Selective elimination of donor bacteria to analyze plasmid reactions during conjugative transfer

Abstract

Bacterial conjugation is an important means of horizontal gene transfer in which DNA is transferred from a donor to a recipient cell by direct cell-to-cell contact. Dissemination of antibiotic resistance in bacteria is very often driven by conjugative plasmids harboring antibiotic resistance genes. Recent research highlighted the temporal cascade of DNA reactions that are critical for the establishment of a plasmid in the new host, which include prompt and robust induction of anti-defense genes as well as re-formation of double-stranded circular form of the plasmid after transfer as single-stranded linear DNA. The nature of conjugation which occurs in a mixture of cell populations, constrained the research on plasmid establishment. Since plasmid molecules before and after transfer are indistinguishable, distinction between donor and recipient/transconjugant cells is required, and physical separation is particularly necessary for the genomic approaches. We established a novel method exploiting a mutant donor which can be expeditiously eliminated from the conjugation, mixture with simple manipulation. This method, dubbed ED-TA for elimination of donor cells for transconjugant analysis, was shown to be a powerful tool to unveil plasmid gene expression profile at the early stages of conjugation (Wen et al. 2025, Nucleic Acids Res, doi: 10.1093/nar/gkaf1299). Here we present an optimized protocol for the ED-TA method which will help our study on plasmid actions during establishment in new host cell, including emerging interests between host defense and plasmid anti-defense systems.

Introduction

Horizontal gene transfer (HGT) plays an important role for bacterial evolution as it facilitates acquisition of new genetic traits. Particularly, antibiotic resistance and virulence are often transmitted by conjugation, whose corresponding genes are encoded on conjugative mobile genetic elements such as plasmids. During conjugation, plasmid DNA is transferred in a single-stranded (ss) linear form from the donor cell to the recipient cell. Subsequently, original double-stranded (ds) circular plasmid DNA is reconstituted in the recipient. Once the plasmid is sustained, the resulting cell is referred to as transconjugant. Since conjugative plasmids also encode genes necessary for their transfer, these transconjugant cells eventually act as donor cells, thus threatening exponential spreading of antibiotic-resistant bacteria.

However, successful dissemination of conjugative plasmids can be somewhat puzzling, knowing that many bacteria have defense mechanisms against alien DNA such as plasmids and phages. In addition to well-known restriction-modification and CRISPR-Cas systems, diverse defense mechanisms have been recently discovered by genomic approaches, and they are becoming an emerging topic [1–3]. On the other hand, it is also recently been appreciated that many conjugative plasmids harbor anti-defense systems [4]. However, these anti-defense systems must function before plasmid is challenged by defense systems. To this end, plasmids encode anti-defense genes in the leading region, the first region to be transferred during the conjugation, along with an ssDNA promoter so that they can be expressed at early stages of conjugation even before the conversion to dsDNA [4–6]. In addition, ssDNA promoter was suggested to be involved in the replication to dsDNA through primer RNA synthesis [7]. Recent study also showed that host-encoded UvrD helicase is involved in the ss to dsDNA conversion process [8]. Together, it is clear that there are intriguing plasmid reactions in the relatively limited time frame of the early stages of conjugation, which could determine abortive or successful conjugation.

In a paradigmatic F plasmid, a handful of leading region genes are examined with their protein production during conjugation as GFP fusion under time-lapse microscopy [5]. While the single-cell level analysis gives great temporal resolution, it sacrifices plasmid-wide throughput. Genomic approaches enable such plasmid-wide analyses; however, it suffers from the technical hurdle of cell separation. Conjugation takes place as a mixture of cells: donor, recipient and eventually transconjugant, and among them transconjugants are the cells of interest. An experimental setup can make donors and recipients distinguishable (eg, resistance markers, fluorescent protein labels, or even different bacterial species), however, a plasmid in a donor or recipient cell is indistinguishable. While microscopic approach enables separation of these cell types at the data analysis stage, genomic approaches require physical separation of cell types at sample (DNA/RNA) preparation. Traditional isolation of transconjugant cells on selective media requires approx. 30 cellular generations after the transfer and establishment of the plasmid. Flow cytometry has been appreciated for a means to distinguish donor, recipient and transconjugant cells from mixed population without cultivation, and its coupling to cell sorting allows collection of transconjugant cells for downstream analysis [9]. However, it relies on the expression of fluorescent protein (eg, GFP) inserted on the plasmid of interest, so that there will be an inevitable maturation time for its expression and folding before the protein becomes fluorescent [10]. Furthermore, it can require hours to collect 10⁸ cells. Indeed, shorter processing time is critical to capture timely events. An alternative approach to cell sorting is elimination of donor cells. This has an advantage which, does not require engineering of “reporter” on the plasmid. However, many approaches, such as challenging antibiotics or phages, or induction of toxic genes, will also require a lot of time for selective killing. Miyakoshi et al. used rifampicin-sensitive and resistant strains to investigate early transcriptional responses after conjugation [11]. It was efficient to investigate transcriptional responses at very early time point after conjugation; however, it is not widely applicable for other plasmid reactions. Therefore, it is apparent that a new method is required to investigate reactions happening in the early stages of conjugation.

Overview of the technique and expected outcomes

We introduced a new method which takes an alternative approach to separate recipient from donor population: elimination of donor cells, by exploiting a genetically modified Escherichia coli strain which can be selectively killed by hypoosmotic shock, in a couple of minutes [12]. In brief, we used this outer membrane-periplasm “disconnected” (DISCO) mutant as the donor and optimized the condition of killing. The resulting method, dubbed ED-TA for elimination of donor for transconjugant analysis, was successfully applied to unveil the transcriptome of pESBL at early stages of plasmid establishment or in abortive conjugation [13]. Here, we describe a step-by-step, hands-on protocol for the ED-TA method. We used the superspreader mutant of pESBL [14–15] and F as test conjugative plasmids but other conjugative plasmids are certainly applicable, yet higher conjugation efficiency is preferred. Furthermore, this protocol uses E. coli as recipient organism, but it can be adapted for other species [13]. Collected recipient/transconjugant cells can be used for downstream experiments to investigate fascinating reactions during conjugation, such as re-formation of circular plasmid molecules and characterization of anti-defense mechanisms.

General solutions and reagents

• Lysogeny broth (LB; Miller)

• LB agar (1.6% w/vol)

• Kanamycin (Km, 50 mg/mL)

• Chloramphenicol (Cm, 25 mg/mL)

• Ampicillin (Amp, 100 mg/mL) or Tetracycline (Tet, 15 mg/mL)

• Magnesium chloride (MgCl₂, 1 M)

• Ethylenediaminetetraacetic acid (500 mM)

• Sodium dodecyl sulfate (SDS, 20%)

Note: The donor strain confers Km. pESBL and F confer Amp and Tet, respectively so that in case of testing other conjugative plasmids use different antibiotics accordingly. This protocol used a recipient strain resistant to Cm, but it can be changed. The antibiotic stock solutions are 1000 × concentrated.

Bacterial strains

• Outer membrane-periplasm ‘disconnected’ (DISCO) mutant E. coli, such as EP272 [12] and bEYY2900 (MG1655 ΔompA::kan Δlpp).

• DISCO mutant harboring the test conjugative plasmid as donor. For example, bEYY2743 for pESBL and bEYY2901 for F.

• Recipient strain exhibiting compatible antibiotic resistance. For example, bEYY1100 (MG1655 ΔlacZ::cat).

Note: The DISCO mutant shows somewhat lower viability, which is amended by addition of 1 mM MgCl₂ [12].

Equipment and other small items

• Shaker and static incubators

• Spectrophotometer

• Centrifuge (eg, Eppendorf, 5804R) which holds 50-mL centrifuge tubes and centrifuges at 5000×g. Temperature control (refrigeration) is preferred but not obligate.

• Vortex mixer

• Cellulose filter membranes (25 mm diameter with 0.45 µm pore size, eg, Millipore HAWP02500).

• 50-mL Disposable conical centrifuge tubes

• (optional) 125-mL disposable sterile flasks (eg, Thermo PETG Flask #4112-0125)

Methods

Preparation of cells

1. Prepare overnight culture of donor (in LB supplemented with 1 mM MgCl₂ and appropriate antibiotics) and recipient (in LB supplemented with appropriate antibiotic) grown at 37°C.

2. In a 125-mL Erlenmeyer, prepare 20 mL LB supplemented with 1 mM MgCl₂ and appropriate antibiotics for the donor. Inoculate 200 µL of overnight culture of the donor cells (1:100 dilution). Incubate in a shaker incubator at 37°C and set t = 0.

   Note 1: Leftover of residential detergents in Erlenmeyer can cause surprising lysis of the donor cells. When applicable, use of disposable sterile flask is advised.

3. In another 125-mL Erlenmeyer flask, prepare 20 mL of LB supplemented with appropriate antibiotic for the recipient cells. At t = 30 minutes, inoculate 20 µL of overnight culture of the recipient (1:1000 dilution) and incubate in the 37°C shaker.

   Note 2: For liquid conjugation, supplement of antibiotics is required for overnight culture but not for the following subculture steps.

4. Monitor the Optical Density (OD) routinely until OD reaches around 1.0 (suggested timings of measurement are t = 3.5, 4.0 and 4.5 hours).

   Note 3: To obtain constant results among experiments, it is recommended to use log phase culture rather than directly using the overnight culture.

Conjugation experiment (solid surface conjugation)

1. Before cells reach OD ∼1.0, Prepare an LB agar plate supplemented with 1 mM MgCl₂. Let the plate dry well and place 2 filter membranes on the plate.

2. Put 16 mL of donor cells in a 50 mL centrifuge tube and 10 mL of recipient cells in another tube.

3. Centrifuge at 5000×g for 3 minutes at 16°C.

4. Remove the supernatant by decantation and pipetting.

5. Resuspend the donor and recipient cells with 200 µL of LB supplemented with 1 mM MgCl₂ and 100 µL of LB, respectively. Mix well by pipetting. Take entire cell suspension of donor cells and put it in the recipient tube.

6. Mix well the donor and recipient cells with pipetting, then put cell mixture on the filter membranes (150 µL per membrane).

7. Start the timer (in this protocol, 30 minutes). Wait a few minutes to dry the membrane then place the plate in 37°C static incubator.

Conjugation experiment (liquid conjugation)

1. Put 16 mL of donor cells and 10 mL of recipient cells in a 50 mL centrifuge tube and mix well.

2. Start the timer (in this protocol, 30 minutes) and place the centrifuge tube in 37°C static incubator.

IMPORTANT: As shown in Fig. 1, conjugation frequency on solid surface and in liquid can be very different depending on the conjugative plasmid of interest. For the best practice, it is recommended that conjugation conditions are determined by pilot experiments. It is also suggested to adjust input donor: recipient ratio, if necessary, by changing the timing of inoculation, dilution factor, and volume of exponential phase cells to be used.

Figure 1.

Elimination of donor cells by the ED-TA method. Colony-forming units (CFUs) of Donor (D), Recipient (R) and Transconjugant (T) cells before (Input) and after conjugation followed by EDTA treatment are shown. (A) Transfer of superspreader pESBL. (B) Transfer of F. The conjugation reaction was carried out on filter membrane (Solid) or in liquid (Liquid) for 30 minutes. R* was measured by Cm-R colonies so that it does not consider whether it received the plasmid or not. The average and standard deviation of at least 3 independent experiments were shown, along with individual results as closed dots. When no viable cells were recovered, the limit of detection was shown as an open dot. (A) Input and Solid data were recapitulated from [13].

Measure the number of cells of input (optional but recommended)

1. Prepare plates in advance for selective growth of donor and recipient.

2. Prepare serial dilutions of donor and recipient cultures grown in Erlenmeyer. Spread 100 µL of 10⁻⁴, 10⁻⁵ and 10⁻⁶ dilutions for donor cells and 10⁻⁵ and 10⁻⁶ dilutions for recipient cells, respectively. Incubate plates overnight at 37°C.

3. Calculate Colony forming units (CFUs; per mL) from plates. Then multiply by the volume used (eg, 16 for donor and 10 for recipient) to measure the number of cells applied for conjugation.

Elimination of donor cells (solid surface conjugation)

1. Prepare 50 mL of 2 mM EDTA with 0.1% SDS and 10 mL 2 mM EDTA solution

2. Once timer goes off, take out the conjugation plate from the incubator. Using sterile forceps, pick up one filter membrane and put it in a new 50-mL centrifuge tube.

3. Add 2 mL of a 2 mM EDTA with 0.1% SDS solution and wash cells out from the membrane by pipetting.

4. Add the other filter membrane in the centrifuge tube and an additional 2 mL of 2 mM EDTA with 0.1% SDS solution.

5. Similarly, wash cells out from the second membrane by pipetting. Vortex for a few seconds.

6. Add 16 mL of 2 mM EDTA with 0.1% SDS solution into the mixture and initiate a 2-minute timer. Meanwhile, vortex at full speed for 2 times for 10-15 seconds. Then, pick up the filter membranes with forceps and discard them.

7. When timer goes off, centrifuge the tube at 5000×g for 3 minutes at 16°C. Discard the supernatant.

8. Resuspend the cells with 20 mL of 2 mM EDTA with 0.1% SDS solution.

9. Centrifuge at 5000×g for 3 minutes at 16°C. Discard the supernatant.

10. Resuspend the cells with 1 mL of 2 mM EDTA solution and transfer the cells into an Eppendorf tube.

Elimination of donor cells (liquid conjugation)

1. Prepare 50 mL of 2 mM EDTA with 0.1% SDS and 10 mL 2 mM EDTA solution

2. Once timer goes off, take out the conjugation mixture from the incubator.

3. Centrifuge at 5000×g for 3 minutes at 16°C. Discard the supernatant.

4. Resuspend the cells with 20 mL of 2 mM EDTA with 0.1% SDS solution and initiate a 2-minute timer. Meanwhile, vortex at full speed for 2 times for 10–15 seconds.

5. When timer goes off, centrifuge the tube at 5000×g for 3 minutes at 16°C. Discard the supernatant.

6. Resuspend the cells with 20 mL of 2 mM EDTA with 0.1% SDS solution

7. Centrifuge the tube at 5000×g for 3 minutes at 16°C. Discard the supernatant.

8. Resuspend the cells with 1 mL of 2 mM EDTA solution and transfer the cells into an Eppendorf tube.

Measure the efficiencies of donor killing and conjugation

1. Prepare plates in advance for selective growth of donor, recipient and transconjugant.

2. Take 100 µL of resuspended cells to prepare serial dilutions. Spread 100 µL of 10⁻¹ dilution for donor, 10⁻⁵ and 10⁻⁶ dilutions for recipient, and 10⁻⁴, 10⁻⁵ and 10⁻⁶ dilutions for transconjugant cells. Incubate plates overnight at 37°C.

3. Calculate the CFU from plates.

Snap-freeze cells for downstream experiments

1. Prepare a small bath of liquid nitrogen (alternatively, dry ice-ethanol).

2. (optional) Aliquot the resuspended cells into 2-3 Eppendorf tubes.

3. Centrifuge the Eppendorf tube(s) at max speed for 1 minute. Discard the supernatant.

4. Snap-freeze the cell pellet and keep the tubes at −80°C.

It was reported that addition of 1 mM MgCl₂ alleviated the DISCO mutant sensitivity to hypoosmotic shock, allowing 2-3 logs better survival rate. On the other hand, addition of 1 mM EDTA enhanced sensitivity, resulting in virtually no survivors [12]. It is also confirmed that 2 mM EDTA treatment did not affect viability of recipient cells. Therefore, conjugation mixture is challenged by 2 mM EDTA to ensure the selective killing of donor at utmost efficiency.

However, gDNA extracted from the EDTA-treated cell pellet contained certain levels of contamination of donor-derived DNA, which may not be neglectable when the transfer frequency was not high enough. Time-lapse microscopy observation of DISCO mutant indicated that the cells do not really burst after the hypoosmotic shock [12]. It is very plausible that some donor DNA and RNA were not released to the solution but retained in the cell debris so that they were sedimented with recipient cells and eventually extracted. Indeed, addition of SDS in the EDTA solution solved the contamination issue (Table 1). E. coli wild type recipient was confirmed to be resistant up to 1% SDS (even in combination with 2 mM EDTA). However, higher SDS concentration makes cell pellet loose after centrifugation and we recommend using 0.1%.

Table 1.

Quantification of DNA.

Conjugation	Donora	Recipientb	pESBLc	n
Mock donor	1d	0.001 ± 0.000	2.83 ± 0.62	3
E. coli (EDTA)	0.061 ± 0.054	1d	0.81 ± 0.69	5
E. coli (EDTA + 0.1% SDS)	0.005 ± 0.006	1d	0.58 ± 0.44	3

Real-time quantitative PCR was used to measure the ratio of donor-, recipient- and plasmid-derived DNA. Average and standard deviation of multiple independent experiments (n) are shown. Mock donor experiment did not include recipient cells in the conjugation mixture and the donor cells were collected in LB. Target genes used are

^a kan,

^b ompA,

^c hp23,

^dSet as 1. Extracted from ref. [13].

Advantages and limitations

While selection of recipient and/or transconjugant cells can be done in other methods including cell sorting, the main advantage of the ED-TA method is its minimum modification to the conventional conjugation experiment: after conjugation reaction, cells are recovered/washed with EDTA/SDS solution instead of growth media (eg, LB) or saline. ED-TA method also requires a couple of rounds of washing steps, but they can be done in <12 minutes (including handling time) with high yield of cells, which are incomparable to cell sorting. Another advantage of the ED-TA method is that it can be applied to abortive conjugation. While few or no transconjugant colonies will be formed on the selective media, this method allows us to investigate how conjugation failed. Lastly, it is also noted that the ED-TA method requires only basic laboratory reagents and equipment. Therefore, the ED-TA method greatly facilitates the study of the process of plasmid establishment in recipient cells during conjugation.

However, there are still certain limitations. First, this method is ideal to investigate ‘plasmid’ reaction in recipient cells, however, it is not always the case to study the ‘host’ reactions during conjugation. In theory, there will be mixed population of recipient cells: one whose plasmid is (will be) established, one’s plasmid will not be established by any means (including host defense mechanisms), and one’s conjugation was not challenged. Unfortunately, current protocol is not applicable to distinguish these 3 types of recipient cells. If almost all recipient cells became transconjugant cells (judged by colony formation on selective media), host reactions such as change of chromosomal gene expression can be analyzed. However, in this case ‘Mock recipient’ control experiment to confirm the effects of EDTA/SDS treatment is strongly suggested. Second, while the ED-TA method can be used to study abortive conjugations, some cautions are required in case of out-of-host range bacteria as recipient. Certain bacterial species are sensitive to the washing condition used in this protocol (2 mM EDTA + 0.1% SDS) so that adjustment of the buffer composition, which followed by re‑validation of donor elimination and recipient survival, is required. For example, Vibrio cholerae is sensitive to low salt washing and supplement of 100 mM NaCl was necessary. Lastly, speed of DNA transfer was calculated as 770 nucleotides per second [16], suggesting that transfer of the conjugative plasmid with 90 kb in size will be done in ∼2 minutes. The protocol and its nature of studying a population of cells are unlikely to provide a time resolution to study the DNA transfer event. Combination with time-lapse microscopy may complement these limitations.

Troubleshooting

The most common issue one may encounter is the contamination of donor, particularly DNA or RNA, which usually comes from poor washing. This will be more critical in case of lower transfer frequency. We highly recommend carrying out quantitative PCR to calculate ratio of donor, recipient, and plasmid for each experiment before performing downstream applications. If rigid washing did not improve the contamination rate, some adjustments on washing conditions will be considered: composition and volume of washing buffer, time and vortexing, speed of centrifugation and so on.

While we found supplementation with 0.1% SDS works best to remove contamination of donor nucleic acids while not making cell pellet too loose, it can make cell pellet transparent after centrifugation. Even if cell pellet was not apparent after the second centrifugation, we strongly encourage to continue the sampling.

Author contributions

Meng Wen (Investigation [equal], Methodology [equal], Writing—original draft [equal], Writing—review & editing [equal]), Emma Mettouchi (Investigation [equal], Methodology [equal], Writing—original draft [equal], Writing—review & editing [equal]), Alejandra Lopez Sanchez (Investigation [supporting], Methodology [supporting], Writing—review & editing [supporting]), and Yoshiharu Yamaichi (Conceptualization [lead], Formal analysis [lead], Funding acquisition [lead], Investigation [supporting], Methodology [supporting], Supervision [lead], Visualization [lead], Writing—original draft [equal], Writing—review & editing [equal])

Conflicts of interest

None declared.

Funding

This work was supported by the French National Research Agency under Grant No. ANR-22-CE12-0032. The M.W. is supported by the China Scholarship Council.