gametes are exchanged and no offspring are formed.
The second step is delivery of DNA to the recipient cell. After the sex pilus establishes contact between two cells, a conduit called the Type IV secretion system is built allowing for the transfer of DNA. The donor cell then begins to replicate the extrachromosomal DNA that will be transferred selected based on the presence of a genetic element known as the OriT or origin of transfer. One end of the newly replicated DNA is threaded into the conduit through DNA protein binding. As the DNA is further replicated, it is pumped through the channel, facilitated by a complex of proteins encoded by genes located close to the OriT. Once the DNA is fully transferred, it will either form an extra chromosomal plasmid, or it may integrate into the chromosome of the recipient cell. Whichever the endpoint of the transferred DNA, the genes it encodes will then be expressed. This gene expression can be used to confirm successful conjugation.
For example, consider a scenario where the donor strain expresses ampicillin resistance and passes this on in the conjugated DNA to the recipient bacterium, but the recipient strain also has a tetracycline resistance gene not present in the donor. In this event, when the cells are plated on LB media containing both tetracycline and ampicillin, colonies should grow only from successfully conjugated bacteria, which will be expressing both resistance phenotypes. To further confirm successful conjugation, plasmid DNA from these colonies can be harvested and then a section of DNA specific to the transferred plasmid can be amplified using polymerase chain reaction, or PCR. When the PCR product is run on an electrophoresis gel alongside a ladder of standard sizes, a PCR fragment of a known size should be visible on the gel, further confirming successful conjugation. In this experiment, a plasmid will be used to transfer the ampicillin resistance gene via conjugation from a donor strain to a tetracycline-resistant recipient strain. After this, to confirm conjugation, the conjugation mixture will be incubated on a plate containing both antibiotics leaving only the transformed bacteria. Finally, successful conjugation will be further confirmed with PCR.
Before starting the procedure, put on the appropriate personal protective equipment, including a lab coat and gloves. Next, sterilize the workspace using 70% ethanol to wipe down the surface.
In this procedure, the ampicillin resistance gene will be transferred from the WM3064 strain of E. coli to the J53 strain of E. coli via conjugation. The donor strain WM3064 is resistant to tetracycline and ampicillin and it requires diaminopimelic acid, or DAP, to grow. The recipient strain J53 is only resistant to tetracycline and it does not require DAP to grow. This means that successfully conjugated cells should be resistant to tetracycline and ampicillin and can grow without DAP.
Prepare the donor strain culture by inoculating five milliliters of LB containing 0.3 millimoles of DAP with a scrap of the frozen donor strain glycerol stock. Then, prepare the recipient strain by inoculating five milliliters of LB broth without DAP with a scrap of the frozen recipient strain glycerol stock. Grow these cultures overnight at 37 degrees Celsius with aeration and shaking at 220 RPM in a shaking incubator. Once the cultures have grown to an OD 600 of two, remove one milliliter of culture from each and place this into two new separate 1.5 milliliter microcentrifuge tubes. Then, centrifuge these aliquots at 3000 RPM for five minutes to pellet the bacterial cells. Discard the supernatant and wash each pellet with 250 microliters of 1X PBS. Centrifuge the samples again and, after discarding the supernatant, resuspend each pellet in 500 microliters of PBS.
To begin the conjugation procedure, first combine 50 microliters of recipient cells with 50 microliters of donor cells in a 1.5 milliliter microcentrifuge tube and mix by pipetting up and down gently. Next, pipette 100 microliters of the recipient cell culture onto another 1X tetracycline plate containing DAP. Next, prepare your negative control by pipetting 100 microliters of the recipient cell culture only onto a non-selective agar plate containing DAP. Then, incubate the conjugation and negative control plates overnight at 37 degrees Celsius.
The next day, take a sterile cell scraper and harvest cells from the conjugation plate by collecting colonies. Then, transfer the colonies to a sterile 1.5 milliliter microcentrifuge tube containing one milliliter of 1X PBS. Repeat this process to collect the recipient cells from the other plate.
After this, vortex the samples to mix. After mixing, transfer the tubes to a centrifuge to gently pellet the cells. Discard the supernatant, then wash the cell pellets in one milliliter of PBS and vortex the tubes to resuspend the cells. Pellet the cells again by centrifuging. Discard the supernatant again and resuspend both cell pellets in one milliliter of PBS. Now, using a sterile pipette tip, plate 100 microliters of the conjugation reaction cell mixture onto an LB agar plate without DAP containing 1X tetracycline and 1X ampicillin. Repeat the plating method using 100 microliters of a ten-fold dilution of the same cell mixture in PBS onto another LB agar plate without DAP containing 1X tetracycline and 1X ampicillin.
Finally, pipette 100 microliters of the negative control cell mixture onto a single LB agar plate with 1X tetracycline only. After overnight incubation at 37 degrees Celsius, the colonies should be visible. Using a sterile pipette tip, pick a single colony from the conjugation reaction plate and add it to a tube containing five milliliters of selective LB media containing both antibiotics. Then, repeat the colony isolation by selecting a single colony from the recipient cell plate. Grow these cultures overnight at 37 degrees Celsius with aeration at 220 RPM.
The next day, wipe down the bench top with 70% ethanol and remove the plates from the incubator. Use a DNA mini prep kit to isolate DNA from 4. 5 milliliters of each culture according to the manufacturer’s instructions. After completing the DNA mini prep, elute the DNA using 35 microliters of nuclease-free water. Finally, use the remaining 0. 5 milliliters of each culture to prepare one milliliter glycerol stocks by adding 0.5 milliliters of 100% glycerol for a one-to-one dilution. Place these aliquots at minus 80 degrees Celsius for storage until needed.
To confirm successful conjugation by PCR, first prepare a PCR master mix by adding 75 microliters of 2X PCR master mix to a microcentrifuge tube. Then, add 7.5 microliters each of a 10 micromolar forward primer and a 10 micromolar reverse primer designed to amplify the ampicillin resistance gene from the plasmid. Next, prepare a second PCR master mix by adding 75 microliters of 2X PCR master mix to a microcentrifuge tube and then adding 7.5 microliters each of a 10 micromolar forward primer and 10 micromolar reverse primer designed to amplify a housekeeping gene, in this case DNA gyrase B.
Now, add 15 microliters of the first master mix to a PCR tube and then add 10 nanograms, approximately two microliters of the template experimental DNA to the same tube. Bring the reaction up to a final volume of 25 microliters with nuclease-free water. Repeat these steps to produce the remaining five reactions, so that the tubes contain the components shown here. Now, transfer these reactions to a thermocycler with the block pre-heated to 98 degrees Celsius and then initiate the program. After completion of the PCR, remove the tubes from the machine. Then, load two microliters of each reaction mixed with two microliters of loading dye and four microliters of a molecular weight marker into consecutive wells of a 1% agarose gel. Set the gel to run at 150 volts for 20 minutes. Finally, visualize the gel using a UV illuminator.
In this experiment, the successful transfer of the ampicillin resistance gene via conjugation was confirmed via PCR. Here, a roughly 500 base pair sized band should be observed in the well containing the conjugated DNA and ampicillin primers, well two in this example. A housekeeping gene, DNA gyrase B, was loaded into wells three and five with conjugated DNA and recipient cell DNA, respectively. Bands observed in these wells act as a positive control to ensure the DNA template was present and that PCR was successful. Bands should not be observed in the well containing the reaction for recipient cell DNA and the ampicillin primer pair, well four in this example, because the recipient cells are not ampicillin-resistant. Additionally, no bands should be observed in the reactions lacking template DNA, wells six and seven here. If these conditions are met, this will confirm the successful transfer of the ampicillin resistance gene, conferring ampicillin resistance from the WM3064 strain of E. coli to the J53 strain of E. coli.