Name: detection of horizontal gene transfer mediated by natural conjugative plasmids in e. coli
Uploaded: 2023-03-24T13:45:00
Duration: 6 min 56 s
Description: Watch this Scientific Journal Video about Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli at JoVE.com

LM-B, IMG, AT, and IS were supported by NIH grant GM055246 awarded to LM-B. GC-C was awarded the UC MEXUS-CONACYT Postdoctoral Fellowship for two consecutive years: AY 2017-18 and 2018-19. We greatly thank students Sage Chavez and Pepper St. Clair from the Genentech-Foundation sponsored Academic Inspiration Network (GAIN) mentoring program for their technical support in experiments.

     

<ol>
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The authors have nothing to disclose.

Conjugative plasmids provide access to a communal pool of genes within a particular environmental setting through recombination and horizontal gene transfer34. Thus, conjugative plasmids are evolutionary entities capable of acquiring and conferring functions that allow bacteria to adapt to multiple conditions (including resistance to antibiotics, resistance to metals, acquisition of metals, biofilm formation, and pathogenic genes, among others) within a temporal scale of hours.
This work presents a protocol for the identification of conjugative plasmids in bacteria. For the protocol to work, the markers used need to differentiate donor and recipient strains, as shown by the controls in media A and B. They also need to effectively select cells carrying the plasmid. The mating reaction is critical. In this reaction, donors and recipients need to make prolonged contact (through pili) for conjugation to occur. Anything that can disrupt cell-to-cell contact, such as an insufficient incubation time or shaking or stirring the culture, thus decreases the efficiency of conjugation. The choice of recipient is also critical, as some recipient strains are refractory to conjugation35,36. LMB100 is proposed as the recipient of choice, as it is able to take plasmids of multiple incompatibility types.
The conjugation rate is specific for each plasmid-donor chromosome pair, recipient, and environmental condition. The conjugation of specific plasmids has been found to be sensitive to a large number of variables, including growth phase, cell density, donor-to-recipient ratio, whether conjugation is conducted in liquid or solid media, carbon, oxygen, bile salts, metal concentrations, presence of mammalian cells, temperature, pH, and mating time13,14,15. The study of these interactions depends on the initial detection of a conjugative plasmid to be studied in depth. Thus, the protocol described here can also be modified to explore the impact of different experimental variables, although this comes at the cost of restricting the number of screened donors. It can also identify mobilizable plasmids in hosts that have helper plasmids (i.e.,&#160;plasmids that enable conjugation by providing functions missing from the mobilizable plasmids in trans6).
Knowing the sequence of the conjugative plasmid is recommended (but not necessary to detect conjugation, as discussed above). When the donors are lactose-negative (producing yellow colonies on MacConkey agar), a lactose-positive recipient (producing pink colonies on MacConkey agar) can be used to distinguish true transconjugants from donor cells that are able to grow on the media to select transconjugants. The protocol described here is designed to detect conjugative plasmids from environmental, commensal, and pathogenic members of the family Enterobacteriaceae; however, it can be used with any bacterial species with a suitable donor, recipient, antibiotic(s), and color markers. The identification of these critical components in other bacteria requires systematic studies using multiple donors, recipients, and markers (antibiotics and colors). This protocol has not been tested in Gram-positive bacteria.
Several variants of the method presented here have been reported in the literature, recently reviewed20. The qualitative outcome can also be referred to in different ways (e.g., exconjugant frequency and gene transfer frequency), and dimensionless units can be used to report conjugation efficiency (mL/CFU x h)20.
The protocol described here solves several limitations previously not addressed by existing methods16,18,19,20. First, a suitable recipient has been identified. Second, the use of two antibiotics (rifampin and streptomycin) to select true transconjugants minimizes the possibility that donors evolve resistance to one of the antibiotics used to select transconjugants through spontaneous mutagenesis. False transconjugants can also result from bystander protection by hydrolysis of the antibiotic used to select transconjugants by enzymes (e.g., beta-lactamases) produced in large quantities by the donor. Third, several experimental controls are included to ensure that the product of mating is a true transconjugant (i.e., a recipient cell that has stably incorporated the conjugative plasmid of the donor). Here, we presented two independent methods to test the authenticity of the transconjugants, namely a colorimetric marker in MacConkey agar, and PCR detection of the replicons and antibiotic resistance genes of the conjugative plasmid in the recipient. Also, the protocol described here is designed to isolate and characterize conjugative plasmids from environmental, commensal, and pathogenic members of the family Enterobacteriaceae (Escherichia, Klebsiella, Enterobacter, Citrobacter, Salmonella, Shigella, and other species).
To understand the mechanics of conjugation, experimental observations have been modeled using computer simulations. These predictive models estimate the frequency of plasmid transfer for given growth densities of donors, recipients, and transconjugants. A model known as the endpoint model found thresholds above which the rate of plasmid transfer in liquid culture, and concluded that the transfer rate is unaffected by&#160;cell density, donor:recipient ratio, and mating time19. Fluorescence in situ hybridization (FISH) has been used as an alternative method of transconjugant plasmid detection. FISH allows plasmid visualization using fluorescence microscopy through the hybridization of a DNA probe and a target DNA. Thus, FISH allows the visual detection of plasmid flows across different cell populations37, although it does not have the same level of sensitivity as the method presented here if transconjugants are detected by visual screening as opposed to selection.
There is an enormous need to understand the biology of conjugative plasmids that are dispersing antibiotic resistance genes through different components of the ecosystem (clinic, agriculture, sewage, wildlife, domestic animals, soils, rivers, and lakes). In sum, the simplified experimental conditions presented in the protocols described here facilitate the screening of donors at scale, and thus represent a key tool for the study of horizontal gene transfer originating in conjugative plasmids from a variety of sources. They can be used to investigate the prevalence of antibiotic-resistance genes or other clinically-relevant genes in conjugative plasmids from multiple sources and bacteria. They can also be adapted for the study of conjugation in vivo (e.g., in the gut of vertebrates) and to study the conditions that modulate the efficiency of conjugation. All these studies will greatly add to understanding how the mobilization of multidrug-resistant conjugative plasmids contributes to the spread of multidrug resistance.

In 1946, Lederberg and Tatum1 described a sexual process in Escherichia coli K-12 that is now known as conjugation. Bacterial conjugation is the process by which a bacterial cell (the donor) transfers unidirectionally genetic material to another cell (the recipient) by direct cell-to-cell contact. Conjugation is broadly distributed in bacteria2,3, although the fraction of donor cells expressing the conjugation machinery is typically very small4.
Plasmids are autonomously-replicating extrachromosomal DNA elements. In addition to genes involved in plasmid replication and maintenance, plasmids frequently carry a cargo of genes involved in adaptation to environmental challenges, such as heavy metals or exposure to antibiotics5. Conjugative plasmids are a class of plasmids with a set of specialized genes that allow their transfer to recipient cells and support their persistence following the transfer6. Conjugative plasmids vary in size from 21.8 kb to 1.35 Mb in bacteria in the phylum Pseudomonadota (synonymous with Proteobacteria), with the median around 100 kb5,7. They also generally have a low copy number, possibly to keep the metabolic burden on the host low8,9.
The typical conjugative apparatus consists of four components: an origin of transfer (oriT), a relaxase, a type IV coupling protein, and a type IV secretion system (a tube-like structure called pilus that allows donors to contact recipients)6. Only a very small fraction of cells carrying conjugative plasmids express the conjugation machinery4, but if the plasmids provide a fitness advantage, the transconjugants can rapidly expand in the population. Between 35% and over 80% of E. coli isolates collected from different habitats have conjugative plasmids with genes that confer resistance to at least one antibiotic10,11; therefore, horizontal gene transfer mediated by conjugative plasmids is a major mechanism driving the global spread of antibiotic resistance genes12.
Mating experiments conducted in laboratory culture&#160;have shown that the conjugation frequency is affected by multiple factors, including the nature of the recipient cells, growth phase, cell density, donor-to-recipient ratio, whether conjugation is conducted in liquid or solid media, carbon, oxygen, bile salts, metal concentrations, presence of mammalian cells, temperature, pH, and mating time13,14,15.
This work describes protocols to detect the presence of conjugative plasmids in a given host strain, to quantify the conjugation rates in solid culture, and to double-check their transfer to recipient cells. These protocols can be used as a first step for the identification of natural conjugative plasmids suitable for research. They use a minimum number of simplified steps because they are designed to screen the presence of conjugative plasmids in bacteria obtained from multiple sources (environmental, commensal, and pathogenic) at scale (dozens to hundreds of donors).
In addition, tests to detect whether the mobilization of a given conjugative plasmid is independent of the antibiotic used for detection (i.e., the relevance of the antibiotic resistance gene under selection) and to compare the conjugation rates of two conjugative plasmids found in different environmental isolates are shown.
Based on the genetic composition of the relevant conjugative plasmids (plasmid replicon and antibiotic resistance gene makeup), each step of the protocol can be modified to study the impact of a variety of factors likely to affect the conjugation rate.
General experimental design: 
The essential components needed to set up a mating experiment are donor cells, a recipient strain, and solid media to select donors (antibiotic A), recipients (antibiotic B), and transconjugants (antibiotic A and B). Transconjugants are recipient cells that stably maintain the conjugative plasmid of the donor.
Donor cells are resistant to an antibiotic (antibiotic A) and are susceptible to the marker or markers used to select recipient cells (antibiotic B). The genomic location of the antibiotic resistance gene (i.e., whether it is located in the chromosome or a plasmid of the donor cell) does not have to be known a priori, because mobilization of antibiotic resistance markers to the recipient (after a direct donor-recipient contact) implies that the donor-provided markers were in a plasmid.
A recipient strain (known to take conjugative plasmids) needs to have a stable selectable marker not present in the donor; this selectable marker is generally resistant to an antibiotic or biocide located in the chromosome. The selection of the recipient to be used in mating experiments is critical, because some E. coli strains vary in their ability to take conjugative plasmids16.
Once these components have been established, any colony that grows on media with both antibiotics (A and B) after a donor-recipient pair contact is a putative transconjugant (Figure 1). This is assuming that donors can grow in media with antibiotic A but cannot grow in media with antibiotic B, and that recipients are able to grow in media with antibiotic B, but not able to grow with antibiotic A. Transconjugation can be confirmed using two diagnostic tests. The first test consists of the detection (by polymerase chain reaction [PCR] amplification of genes, or other methods) of genes found in the conjugative plasmid in transconjugant colonies. The second test involves the use of differential colony color markers based on lactose metabolism. The differential colony color is revealed by the use of MacConkey agar; the lactose in the agar can be used as a fermentation source by lactose-fermenting (lac+) microorganisms. These microorganisms produce organic acids, particularly lactic acid, which lower the pH. Neutral red is a pH indicator included in the media that turns from off-white to bright red/pink as the pH drops below 6.817. Thus, E. coli lactose-positive strains produce larger pink colonies on MacConkey agar, whereas lactose-negative strains produce pale yellow and smaller colonies on MacConkey agar.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64523/64523fig01.jpg" /> 
Figure 1: Experimental design used to detect the presence of conjugative plasmids in donor strains. In this example, the donor carries a conjugative plasmid with an antibiotic resistance gene that confers resistance to antibiotic A, but they are susceptible to antibiotic B. Conversely, the recipient has a chromosomal resistance determinant that confers protection from antibiotic B, but it is susceptible to antibiotic A. Transconjugants are resistant to both antibiotics (A and B), because they have the conjugative plasmid of the donor that confers resistance to antibiotic A and the chromosome of the recipient that confers protection from antibiotic B. <a href="https://www.jove.com/files/ftp_upload/64523/64523fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The conjugation rate for a donor-recipient pair (under given experimental conditions) can be calculated by dividing the number of transconjugants by the number of donors or by the number of recipients; the first rate indicates the fraction of donor cells exhibiting functional expression of the conjugation machinery by the donor16,18, while the second rate indicates the ability of the recipient to take conjugative plasmids19,20. In this study, unless otherwise stated, the conjugation rate represents the fraction of recipient cells that become transconjugant (i.e., rate per recipient).
Here, two independent mating experiments are reported, involving one E. coli recipient and two E. coli donors. In addition, different antibiotics were used to select transconjugants for one of the donors to confirm that a single multidrug-resistant plasmid can be selected with any of the antibiotic resistance genes found in the conjugative plasmid.
The donor and recipient strains used in this work have been fully sequenced to understand all components of this experimental system; however, these protocols were designed to screen for the presence of conjugative plasmids in hosts of unknown sequence, and can be used in this experimental context as well; however, in this case, the relevant genes are sequenced first.
The donor and recipient strains used in the protocol are the following:
Donor 1. E. coli SW4955 was collected in a lake in Baton Rouge (LA, USA). It has a 134,797 bp conjugative plasmid (p134797) with IncFIC(FII) and IncFIB (AP001918) replicons. This conjugative plasmid has genes that confer resistance to third-generation cephalosporins (blaCTX-M-55), aminoglycosides (aac(3)-IIa and aadA1), phenicols (catA2), tetracyclines (tet(A)), trimethoprim (dfrA14), and sulphonamides (sul3). For a complete map of p134797, please see Figure 2A. E. coli SW4955 is lactose-positive, producing pink colonies on MacConkey agar.
Donor 2. E. coli SW7037 was collected in Lake Erie (Ottawa County, OH, USA). It carries a 101,718 bp conjugative plasmid (p101718) with an IncI1-I(Alpha) replicon. This conjugative plasmid has a gene that confers resistance to beta-lactams (blaCMY-2). For a complete map of p101718, please see Figure 2B. E. coli SW7037 is also lactose-positive, producing pink colonies on MacConkey agar.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64523/64523fig02.jpg" /> 
Figure 2: Genetic map of the conjugative plasmids used in this study. (A) Plasmid p134797, the conjugative plasmid found in E. coli strain SW4955. (B) Plasmid p101718, the conjugative plasmid found in E. coli strain SW7037. Antibiotic resistance genes are highlighted in blue, and genes belonging to the conjugative apparatus&#160;are highlighted in red. <a href="https://www.jove.com/files/ftp_upload/64523/64523fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Recipient. E. coli LMB100 is used as recipient. This is a plasmidless strain that is resistant to rifampin (100 mg/L) and to streptomycin (100 mg/L). Having resistance to two antibiotics reduces the possibility of resistance mutations arising in the donor that would interfere with the interpretation of results. In addition, E. coli LMB100 is lactose-negative, and can be distinguished from the two donor strains because it produces pale yellow and small colonies (as opposed to larger, pink colonies) on MacConkey agar.
When the donor is lactose-negative, we recommend to use of a lactose-positive recipient (e.g., E. coli J53). The LMB100 and J53 strains are available to other labs for use. Please send a request to Dr. Gerardo Cort&#233;s-Cort&#233;s along with an address and FedEx number.
The solid media needed to select and count donors, recipients, and transconjugants is MacConkey agar in Petri dishes 100 mm in diameter. The addition of the following antibiotics is needed: (i) Media A: carbenicillin (100 mg/L) to count donors and to ensure that recipients cannot grow with this antibiotic. (ii) Media B: rifampin (100 mg/L) + streptomycin (100 mg/L) to count recipients and to ensure that donors cannot grow in these two antibiotics. (iii) Media AB: carbenicillin (100 mg/L) + rifampin (100 mg/L) + streptomycin (100 mg/L) to obtain and count transconjugants. (iv) Media C: no antibiotics to streak all isolates studied.
The conjugation rates of conjugative plasmids from E. coli SW4955 and E. coli SW7037 to E. coli LMB100 are compared. In addition, in the case of the p134797 conjugative plasmid (SW4955 strain), the antibiotic carbenicillin (100 mg/L) is replaced by gentamicin (2 mg/L), chloramphenicol (25 mg/L), tetracycline (10 mg/L), trimethoprim (20 mg/L), or sulfamethoxazole (100 mg/L) in subsequent experiments to establish if the antibiotic resistance marker used for selection has any impact on the results.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL tube racks</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-313630</td>
			<td>It can be replaced for another brand</td>
		</tr>
		<tr>
			<td>2.0 mL Cryogenic vials</td>
			<td>Corning</td>
			<td>430659</td>
			<td>It can be replaced for another brand</td>
		</tr>
		<tr>
			<td>14 mL conical tubes</td>
			<td>FALCON</td>
			<td>149597</td>
			<td>It can be replaced for another brand</td>
		</tr>
		<tr>
			<td>14 mL tube racks</td>
			<td>Thermo Fisher Scientific</td>
			<td>8850</td>
			<td>It can be replaced for another brand</td>
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		<tr>
			<td>1 kb DNA ladder</td>
			<td>New England Biolabs</td>
			<td>N0552G&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL sterile flasks&#160;</td>
			<td>PYREX</td>
			<td>5320</td>
			<td>It can be replaced for another permanent marker brand</td>
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			<td>Acetic acid</td>
			<td>Fisher Scientific</td>
			<td>A38SI-212</td>
			<td>It can be replaced for another brand</td>
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			<td>Agarose (molecular biology grade, multipurpose)</td>
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			<td>BP160-500</td>
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			<td>Carbenicillin</td>
			<td>GOLD BIOTECHNOLOGY</td>
			<td>C-103-50</td>
			<td>It can be replaced for another brand</td>
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			<td>Disposable inoculating loops: 1 &#181;L</td>
			<td>Fisherbrand</td>
			<td>22-363-595</td>
			<td>It can be replaced for another brand</td>
		</tr>
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			<td>DNA gel loading dye 6x&#160;</td>
			<td>New England Biolabs</td>
			<td>B7024S</td>
			<td>It can be replaced for another brand</td>
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			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP119-500</td>
			<td>It can be replaced for another brand</td>
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			<td>Electronic digital scale</td>
			<td>Denver Instrument</td>
			<td>APX-2001</td>
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			<td>Eppendorf conical tubes: 1.5 mL</td>
			<td>Eppendorf</td>
			<td>14-282-302</td>
			<td>It can be replaced for another brand</td>
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			<td>Equipment for agarose gel electrophoresis</td>
			<td>Fisher Scientific</td>
			<td>FP300</td>
			<td>It can be replaced for another brand</td>
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		<tr>
			<td>Ethanol-resistant markers</td>
			<td>Sharpie</td>
			<td>37001; 37002; 37003</td>
			<td>It can be replaced for another brand</td>
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		<tr>
			<td>Gel documentation systems (UVP GelSolo)</td>
			<td>Analytakjena</td>
			<td>SP-1108; 849-97-0936-01; 95-0612-01</td>
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			<td>Gloves</td>
			<td>X-GEN</td>
			<td>44-100M</td>
			<td>Any nitrile gloves brand works</td>
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			<td>Ice bucket</td>
			<td>Thermo Fisher Scientific</td>
			<td>432128</td>
			<td>It can be replaced for another brand</td>
		</tr>
		<tr>
			<td>MacConkey agar&#160;</td>
			<td>BD DIFCO</td>
			<td>212123</td>
			<td></td>
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		<tr>
			<td>Master Mix&#160;</td>
			<td>BioLabs</td>
			<td>M0496S&#160;</td>
			<td>It can be replaced for another brand</td>
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			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A452-4</td>
			<td>It can be replaced for another brand</td>
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		<tr>
			<td>Microcentrifuge tubes: 2.0 mL</td>
			<td>Fisherbrand</td>
			<td>05-408-138</td>
			<td>It can be replaced for another brand</td>
		</tr>
		<tr>
			<td>Mueller Hinton agar</td>
			<td>BD DIFCO</td>
			<td>DF0479-17-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Mueller Hinton broth</td>
			<td>BD DIFCO</td>
			<td>275730</td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoSpin Plasmid, Mini kit for plasmid DNA</td>
			<td>Takara Bio USA, Inc., MACHEREY-NAGEL</td>
			<td>740588. 250</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tube racks</td>
			<td>AXYGEN&#160;</td>
			<td>&#160;R96PCRFSP</td>
			<td>It can be replaced for another brand</td>
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		<tr>
			<td>PCR tubes (0.2 mL)</td>
			<td>AXYGEN&#160;</td>
			<td>PCR-02-C</td>
			<td>It can be replaced for another brand</td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Fisher Scientific</td>
			<td>50-164-7517</td>
			<td></td>
		</tr>
		<tr>
			<td>Set of micropipettes that dispense between 1 - 10 &#956;L (P10), 2&#8211;20 &#956;L (P20), 20&#8211;200 &#956;L (P200) and 200&#8211;1000 &#956;L (P1000)</td>
			<td>RAININ</td>
			<td>17008648; 17008650; 17008652; 17008653</td>
			<td>Micropipettes should be calibrated; can be replaced for another brand</td>
		</tr>
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			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S671-3</td>
			<td>It can be replaced for another brand</td>
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			<td>Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>335905P</td>
			<td>It can be replaced for another brand</td>
		</tr>
		<tr>
			<td>Sterilized tips for 10 &#956;L, 200 &#956;L and 1000 &#956;L&#160;</td>
			<td>Eclipse</td>
			<td>1011-260-000-9; 1018-260-000; 1019-260-000-9</td>
			<td>It can be replaced for another brand</td>
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			<td>Streptomycin</td>
			<td>Fisher Scientific</td>
			<td>BP910-50</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Safe DNA gel stain&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>S33102</td>
			<td></td>
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		<tr>
			<td>Taq DNA Polymerase</td>
			<td>BioLabs</td>
			<td>M0273S</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal cycler C1000 Touch&#160;</td>
			<td>Bio-Rad</td>
			<td>1851196</td>
			<td>It can be replaced for another brand</td>
		</tr>
		<tr>
			<td>Tris&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP153-500</td>
			<td>It can be replaced for another brand</td>
		</tr>
	</tbody>
</table>

detection of horizontal gene transfer mediated by natural conjugative plasmids in e. coli

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the study of the mobilization of naturally occurring conjugative plasmids, using two naturally-occurring plasmids as an example. These protocols rely on the differential presence of selectable markers in donor, recipient, and conjugative plasmid. Specifically, the methods described include 1) the identification of natural conjugative plasmids, 2) the quantification of conjugation rates in solid culture, and 3) the diagnostic detection of the antibiotic resistance genes and plasmid replicon types in transconjugant recipients by polymerase chain reaction (PCR). The protocols described here have been developed in the context of studying the evolutionary ecology of horizontal gene transfer, to screen for the presence of conjugative plasmids carrying antibiotic-resistance genes in bacteria found in the environment. The efficient transfer of conjugative plasmids observed in these experiments in culture highlights the biological relevance of conjugation as a mechanism promoting horizontal gene transfer in general and the spread of antibiotic resistance in particular.

Given that donors SW4955 and SW7037 have been sequenced, these two donor strains are expected to have the resistance phenotypes corresponding to the resistance gene profile identified in their genomic sequence. Plasmid p134797 (from SW4955) has genes that confer&#160;resistance to third-generation cephalosporins (blaCTX-M-55), and resistance to aminoglycosides (aac(3)-IIa and aadA1), phenicols (catA2), tetracyclines (tet(A)), trimethoprim (dfrA14), and sulphonamides (sul3); no antibiotic resistance genes were found in the SW4955 chromosome. Plasmid p101718 (from SW7037) only carries one antibiotic resistance gene (blaCMY-2), expected to confer resistance to third-generation cephalosporins (blaCTX-M-55). Again no antibiotic resistance genes were found in the SW7037 chromosome.
Following mating, detection of the transfer of the two conjugative plasmids bearing all the resistance genes mentioned above was expected. Positive conjugation detection implies that the recipients identified as transconjugants should have acquired the conjugative plasmids. The presence of the diagnostic resistance genes for the conjugative plasmids in the transconjugants (as detected by PCR) was also expected.
To illustrate the methods described here, the ability of two E. coli environmental isolates to transfer plasmid DNA by conjugation was tested. A schematic representation of the experimental setting is shown in Figure 1. Two donors (E. coli SW4955 and SW7037) and one recipient (E. coli LMB100) were mated independently. The gene map of conjugative plasmids found in E. coli SW4955 (p134797) and SW7037 (p101718) is shown in Figure 2.
Conjugative plasmids were selected with carbenicillin and counter-selected using rifampin and streptomycin, whose resistance determinants are in the chromosome of the recipient. MacConkey agar was used to distinguish the lactose-positive donors (which produce large, pink colonies) from the recipient and transconjugants (which are lactose negative and produce smaller, pale yellow colonies) (Figure 3).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64523/64523fig03.jpg" /> 
Figure 3: Illustration of the differential color markers for donor and recipient colonies on MacConkey agar. The colonies corresponding to donors are pink on MacConkey agar because they are lactose positive. This distinguishes donor from recipient colonies, which are pale yellow and smaller (lactose negative). <a href="https://www.jove.com/files/ftp_upload/64523/64523fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The mobilization of the conjugative plasmid p134797 was detected, with each of the five antibiotics corresponding to the five different classes of resistance genes found in the plasmid.
An example of how to calculate the conjugation efficiency (CE) for strain SW4955 is presented. From the assay with strain SW4955, 172 CFU of transconjugants were counted in the plate from dilution 10-2; the count of the recipient from the corresponding dilution (10-2) was 10,300,000 CFU/mL (Table 4).
The CE is calculated as follows:
CE = (174 CFU/mL)/(10,300,000 CFU/mL)
CE= 1.67 x 10-5&#160;transconjugants/recipient
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">LMB100</td>
			<td>Transconjugants from strain SW4955 selected with Carbelicillin</td>
			<td rowspan="2">Conjugation Efficiency</td>
		</tr>
		<tr>
			<td>Dilution</td>
			<td>CFU (countable plate)</td>
			<td>Approx. CFU/mL</td>
			<td>CFU (countable plate)</td>
		</tr>
		<tr>
			<td>100</td>
			<td>confluent</td>
			<td>1.03E+09</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10-1</td>
			<td>confluent</td>
			<td>1.03E+08</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10-2</td>
			<td>confluent</td>
			<td>10300000</td>
			<td>172</td>
			<td>1.67 x 10-5 transconjugants/recipient</td>
		</tr>
		<tr>
			<td>10-3</td>
			<td>confluent</td>
			<td>1030000</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10-4</td>
			<td>confluent</td>
			<td>103000</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10-5</td>
			<td>confluent</td>
			<td>10300</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10-6</td>
			<td>confluent</td>
			<td>1030</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10-7</td>
			<td>103</td>
			<td>103</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="5">Values used to calculate the CE are highlighted in bold.</td>
		</tr>
	</tbody>
</table>
Table 4: An example of how to calculate the conjugation efficiency (CE) for strain SW4955.
The results are shown in Table 5, expressed as conjugation efficiency per recipient. The five conjugation efficiencies obtained using strain SW4955 as a donor were all within the same order of magnitude. These results indicate that the mobilization of the conjugative plasmid can be detected regardless of the selection used for transconjugant identification.
Using the strain SW7037 as a donor, the conjugation efficiency obtained was three orders of magnitude lower; these results allow the comparison of conjugation efficiencies of different donors and plasmid types with the same recipients.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="5">Conjugation efficiency</td>
		</tr>
		<tr>
			<td>Strain</td>
			<td>Carbenicillin (100 mg/L)</td>
			<td>Gentamicin (2 mg/L)</td>
			<td>Chloramphenicol (25 mg/L)</td>
			<td>Tetracycline (10 mg/L)</td>
			<td>Trimethoprim (20 mg/L)</td>
		</tr>
		<tr>
			<td>SW4955</td>
			<td>1.67 x 10-5</td>
			<td>5.67 x 10-5</td>
			<td>2.17 x 10-5</td>
			<td>7.62 x 10-5</td>
			<td>1.36 x 10-5</td>
		</tr>
		<tr>
			<td>SW7037</td>
			<td>2.14 x 10-6</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 5: Conjugation efficiencies of E. coli donor strains SW4955 and SW7037 depending on the&#160;antibiotic used for selection.
In transconjugants, the presence of replicons and all the resistance genes encoded in two conjugative plasmids tested, and their corresponding replicons, was checked by PCR. The conditions used for these diagnostic PCR reactions are shown in Table 3.
The diagnostic gels are shown in Figure 4. These gels confirm the presence of the expected replicons in transconjugants (IncFIC and IncFIB in p1347975, and IncI1 in p101718). They also confirm the presence of the expected antibiotic resistance genes, namely the blaCTX-M-55 group (beta-lactam), aac(3)-II and aadA (aminoglycoside), catA2 (phenicol), tet(A) (tetracycline), dfrA14 (trimethoprim), and sul3 (sulphonamide) for p1347975, and blaCMY-2 for the p101718 (Figure 4).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64523/64523fig04.jpg" /> 
Figure 4: Diagnostic electrophoresis gels. Gel electrophoresis of PCR products of antibiotic resistance genes and plasmid replicons found in conjugative plasmids p134797 and p101718 in the donor cells (strains SW4955 and SW7037, respectively) and in the corresponding LMB100 transconjugants. Carbenicillin was used for plasmid selection. Abbreviations. -: negative control (DNA of strain LMB100 was used as a negative control); D: donor; T: transconjugant. Expected amplicon sizes:&#160;blaCTX-M-55: 864 pb; aac(3)-IIa: 237 bp; aadA1: 283 bp; catA2: 225 bp; tet(A): 957 bp; dfrA14: 302 bp; sul3: 799 bp; IncFIC(FII): 262 bp; IncFIB: 683 bp; blaCMY-2:&#160;1,855 bp; IncI1-I: 139 bp. The gel has 1% agarose. <a href="https://www.jove.com/files/ftp_upload/64523/64523fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli at JoVE.com

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation mediates horizontal gene transfer by mobilizing plasmid DNA across two different cells, facilitating the spread of beneficial genes. This work describes a widely-used method for the efficient detection of conjugative plasmid transfer, based on the use of differential markers in the conjugative plasmid, donor, and recipient to detect transconjugation.

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the ...

Bacteria like E.coli carry antibiotic resistant genes in conjugative plasmids. This bacteria can be found in hospitals, living as commensals in animals, and in aquatic environments. Natural conjugative plasmids horizontal transfer of genes between different bacterial lineages.We present metals for the detection of conjugative plasmids and formation conjugation efficiency. This technique is simple and sensitive. It is sensitive because it uses markers for the plasmid, the donor, and the recipient.These markers are selectable, which means that they can identify single events in very large populations. The protocols we present are useful to study the evolution of antibiotic resistance and for epidemiological surveillance, that is to monitor the flow of drug-resistant genes. Demonstrating the procedure will be Caison Warner, a PhD student from my laboratory, and Pepper St.Clair, an undergraduate student from CAMS lab.On day one, separately streak the donors and recipient from glycerol stocks on media C which is MacConkey agar without antibiotics and incubate overnight at 37 degrees Celsius. On day two, label a 14 milliliter culture tube for each donor and recipient. Select a single colony of each donor and recipient and grow them in two milliliters of Mueller Hinton Broth at 200 RPM for 18 hours at 37 degrees Celsius.On day three, measure the optical density of 10 times saline diluted overnight grown culture at 600 nanometers without vortexing. If required, use sterilized saline solution to adjust the optical density to two. Label a 1.5 milliliter microcentrifuge tube as a mating tube, indicating the donor and recipient strains.Transfer 500 microliters of the adjusted suspension of each donor and recipient in the mating tube and gently mix the suspension by inversion. Centrifuge the mating tube at room temperature for 10 minutes at 500 G.Pipette out 800 microliters of supernatant from the mating tube without disturbing the pellet, leaving 200 microliters in the tube. Incubate the mating tube for 18 hours at 37 degrees Celsius.Further, as a negative control, streak the overnight culture of donors on media B and recipients on media A and incubate the plates overnight at 37 degrees Celsius. After incubation, on day four, add 800 microliters of saline solution to the mating tube and resuspend by vortexing. Make serial dilutions of the mating suspension and add 100 microliters of each dilution on media plates.Once the plates are dried, incubate them for 18 hours at 37 degrees Celsius and calculate the frequency of conjugation per donor or per recipient as described in the manuscript. After extracting DNA from the bacterial cell walls using a simple boil prep extraction method, label a 1.5 milliliter tube as a pool in the required PCR tubes with the name of the gene and sample. In the tube labeled as pool, add sterile water, PCR master mix, primers, and polymerase.Mix gently by pipetting up and down at least 10 times in a chilled environment. Add the template DNA to the corresponding tube and secure the PCR tubes with caps. Place the PCR tubes into the thermocycler and start the program for resistance genes and replicons as described in the manuscript.For amplicon confirmation, prepare a 1%agarose gel by adding agarose and TAE buffer in a 250 milliliter flask and melt the agarose using a microwave. Once the agarose solution has cooled to approximately 55 degrees Celsius, under a fume hood, add six microliters of SYBR Green and mix. Pour the gel into a tray sealed with adhesive tape to avoid spillage and allow the gel to set for 15 to 25 minutes until turbidity appears.After removing the adhesive tape, place the gel into the electrophoresis chamber and add TAE buffer to cover the gel. Mix five microliters of six times DNA gel loading dye with 25 microliters of each reaction by pipetting up and down at least 10 times. Then load the mixture into wells by avoiding bubbles.After adding four microliters of one kilobase pair DNA ladder into the first well, close the chamber lid and run the gel for 60 minutes at 120 volts and 400 milliamperes. Visualize the gel under UV light and record the image. MacConkey agar distinguished the lactose positive donors which produced large pink colonies from the recipient and transconjugants, which were lactose negative and produced smaller pale yellow colonies.The five conjugation efficiencies of a donor strain SSW4955 were all within the same order of magnitude, indicating the mobilization of conjugative plasmid can be detected regardless of the selection used for transconjugant identification. Whereas the donor strain SSW7037 showed a conjugation efficiency three times lower, which allows the comparison of conjugation efficiencies of different donors and plasmid types with the same recipients. The gel electrophoresis of PCR products confirmed the presence of expected replicons in transconjugants.This assay completely depends on selectable markers. Controls must be in place to confirm that each selection is working and transconjugants need to be confirmed colorimetrically or by PCR. The protocols presented here can be adapted for the study of conjugation in vivo or biofilm models, and for a studying variables modulating conjugation efficiency.This protocol for detecting mobilizable plasmids at scale will greatly add to our understanding of the flow of genes across the environment, and to study the variables affecting the efficiency of conjugation.

Research

JoVE Journal

Genetics

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

Manipulation of Gene Function in Mexican Cavefish

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...