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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M., Stewart, F. M., Levin, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+rate+of+plasmid+transfer:+an+end-point+method.">Estimating the rate of plasmid transfer: an end-point method.</a> Journal of General Microbiology. 136 (11), 2319-2325 (1990).</li><li>Huisman, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+plasmid+conjugation+rates:+A+new+computational+tool+and+a+critical+comparison+of+methods.">Estimating plasmid conjugation rates: A new computational tool and a critical comparison of methods.</a> Plasmid. 121, 102627 (2022).</li><li>Jung, B., Hoilat, G. 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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><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></tr><tr><td>1 kb DNA ladder</td><td>New England Biolabs</td><td>N0552G&amp;nbsp;</td><td></td></tr><tr><td>250 mL sterile flasks&amp;nbsp;</td><td>PYREX</td><td>5320</td><td>It can be replaced for another permanent marker brand</td></tr><tr><td>Acetic acid</td><td>Fisher Scientific</td><td>A38SI-212</td><td>It can be replaced for another brand</td></tr><tr><td>Agarose (molecular biology grade, multipurpose)</td><td>Fisher Scientific</td><td>BP160-500</td><td>It can be replaced for another brand</td></tr><tr><td>Carbenicillin</td><td>GOLD BIOTECHNOLOGY</td><td>C-103-50</td><td>It can be replaced for another brand</td></tr><tr><td>Disposable inoculating loops: 1 &amp;micro;L</td><td>Fisherbrand</td><td>22-363-595</td><td>It can be replaced for another brand</td></tr><tr><td>DNA gel loading dye 6x&amp;nbsp;</td><td>New England Biolabs</td><td>B7024S</td><td>It can be replaced for another brand</td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>BP119-500</td><td>It can be replaced for another brand</td></tr><tr><td>Electronic digital scale</td><td>Denver Instrument</td><td>APX-2001</td><td>It can be replaced for another brand</td></tr><tr><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></tr><tr><td>Equipment for agarose gel electrophoresis</td><td>Fisher Scientific</td><td>FP300</td><td>It can be replaced for another brand</td></tr><tr><td>Ethanol-resistant markers</td><td>Sharpie</td><td>37001; 37002; 37003</td><td>It can be replaced for another brand</td></tr><tr><td>Gel documentation systems (UVP GelSolo)</td><td>Analytakjena</td><td>SP-1108; 849-97-0936-01; 95-0612-01</td><td>It can be replaced for another brand</td></tr><tr><td>Gloves</td><td>X-GEN</td><td>44-100M</td><td>Any nitrile gloves brand works</td></tr><tr><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&amp;nbsp;</td><td>BD DIFCO</td><td>212123</td><td></td></tr><tr><td>Master Mix&amp;nbsp;</td><td>BioLabs</td><td>M0496S&amp;nbsp;</td><td>It can be replaced for another brand</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A452-4</td><td>It can be replaced for another brand</td></tr><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&amp;nbsp;</td><td>&amp;nbsp;R96PCRFSP</td><td>It can be replaced for another brand</td></tr><tr><td>PCR tubes (0.2 mL)</td><td>AXYGEN&amp;nbsp;</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 &amp;mu;L (P10), 2&amp;ndash;20 &amp;mu;L (P20), 20&amp;ndash;200 &amp;mu;L (P200) and 200&amp;ndash;1000 &amp;mu;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><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>S671-3</td><td>It can be replaced for another brand</td></tr><tr><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 &amp;mu;L, 200 &amp;mu;L and 1000 &amp;mu;L&amp;nbsp;</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></tr><tr><td>Streptomycin</td><td>Fisher Scientific</td><td>BP910-50</td><td></td></tr><tr><td>SYBR Safe DNA gel stain&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>S33102</td><td></td></tr><tr><td>Taq DNA Polymerase</td><td>BioLabs</td><td>M0273S</td><td></td></tr><tr><td>Thermal cycler C1000 Touch&amp;nbsp;</td><td>Bio-Rad</td><td>1851196</td><td>It can be replaced for another brand</td></tr><tr><td>Tris&amp;nbsp;</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 ...

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5.0K Views.  University of California Irvine. 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.

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

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 coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

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

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

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

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

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

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial populations. This is especially the case under rapidly changing environments such as fluctuating antibiotics exposure. We recently showed that plasmids maintain antibiotic resistance genes in Escherichia coli without positive selection for the plasmid presence. Here we describe an experimental system that allows following both the plasmid genotype and phenotype in long-term evolution experiments. We use molecular techniques to design a model plasmid that is subsequently introduced to an experimental evolution batch system approach in an E. coli host. We follow the plasmid frequency over time by applying replica plating of the E. coli populations while quantifying the antibiotic resistance persistence. In addition, we monitor the conformation of plasmids in host cells by analyzing the extent of plasmid multimer formation by plasmid nicking and agarose gel electrophoresis. Such an approach allows us to visualize not only the genome size of evolving plasmids but also their topological conformation-a factor highly important for plasmid inheritance. Our system combines molecular strategies with traditional microbiology approaches and provides a set-up to follow plasmids in bacterial populations over a long time. The presented approach can be applied to study a wide range of mobile genetic elements in the future.

Our experimental approach provides a strategy to follow plasmid abundance and antibiotic resistance over time in bacterial populations.

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Immunology and Infection

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Bacterial Transformation

<h4>Background</h4>

In early 20th century, pneumonia was accountable for a large portion of infectious disease deaths1. In order to develop an effective vaccine against pneumonia, Frederick Griffith set out to study two different strains of the Streptococcus pneumoniae: a non-virulent strain with a rough appearance (R-strain) and a virulent strain with a smooth appearance (S-strain) due to an outer polysaccharide capsule2. This outer layer of the S-strain bacteria enabled them to withstand the host immune system, eventually leading to a life-threatening illness. When Griffith separately injected mice with heat-killed bacteria of either strain, the mice lived. However, when he injected mice with a combination of the heat-killed S-strain with live R-strain, the mice died2. When he analyzed the samples obtained from dead mice injected with the combination, he observed live S-strain bacteria to be present. In 1928, Griffith noted that a &#8220;transformation&#8221; process must have occurred to change the nonvirulent bacteria into the virulent strain. As the first known discovery of bacterial transformation, his discovery paved the way for the development of an essential tool in genetic engineering - transformation3.

Transformation is the genetic change in a cell due to the intake of DNA from the environment. In Griffith&#8217;s experiment, the DNA that encodes the protective polysaccharide coating of the S-strain bacteria did not break down from the heat shock and was introduced into the R-strain, allowing the latter to bypass the immune system of the mouse. While this process constantly occurs in the wild between organisms and even different species, scientists transform bacteria in laboratory settings for research purposes4.

<h4>Bacterial Transformation using Plasmids</h4>

Bacteria are the ideal organisms for transformation as they can easily take in exogenous genetic material into their genome and quickly amplify it3,5. They have one circular chromosome and multiple small circular pieces of double-stranded DNA called plasmids within the cytoplasm. These plasmids can replicate independently from the chromosomal DNA and generally provide certain functional benefits, such as resistance to antibiotics6,7. In their natural environment, bacteria undergo &#8220;bacterial transformation&#8221; by taking in plasmids from other bacteria in a process called conjugation8. Moreover, when they multiply, each of their progeny receives a copy of the new plasmid.

Plasmids that are used for experimental purposes are called plasmid vectors. In a laboratory setting, scientists can artificially create &#8220;recombinant plasmids&#8221; that are approximately 5,000-10,000 base pairs in length by inserting DNA fragments into a plasmid vector. These recombinant plasmids usually have certain components: an origin of replication (ORI), an antibiotic resistance gene, a multiple cloning site, a promoter, a selection marker, and the gene of interest. The origin of replication is where replication begins. The antibiotic resistance gene allows the bacteria that take in the plasmid to survive on plates in the presence of a certain antibiotic drug. Although plasmids are relatively small pieces of DNA, scientists need to treat the host cells to enable the plasmid penetration through the cell membrane. Hence, the efficiency of the transformation is directly related to porosity of the host membrane. One common approach is to heat-shock the bacteria that have been treated with a calcium chloride solution9. The bacteria that do not incorporate the plasmid will not transform, and therefore have no resistance to survive on the plate and be visible. The multiple cloning sites aid in DNA insertion by containing sites for restriction enzymes to cut the plasmid where the gene of interest can be inserted and ligated. The promoter drives transcription of the gene of interest. It is tagged by a marker, usually a fluorescent protein such as a green fluorescent protein (GFP), or can be an additional antibiotic resistance gene. The antibiotic resistance gene and the other selection markers help us determine whether the collected bacteria contain the plasmid of interest.

<h4>Applications</h4>

Effective transformation methods enabled scientists to isolate and profile genes and gene products and led to many advancements in life sciences and medicine, such as development of effective drugs, generation of genetically-modified crops, and advanced diagnostic tools10. In addition, with technological advancements, new methods of transformation have emerged. For instance, Gateway Cloning allows insertion of multiple DNA fragments into different vectors as well as transfer of DNA sequences between plasmids11. Furthermore, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 is a gene-editing technique that directly modifies nucleotides in the genome and does not require the use of plasmids12. After transformation, researchers often isolate and profile the gene of interest and its products. Subsequently, the whole process of genetic cloning has opened up a new field of genetic manipulation. Thanks to genetic cloning, researchers can manipulate bacteria to produce large amounts of specific human proteins, such as insulin to treat diabetes patients10. Cloning also has served a major importance in modern day agriculture. Genetically modified organisms (GMO) are a direct result of genetic cloning and bacterial transformation10. For instance, scientists are working to generate genetically modified crops with nitrogen-fixing genes incorporated into their genome to boost food production and reduce fertilizer use, thus, diminishing the economic and environmental impact of fertilizers13. In sum, bacterial transformation is the first step of modern-day biotechnology and the foundation of future research discoveries.

<h4>References</h4>

<ol class="references">
	<li>GL, Armstrong, LA, Conn and RW, Pinner. Trends in Infectious Disease Mortality in the United States During the 20th Century. JAMA. 1999, Vol. 281, 1 (61-66).</li>
	<li>F, Griffith. The Significance of Pneumococcal Types. J Hyg (Lond). . 1928 , 2 (113-59).</li>
	<li>Lorenz, MG and Wackernagel, F. Bacterial gene transfer by natural genetic transformation in the environment. Microbiol Rev. . 1994 Sep;, Vol. 58, 3: (563-602).</li>
	<li>S, Domingues, et al. Natural Transformation Facilitates Transfer of Transposons, Integrons and Gene Cassettes between Bacterial Species. PLOS Pathogens. 2012, 8(8): e1002837.</li>
	<li>Dubnau, D. DNA uptake in bacteria. Annu Rev Microbiol. . 1999, 53: (217-44).</li>
	<li>Solar, G del, et al. Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev. . 1998 , Vol. 62, 2: (434-64).</li>
	<li>Bennett, PM. Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria. Br J Pharmacol. . 2008 , Vol. 153, S1: (S347-S357).</li>
	<li>MLlosa, et al. Bacterial conjugation: a two-step mechanism for DNA transport. Mol Microbiol. 2002 Jul;, Vol. 45, 1: (1-8).</li>
	<li>Roychoudhury, A, Basu, S and Sengupta, DN. Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors. Indian J Biochem Biophys. . 2009 , Vol. 46, 5: (395-400).</li>
	<li>Khan, S, et al. Role of Recombinant DNA Technology to Improve Life. I. nt J Genomics. . 2016, 2016:2405954.</li>
	<li>Marsischky, G and LaBaer, J. Many Paths to Many Clones: A Comparative Look at High-Throughput Cloning Methods. Genome Res. 2004, Vol. 14, 2020-28.</li>
	<li>Doudna, JA and Charpentie, E. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014, Vol. 346, 6213-1258096.</li>
	<li>Good, A. Toward nitrogen-fixing plants. Science. 2018, Vol. 359, 6378: 869-70.</li>
</ol>

Bacterial Transformation using Plasmids - Concept

Background

In early 20th century, pneumonia was accountable for a large portion of infectious disease deaths1. In order to develop an effective vaccine ...

Education

JoVE Lab Manual

Biology

Transformation of E. coli Cells Using an Adapted Calcium Chloride Procedure

Source: Natalia Martin1, Andrew J. Van Alst1, Rhiannon M. LeVeque1, and Victor J. DiRita1 
1 Department of Microbiology and Molecular Genetics, Michigan State University
Bacteria have the ability to exchange genetic material (DeoxyriboNucleic Acid, DNA) in a process known as horizontal gene transfer. Incorporating exogenous DNA provides a mechanism by which bacteria can acquire new genetic traits that allow them to adapt to changing environmental conditions, such as the presence of antibiotics or antibodies (1) or molecules found in natural habitats (2). There are three mechanisms of horizontal gene transfer: transformation, transduction, and conjugation (3). Here we will focus on transformation, the ability of bacteria to take up free DNA from the environment. In the laboratory, the transformation process has four general steps: 1) Preparation of competent cells, 2) Incubation of competent cells with DNA, 3) Recovery of cells, and 4) Plating of the cells for growth of the transformants (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10515/10515fig1.jpg" /> 
Figure 1: General steps of the transformation process. The transformation process has four general steps: 1) Preparation of competent cells, 2) Incubation with DNA, 3) Recovery of the cells and 4) Plating cells for growth of the transformants.
For transformation to occur, the recipient bacteria must be in a state known as competence. Some bacteria have the ability to become naturally competent in response to certain environmental conditions. However, many other bacteria do not become competent naturally, or the conditions for this process are yet unknown. The ability to introduce DNA into bacteria has a range of research applications: to generate multiple copies of a DNA molecule of interest, to express large amount of proteins, as a component in cloning procedures, and others. Because of the value of transformation to molecular biology, there are several protocols aimed to make cells artificially competent when conditions for natural competence are unknown. Two main methods are used to prepare artificially competent cells: 1) through chemical treatment of cells and 2) exposing the cells to electric pulses (electroporation). The former uses different chemicals depending on the procedure to create attraction between the DNA and the cell surface, while the latter uses electric fields to generate pores in the bacterial cell membrane through which DNA molecules can enter. The most efficient approach for chemical competence is incubation with divalent cations, most notably calcium (Ca2+) (4,5) Calcium-induced competence is the procedure that will be described here (6). This method is mainly used for transformation of Gram-negative bacteria, and that will be the focus of this protocol.
The procedure of chemical transformation involves a series of steps in which cells are exposed to cations to induce chemical competence. These steps are subsequently followed by a temperature change - heat shock - that favors the uptake of foreign DNA by the competent cell (7). The bacterial cell envelopes are negatively charged. In Gram-negative bacteria like Escherichia coli, the outer membrane is negatively-charged due to the presence of lipopolysaccharide (LPS) (8). This results in repulsion of the similarly negatively-charged DNA molecules. In chemical competence induction, positively-charged calcium ions neutralize this charge repulsion enabling DNA absorbance onto the cell surface (9). Calcium treatment and incubation with DNA are done on ice. Subsequently, an incubation at higher temperatures (42&#176;C), the heat shock, is performed. This temperature imbalance further favors the DNA uptake. Bacterial cells need to be at the mid-exponential growth phase to withstand the heat shock treatment; in other growth stages the bacterial cells are too sensitive to the heat resulting in loss of viability which significantly decreases transformation efficiency.
Different DNA sources can be used for transformation. Typically, plasmids, small circular, double-stranded DNA molecules, are used for transformation in most laboratory procedures in E. coli. For plasmids to be maintained in the bacterial cell after transformation, they need to contain an origin of replication. This allows them to be replicated in the bacterial cell independently from the bacterial chromosome. Not all the bacterial cells get transformed during the transformation procedure. Thus, transformation yields a mixture of transformed cells and non-transformed cells. To distinguish between these two populations, a selection method to identify the cells that have acquired the plasmid is used. Plasmids usually contain selectable markers, which are genes encoding a trait that confers an advantage for growth (i.e. resistance to an antibiotic or chemical or rescue from a growth auxotrophy). After transformation, bacterial cells are plated on selective media, which only allows for growth of the transformed cells. In the case of cells transformed with a plasmid conferring resistance to a given antibiotic, the selective media will be growth media containing that antibiotic. Several different methods can be used to confirm that the colonies grown in the selective media are transformants (i.e. have incorporated the plasmid). For example, plasmids can be recovered from these cells using plasmid preparation methods (10) and digested to confirm plasmid size. Alternatively, colony PCR can be used to confirm the presence of the plasmid of interest (11).
The aim of this experiment is to prepare E. coli DH5&#945; chemically competent cells, using an adaptation of the calcium chloride procedure (12), and to transform them with the plasmid pUC19 to determine transformation efficiency. The E. coli strain DH5&#945; is a commonly used strain in molecular biology applications. Because of its genotype, specifically the recA1 and endA1, this strain allows for increased insert stability and improve the quality of plasmid DNA in subsequent preparations. Since the transformation efficiency decreases with increasing size of the DNA, the plasmid pUC19 was used in this protocol because of its small size (2686 bp) (see https://www.mobitec.com/cms/products/bio/04_vector_sys/standard_cloning_vectors.html for a vector map). pUC19 confers resistance to ampicillin and thus, this was the antibiotic used for selection.

Transformation of E. coli: Adapted Calcium Chloride Procedure

Source: Natalia Martin1, Andrew J. Van Alst1, Rhiannon M. LeVeque1, and Victor J. DiRita1
1 Department of Microbiology and Molecular Genetics, Michigan ...

JoVE Science Education

Advanced Biology

Microbiology

Conjugation: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

Source: Alexander S. Gold1, Tonya M. Colpitts1 
1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases Laboratories, Boston, MA
First discovered by Lederberg and Tatum in 1946, conjugation is a form of horizontal gene transfer between bacteria that relies on direct physical contact between two bacterial cells (1). Unlike other forms of gene transfer, such as transformation or transduction, conjugation is a naturally occurring process in which DNA is secreted from a donor cell to a recipient cell in a unidirectional manner. This directionality and the ability for this process to increase the genetic diversity of bacteria has given conjugation the reputation as a form of bacterial &#34;mating,&#34; which is believed to have contributed greatly to the recent rise in antibiotic resistant bacteria (2, 3). By using selective pressures, for example the use of antibiotics, conjugation has been manipulated for use in the laboratory setting, making it a powerful tool for horizontal gene transfer between bacteria, and in some cases from bacteria to yeast, plant, and animal cells (4). Apart from applications in the laboratory, bacterium-eukaryote gene transfer by conjugation is an exciting avenue of DNA transfer with a multitude of possible biotechnical applications and naturally occurring implications (5).
Conjugation is thought to work by a &#34;two-step mechanism&#34; (6). First, before any DNA can be transferred, the donor cell must make direct cell-to-cell contact with the recipient. This process has been characterized best in gram-negative bacteria, the most studied of which is Escherichia coli. Cell-to-cell contact is established by the presence of a complex network of extracellular filaments on the donor known as the sex pilus, a conjugative element encoded for by the transferable gene known as the F (fertility) factor (7, 8). In addition to establishing contact between donor and recipient, several proteins are transported via the sex pilus to the recipient cytoplasm, forming a type IV secretion system (T4SS) conduit between the two cells, a necessary structure for the second step of conjugation, DNA transfer (6). By combining this function of the sex pilus with rolling circle replication of DNA, the donor cell is able to transfer DNA in the form of a transposable element, such as a plasmid or transposon, to the recipient by a &#34;shoot and pump&#34; model (6). In this case, the &#34;shooting&#34; is the transport of the pilot protein, with linked DNA, by the T4SS into the recipient cell, and the &#34;pumping&#34; is the active transport of DNA to the recipient, a process reliant on the T4SS and catalyzed by coupling proteins (6). The machinery used in this this process is composed of an origin of transfer sequence (oriT), which must be provided by the DNA in cis and trans genes, which encode a relaxase, mate pair formation complex, and type IV coupling protein, and can be present in cis or trans (9). This relaxase cleaves the nic site within the oriT sequence and covalently attaches to the 5&#39; end of the transferred strand to produce the relaxosome, a single-stranded DNA-relaxase complex with other auxiliary proteins (9). Once formed, the relaxosome connects to the mating pair formation complex, via the type IV coupling protein, which allows for the transfer of the ssDNA-relaxase complex into recipient cells by the T4SS (10). Once in the cytoplasm of the recipient, the DNA can integrate into the recipient genome or exist separately in the form of a plasmid, either of which allow for the expression of its genes.
In this experiment, the widely used conjugation donor strain E. coli WM3064 was used to transfer the gene encoding for ampicillin resistance to the recipient strain E. coli J53. While both strains of the gram-negative bacteria were resistant to tetracycline, only the donor strain WM3064 had the gene for ampicillin resistance, encoded for in the pWD2-oriT shuttle vector, and was auxotrophic to diaminopimelic acid (DAP) (11-13). This experiment consisted of two main steps, the preparation of donor and recipient strains, followed by the transfer of the ampicillin resistance gene from donor to recipient by conjugation (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10516/10516fig1.jpg" /> 
Figure 1: Conjugation schematic. This schematic shows the successful transfer of a plasmid, only one example of a transposable DNA element, from a donor cell to a recipient cell using conjugation. Upon contact with the recipient cell by the donor cell via the sex pilus, the plasmid replicates by rolling circle replication, moves through the multiprotein complex joining the two cells, and forms a new full-length plasmid in the recipient cell.
By incubating a mixture of donor and recipient cells, then successively plating these cells in the presence of tetracycline and DAP, this allowed for the successful transfer of the ampicillin resistance gene. Successively plating cells grown from this mixture in the presence of tetracycline and ampicillin, removed all donor cells due to the lack of DAP and any recipient cells that may not have gained the ampicillin resistance gene, yielding strictly recipient J53 strain bacteria that acquired ampicillin resistance (Figure 2). Once carried out, the successful transfer of the ampicillin resistance gene was confirmed by PCR. Since conjugation was successful, the J53 strain of E. coli contained pWD2-oriT and was resistant to ampicillin, and the gene encoding for this resistance is detectable by PCR. However, if unsuccessful there would have been no detection of the ampicillin resistance gene and ampicillin would still function as an effective antibiotic against the J53 strain.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10516/10516fig2.jpg" /> 
Figure 2: Protocol schematic. This schematic shows an overview of the presented protocol.
<img alt="Figure 3A" class="xfigimg" src="/files/ftp_upload/10516/10516fig3a.jpg" /> 
Figure 3A: The confirmation of successful conjugation by PCR. A) Freezer stocks of the conjugated and negative control samples were streaked out on agar plates and a colony was selected (red) for DNA isolation.

Conjugation: Transferring Ampicillin Resistance in E. coli

Source: Alexander S. Gold1, Tonya M. Colpitts1
1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases ...

Phage Transduction: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

Source: Alexander S. Gold1, Tonya M. Colpitts1 
1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases Laboratories, Boston, MA
Transduction is a form of genetic exchange between bacteria that utilizes bacteriophages, or phages, a class of virus that infects exclusively prokaryotic organisms. This form of DNA transfer, from one bacterium to another by way of a phage, was discovered in 1951 by Norton Zinder and Joshua Ledererg, who termed the process &#34;transduction&#34; (1). Bacteriophages were first discovered in 1915 by British bacteriologist Frederick Twort, then independently discovered again in 1917 by French-Canadian microbiologist Felix d&#39;Herelle (2). Since then, the structure and function of these phages have been widely characterized (3), dividing these phages into two classes. The first of these classes are the lytic phages which upon infection multiply within the host bacterium, disrupting the bacterial metabolism, lysing the cell, and releasing progeny phage (4). As a result of this anti-bacterial activity and the increasing prevalence of antibiotic-resistant bacteria, these lytic phages have recently proved useful as a substitute treatment for antibiotics. The second of these classes are the lysogenic phages which can either multiply within the host via the lytic cycle or enter a quiescent state in which their genome is integrated into that of the host (Figure 1), a process known as lysogeny, with the ability for phage production to be induced in multiple later generations (4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10517/10517fig1.jpg" /> 
Figure 1: Infection of host cell by bacteriophage. Adsorption by the phage to the bacterial cell wall via interactions between the tail fibers and receptor (purple). Once on the cell surface, the phage is irreversibly attached to the bacterial cell using the base plate (black) which is moved to the cell wall by the contractile sheath (yellow). Phage genome (red) then enters the cell and integrates into the host cell genome.
While bacterial transduction is a naturally occurring process, using modern technology it has been manipulated for the transfer of genes into bacteria in the laboratory setting. By inserting genes of interest into the genome of a lysogenic phage, such as phage, one is able to transfer these genes into the genomes of bacteria and consequently express them within these cells. While other methods of gene transfer, such as transformation, use a plasmid for gene transfer and expression, the insertion of the phage genome into that of the recipient bacterium not only has the potential to confer new traits to this bacterium, but also allows for naturally occurring mutations and other factors of the cellular environment to alter the function of the transferred gene.
Compared to other methods of horizontal gene transfer, such as conjugation, transduction is fairly flexible in the criteria required for donor and recipient cells. Any genetic element that can fit inside the genome of the phage being used can be transferred from any strain of donor bacteria to any strain of recipient bacteria as long as both are permissive to the phage, requiring the expression of necessary phage receptors on the cell surfaces. Once this gene is moved out of the donor genome and packaged into the phage, it can be transferred to the recipient. Following transduction, it is necessary to select for recipient bacteria that contain the gene of interest have to be selected for. This could be done by use of a genetic marker, such as a FLAG-tag or polyhistidine-tag, to mark the gene of interest, or the intrinsic function of the gene, in the case of genes that encode for antibiotic resistance. Additionally, PCR could be used to further confirm successful transduction. By using primers for a region within the gene of interest and comparing signal to a positive control, bacteria that has the gene of interest, and a negative control, bacteria that underwent the same steps as the transduction reaction with no phage. While bacterial transduction is a useful tool in molecular biology, it has and continues to play an important role in the evolution of bacteria, particularly with regard to the recent rise of antibiotic resistance.
In this experiment, bacterial transduction was used to transfer the gene encoding for resistance to the antibiotic ampicillin from the W3110 strain of E.coli to the J53 strain via the P1 bacteriophage (5). This experiment consisted of two main steps. First, the preparation of P1 phage containing the ampicillin resistance gene from the donor strain. Second, the transfer of this gene to the recipient strain by transduction with the P1 phage (Figure 1). Once carried out, the successful transfer of the ampicillin resistance gene could be determined by qPCR (Figure 2). If transduction was successful, the J53 strain of E. coli would be resistant to ampicillin, and the gene conferring this resistance detectable by qPCR. If unsuccessful, there would be no detection of the ampicillin resistance gene and ampicillin would still function as an effective antibiotic against the J53 strain.

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10517/10517fig2.jpg" /> 
Figure 2: The confirmation of successful transduction by qPCR. By comparing the Cq values detected for the gene of interest from the transduction reaction and the negative control reaction, and normalizing these values against a housekeeping gene, one was able to confirm that bacterial transduction was successful.

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

Summary

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High-Resolution Comparison of Bacterial Conjugation Frequencies

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Bacterial Transformation

Transformation of E. coli Cells Using an Adapted Calcium Chloride Procedure

Conjugation: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

Phage Transduction: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli