We thank Dr. K. Kamachi of the National Institute of Infectious Diseases (Japan) for providing pBP136 and Prof. Dr. H. Nojiri of the University of Tokyo (Japan) for providing pCAR1. We are also grateful to Professor Dr. Molin S&#248;len of the Technical University of Denmark for providing pJBA28. This work was supported by JSPS KAKENHI (Grant Numbers 15H05618 and 15KK0278) to MS (https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15H05618/, https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15KK0278/).

1. Preparation of a Donor with Green Fluorescent Protein (GFP)- and Kanamycin Resistance Gene-Tagged Plasmids
<ol>
	<li>Introduction of marker genes into the target plasmid pBP136 
	Note: The goal of this protocol is to generate pBP136::gfp. The bacterial strains and plasmids used in this study are listed in Table 1.
	<ol>
		<li>Grow cultures of Escherichia coli DH10B harboring pBP13617 in 5 mL of sterile Luria broth (LB) and E. coli S17-1&#955;pir18 harboring pJBA2819 [containing a kanamycin (Km)-resistance gene and gfpmut3* gene with its promoter and terminator in a mini-Tn5] in 5 mL of sterile LB containing 50 &#956;g/mL Km at 37 &#176;C overnight (O/N, 16&#8211;24 h) with shaking at 200 revolutions per minute (rpm).</li>
		<li>Harvesting and washing
		<ol>
			<li>Harvest 1 mL of each culture, place it into a 2 mL microtube, and centrifuge (10,000 &#215; g, room temperature, 2 min). Then, discard the supernatant and resuspend the cell pellet in 2 mL of sterile phosphate buffered saline (PBS).</li>
			<li>Centrifuge again (10,000 &#215; g, room temperature, 2 min), and resuspend in 500 &#956;L of sterile PBS.</li>
		</ol>
		</li>
		<li>Filter mating
		<ol>
			<li>Prepare sterile LB plates (with 1.6% agar), and place a sterile 0.22 &#956;m pore size membrane filter on it. Mix 500 &#956;L of E. coli S17-1&#955;pir harboring pJBA28 with E. coli DH10B harboring pBP136 and spot the mixture on the filter on the LB plate. Incubate the plate O/N at 30 &#176;C. Remove the filter from the LB plate, place it into a sterile 50 mL plastic tube, and add 1 mL of sterile PBS. 
			Note: pJBA28 can replicate in the presence of &#928; protein, encoded by the pir gene18, and can be transferred from S17-1&#955;pir to DH10B. pBP136 carries no marker gene17 and can be transferred from DH10B to S17-1&#955;pir. Therefore, we could not distinguish S17-1&#955;pir harboring pBP136 and pJBA28 from DH10B harboring pBP136 and the mini-Tn5 (transposed into the chromosome or pBP136) at this stage. Then, we used mixtures of them as donors in subsequent steps (1.1.4.&#8211;1.1.5.).</li>
		</ol>
		</li>
		<li>Grow an O/N culture of the above mating mixture in sterile LB containing 50 &#956;g/mL Km at 37 &#176;C with shaking at 200 rpm and a culture of Pseudomonas putida KT2440 [Km-sensitive (Kms), rifampicin-sensitive (Rifs), gentamicin-sensitive (Gms), and tetracycline resistant (Tcr)] in medium containing 12.5 &#956;g/mL Tc at 30 &#176;C with shaking at 200 rpm.</li>
		<li>After harvesting and washing the cells as in step 1.1.2, use them (the mating mixture and KT2440) for filter mating (O/N, 30 &#176;C) as in step 1.1.3.</li>
		<li>Prepare sterile LB plates containing 50 &#956;g/mL Km and 12.5 &#956;g/mL Tc (LB + Km + Tc plates).</li>
		<li>Dilute the resuspended mixture on the membrane filter with sterile PBS (101&#8211;105-fold), and then spread each dilution onto LB + Km + Tc plates and incubate the plates at 30 &#176;C for 2&#8211;3 d.</li>
		<li>Pick colonies from the plates, grow an O/N culture in sterile LB containing Km and Tc as well as P. resinovorans CA10dm4RG (Rifr and Gmr)6 in sterile LB containing Rif (25 &#956;g/mL) and Gm (30 &#956;g/mL) at 30 &#176;C and 200 rpm. 
		Note: As described in the previous note, the colonies on the LB + Km + Tc plates (from 1.1.7.) may be KT2440 harboring pBP136 carrying a mini-Tn5 and KT2440 with a mini-Tn5, because pJBA28 could be directly transferred from S17-1&#955;pir harboring pBP136 and pJBA28 to KT2440. This is why another mating with P. resinovorans CA10RG is required to obtain the target pBP136 with a mini-Tn5 in the following steps.</li>
		<li>After harvesting and washing the cells as in step 1.1.2, use them for filter mating (O/N, 30 &#176;C) as in step 1.1.3.</li>
		<li>Prepare sterile LB plates containing Rif, Gm, and Km (LB + Rif + Km + Gm plates).</li>
		<li>Resuspend the mixture on the filter and then dilute it 101&#8211;105-fold, spread it onto LB + Rif + Km + Gm plates, and incubate the plates for 2&#8211;3 d at 30&#160;&#176;C.</li>
		<li>Pick the colonies and check if they harbor pBP136 by PCR using specific primers for the plasmid.</li>
	</ol>
	</li>
	<li>Introduction of a selective marker gene into the target plasmid pCAR1 
	Note: The goal of this protocol is to generate pCAR1::gfp
	<ol>
		<li>Grow an O/N culture of P. putida KT2440 harboring pCAR1 (Kms, Gms, Rifs, Tcr)20 at 200 rpm and 30 &#176;C and E. coli S17-1&#955;pir18 harboring pJBA28 in 5 mL of sterile LB containing 50 &#956;g/mL Km at 200 rpm and 37 &#176;C.</li>
		<li>After harvesting and washing the cells as in step 1.1.2, use them for filter mating (O/N, 30 &#176;C) as in step 1.1.3.</li>
		<li>Remove the filter from the LB plate, place it into a sterile 50 mL plastic tube, and add 1 mL of sterile PBS.</li>
		<li>Dilute the resuspended mixture with sterile PBS (101&#8211;105-fold), and spread the diluted mixture onto sterile selective LB + Tc + Km plates. 
		Note: pCAR1 does not replicate in E. coli; thus, P. putida KT2440 harboring pCAR1 with a mini-Tn5 can be selected on LB + Tc + Km plates.</li>
		<li>Pick a colony from the plate, and grow an O/N culture in sterile LB containing Km and Tc and a culture of P. resinovorans CA10dm4RG in sterile LB containing Rif and Gm (200 rpm, 30 &#176;C).</li>
		<li>After harvesting and washing as in step 1.1.2, use the cells for filter mating (O/N, 30 &#176;C) as in step 1.1.3.</li>
		<li>Resuspend the mixture on the filter and then dilute it, spread onto LB + Rif + Km + Gm plates, and incubate the plates for 2&#8211;3 d at 30&#160;&#176;C.</li>
		<li>Pick the colonies and check if they harbor pCAR1 by PCR with specific primers for the plasmid.</li>
	</ol>
	</li>
	<li>Confirm the transferability of the tagged-plasmids and prepare the donors for the next steps 
	Note: The goal of this protocol is to confirm the transferability of the above constructed plasmids and prepare the donors for the next steps.
	<ol>
		<li>Grow an O/N culture of P. resinovorans CA10dm4RG harboring pBP136::gfp or pCAR1::gfp in sterile LB containing Km and a culture of P. putida SMDBS [Kms, Gms, Rifr, Tcr, lacIq, in which PA1/O4/O3-gfpmut3* is not expressed because of its chromosomal lacIq gene]21 in 3 mL of LB containing Tc (200 rpm, 30 &#176;C).</li>
		<li>After harvesting and washing the cells as in step 1.1.2, use them for filter mating (O/N, 30 &#176;C) as in step 1.1.3.</li>
		<li>Place the filter into a sterile 50 mL plastic tube, and resuspend with 1 mL of sterile PBS. Dilute the resuspended mixture with sterile PBS (101&#8211;105-fold), spread the diluted mixture onto sterile selective LB + Tc + Km plates.</li>
		<li>Pick the colonies and check if they harbor each of the plasmids by PCR with specific primers. 
		Note: Confirmation of the insertion position of the mini-Tn5 by direct sequencing after plasmid extraction is optional, to confirm that the insertion does not affect the transfer function of the plasmids.</li>
	</ol>
	</li>
</ol>
2. Calculation of Conjugation Frequency by the MPN Method
<ol>
	<li>Prepare sterile LB + Km and LB + Gm plates.</li>
	<li>Grow an O/N culture of P. putida SMDBS harboring pBP136::gfp or pCAR1::gfp in 3 mL of sterile LB containing Km and a culture of P. putida KT2440RGD (Gmr, Rifr) in 3 mL of LB containing Gm (140 rpm, 30 &#176;C).</li>
	<li>After harvesting and washing the cells as in step 1.1.2, use them for filter mating at 30 &#176;C for 45 min as in step 1.1.3.</li>
	<li>Serially dilute the above donor and recipient culture (101 &#8211;107) and spread it onto LB + Km (donor) or LB + Gm (recipient) plates (each in triplicate) to count the colony forming units (CFU). Incubate the plates at 30 &#176;C for 2 d.</li>
	<li>Resuspend the mixture on the filter in sterile LB containing Km and Gm, and serially dilute (from 21 to 224&#8211;107.2) using a 96-well cell culture plate (in quadruplicate).</li>
	<li>Incubate the 96-well plate for the appropriate time (2 d at 30 &#176;C).</li>
	<li>Count the CFU of the donor and recipient on the plates (step 2.4.) and count the number of wells in which the transconjugants grow.</li>
	<li>Calculate the MPN and its deviation by using the MPN calculation program developed by Jarvis et al.22, which is available at http://www.wiwiss.fu-berlin.de/fachbereich/vwl/iso/ehemalige/professoren/wilrich/MPN_ver5.xls.
	<ol>
		<li>Enter the name of the experiment (ex., &#8216;test&#8217;), the date of the experiment (ex., 2018/4/9), the number of test series, and the max. no. of dilutions (enter &#8216;24&#8217;) in row #7 of the &#8216;Program&#8217; sheet of the Excel file (&#8216;MPN_ver5.xls&#8217;).</li>
		<li>Enter &#8216;2-1 (= 0.5) to 2-24 (= 5.96 &#215; 10-8)&#8217; in the &#8216;dilution factor d&#8217; column, &#8216;0.01&#8217; in &#8216;volume in ml or g w&#8217; column, and &#8216;4&#8217; in &#8216;No. of tubes n&#8217; in the automatically produced tables of &#8216;input data&#8217;.</li>
		<li>Enter the number of wells in which the transconjugants grow at each sample dilution (0&#8211;4).</li>
		<li>Push the upper right &#8216;Calculate Results&#8217; button, and then obtain the results (in MPN/&#956;L) and their 95% confidence limits (lower and upper).</li>
	</ol>
	</li>
	<li>Calculate the conjugation frequency of the plasmids by dividing the number of transconjugants (MPN/mL) by the numbers of donor and recipient cells (CFU/mL).</li>
</ol>
3. Preparation for Estimation of the Probability of Donor-Initiated Conjugation
<ol>
	<li>Grow an O/N culture of P. putida SMDBS harboring pBP136::gfp or pCAR1::gfp in 3 mL of sterile LB containing Km and a culture of P. putida KT2440RGD (Gmr, Rifr) in 3 mL of sterile LB containing Gm using 300 mL flasks (140 rpm, 30 &#176;C) as precultures.</li>
	<li>Transfer 200 &#956;L of the preculture into 200 mL of fresh sterile LB containing Km or Gm in 500 mL flasks and incubate at 30 &#176;C with shaking at 140 rpm.</li>
	<li>Measure the turbidity at 600 nm (OD600) of the culture using a UV-VIS spectrophotometer and spot the culture, diluted in LB (101&#8211;108-fold dilutions), onto an LB plate containing Km or Gm. Incubate these LB plates at 30 &#176;C for 1&#8211;2 d, and determine the CFU.</li>
	<li>Plot the OD600 values and the CFU with growth time to generate growth curves of the donor and recipient.</li>
	<li>Grow cultures of the donor and recipient strain to mid-log phase, based on the growth curve.</li>
	<li>After harvesting and washing the cells, prepare 101&#8211;103 CFU of the donor in 10 &#956;L of LB and 105&#8211;107 CFU of the recipient in 100 &#956;L of LB.</li>
	<li>Mix 10 &#956;L of the donor and 100 &#956;L of the recipient cultures at different densities in 96-well plates (in triplicate). For example, mix 101 CFU of the donor and 105 CFU of the recipient and add it to each of the 96 wells, and mix 101 CFU of the donor and 106 CFU of the recipient in another 96-well plate, and so on.</li>
	<li>Incubate the mixture at 30 &#176;C for 45 min, and then add high concentrations of antibiotics (100 &#956;g/mL Km and 60 &#956;g/mL Gm) to each well to inhibit further conjugation.</li>
	<li>Incubate the plate at 30 &#176;C for 2 d.</li>
	<li>Count the number of wells in which transconjugants grow.</li>
	<li>Choose the recipient density that is appropriate for estimation of the probability of donor-initiated conjugation based on the above data (transconjugants should be found in at least 1 well of a 96-well plate). 
	Note: Transconjugants will grow in all wells when the densities of the donor and recipient cells are high and more than one conjugation occurs in a well. In contrast, no transconjugants will be found in any wells when the cell density is too low. In the following section, a single donor cell is sorted into a well. Therefore, the recipient density should be at maximum.</li>
</ol>
4. Estimation of the Probability of Donor-Initiated Conjugation
<ol>
	<li>Prepare 200 mL of a mid-log phase culture of donor P. putida SMDBS harboring pBP136::gfp or pCAR1::gfp and that of recipient P. putida KT2440RGD, as described in 3.6&#8211;3.7.</li>
	<li>Place 106 CFU of the recipient in 100 &#956;L of LB in each well of a 96-well plate.</li>
	<li>Set up the FACS system (flow cytometry and cell sorter with a robotic arm, a 488 nm argon laser, and a 70 &#956;m nozzle orifice). Set to forward scatter (FSC), with a 1% threshold as the acquisition trigger. Tune the H gain and A gain of the FSC and side scatter (SSC) at maximum sensitivity, which can exclude false positive signals, using PBS as a negative control. Set the sort gate based on FSC and SSC and 0.5 drop sort mode for maximal sort purity.</li>
	<li>Sort a single donor cell by FACS on an LB plate (384 different spots), incubate the plate at 30 &#176;C for 2 d, and then count how many colonies appear on the plate from the sorted cells. 
	Note: This procedure is for validation of the set gate. If there are 384 colonies on the plate, it means that 100% of the sorted cells could form colonies. The average validity of the sorting is always 90&#8211;95%.</li>
	<li>Sort a single donor cell by FACS into each well of a 96-well plate with the recipient (4.2).</li>
	<li>Incubate the plate for 45 min at 30 &#176;C, and then add high concentrations of antibiotics (100 &#956;g/mL Km and 60 &#956;g/mL Gm) to each well to prevent further conjugation.</li>
	<li>Incubate the plate at 30 &#176;C for 2 d.</li>
	<li>Count the number of wells in which transconjugants grew as determined by visual inspection with the naked eye.</li>
	<li>Calculate the probability of donor-initiated conjugation by dividing the number of wells with transconjugants by the total number of wells in which the donor was sorted.</li>
</ol>

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<li>Herrero, M., de Lorenzo, V., Timmis, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transposon+vectors+containing+non-antibiotic+resistance+selection+markers+for+cloning+and+stable+chromosomal+insertion+of+foreign+genes+in+gram-negative+bacteria%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria.</a> Journal of Bacteriology. 172 (11), 6557-6567 (1990).</li>
<li>Bagdasarian, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specific-purpose+plasmid+cloning+vectors.+II.+Broad+host+range%2C+high+copy+number%2C+RSF1010-derived+vectors%2C+and+a+host-vector+system+for+gene+cloning+in+Pseudomonas%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas.</a> Gene. 16 (1-3), 237-247 (1981).</li>
<li>Maeda, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complete+nucleotide+sequence+of+carbazole%2Fdioxin-degrading+plasmid+pCAR1+in+Pseudomonas+resinovorans+strain+CA10+indicates+its+mosaicity+and+the+presence+of+large+catabolic+transposon+Tn4676%5BTitle%5D)+AND+%22Journal+of+Molecular+Biology%22%5BJournal%5D)">Complete nucleotide sequence of carbazole/dioxin-degrading plasmid pCAR1 in Pseudomonas resinovorans strain CA10 indicates its mosaicity and the presence of large catabolic transposon Tn4676.</a> Journal of Molecular Biology. 326 (1), 21-33 (2003).</li>
<li>Takahashi, Y., Shintani, M., Yamane, H., Nojiri, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+complete+nucleotide+sequence+of+pCAR2%3A+pCAR2+and+pCAR1+were+structurally+identical+IncP-7+carbazole+degradative+plasmids%5BTitle%5D)+AND+%22Bioscience%2C+Biotechnology%2C+and+Biochemistry%22%5BJournal%5D)">The complete nucleotide sequence of pCAR2: pCAR2 and pCAR1 were structurally identical IncP-7 carbazole degradative plasmids.</a> Bioscience, Biotechnology, and Biochemistry. 73 (3), 744-746 (2009).</li>
</ol>

The authors have nothing to disclose.

Here, we present a high-resolution protocol for detecting differences in conjugation frequency under different conditions, using a MPN method to estimate the number of transconjugants. One important step in the protocol is diluting the mixture of donor and recipient after mating until no transconjugants grow. Another step is adding high concentrations of antibiotics to the selective liquid medium to prevent further conjugation. These procedures can reduce the background caused by further conjugation in the selective medi...

Bacterial conjugation of mobile genetic elements, conjugative plasmids, and integrative and conjugative elements (ICEs) is important for the horizontal spread of genetic information. It can promote rapid bacterial evolution and adaptation and transmit multidrug resistance genes1,2. The conjugation frequency can be affected by proteins encoded on the conjugative elements for mobilization of DNA (MOB) and mating pair formation (MPF), including sex pili, which are classified according to MOB and MPF type3,4,5. It can also be affected by the donor and recipient pair6 and the growth conditions of the cells7,8,9,10,11,12 (growth rate, cell density, solid surface or liquid medium, temperature, nutrient availability, and the presence of cations). To understand how the conjugative elements spread among bacteria, it is necessary to compare conjugation frequency in detail.
The conjugation frequency between donor and recipient pairs after mating are usually estimated by conventional methods as follows. (i) First, the numbers of donor and recipient colonies are counted; (ii) then, the recipient colonies, which received the conjugative elements (= transconjugants) are counted; (iii) and finally, the conjugation frequency is calculated by dividing the colony forming units (CFU) of the transconjugants by those of the donor and/or recipient13. However, when using this method, the background is high due to additional conjugation events that can also occur on the selective plates used to obtain transconjugants when the cell density is high10. Therefore, it is difficult to detect small differences in frequency (below a 10-fold difference). We recently introduced a most probable number (MPN) method using liquid medium containing a higher concentration of antibiotics. This method reduced the background by inhibiting further conjugation in selective medium; thus, the conjugation frequency could be estimated with higher resolution.
Conjugation can be divided into three steps: (1) attachment of the donor-recipient pair (2) initiation of conjugative transfer, and (3) dissociation of the pair14. During steps (1) and (3), there is physical interaction between the donor and recipient cells; thus, cell density and the environmental conditions can influence these steps, although the features of the sex pili are also important. Step (2) is likely regulated by the expression of several genes involved in conjugation in response to external changes, which could be affected by various features of the plasmid, donor, and recipient. Although the physical attachment or detachment of donor-recipient pairs can be mathematically simulated using an estimation of cells as particles, the frequency of step (2) should be experimentally measured. There have been a few reports on direct observations of how often donors can initiate conjugation [step (2)] using fluorescence microscopy15,16; however, these methods are not high-throughput because a large number of cells must be monitored. Therefore, we developed a new method to estimate the probability of the occurrence of step (2) by using fluorescence activated cell sorting (FACS). Our method can be applied to any plasmid, without identification of the essential genes for conjugation.

<table><tbody><tr><td>MoFlo XDP</td><td>Beckman-Coulter</td><td>ML99030</td><td>FACS</td></tr><tr><td>IsoFlow</td><td>Beckman-Coulter</td><td>8599600</td><td>Sheath solution</td></tr><tr><td>Fluorospheres (10 &amp;mu;m)</td><td>Beckman-Coulter</td><td>6605359</td><td>beads to set up the FACS</td></tr><tr><td>Incubator</td><td>Yamato Scientific Co. Ltd</td><td>211197-IC802</td><td></td></tr><tr><td>UV-VIS Spectrophotometer UV-1800</td><td>SIMADZU Corporation</td><td>UV-1800</td><td></td></tr><tr><td>96-well plates</td><td>NIPPON Genetics Co, Ltd</td><td>TR5003</td><td></td></tr><tr><td>microplate type Petri dish</td><td>AXEL</td><td>1-9668-01</td><td>for validation of sorting</td></tr><tr><td>membrane filter</td><td>ADVANTEC</td><td>C045A025A</td><td>for filter mating</td></tr><tr><td>pippettes</td><td>Nichiryo CO. Ltd</td><td>00-NPX2-20,&lt;br/&gt; 00-NPX2-200,&lt;br/&gt; 00-NPX2-1000</td><td>0.5-10 &amp;mu;L, 20-200 &amp;mu;L, 100-1000 &amp;mu;L</td></tr><tr><td>multi-channel pippetes</td><td>Nichiryo CO. Ltd</td><td>00-NPM-8VP,&lt;br/&gt; 00-NPM-8LP</td><td>0.5-10 &amp;mu;L, 20-200 &amp;mu;L</td></tr><tr><td>Tryptone</td><td>BD Difco</td><td>211705</td><td></td></tr><tr><td>Yeast extract</td><td>BD Difco</td><td>212750</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S-5886</td><td></td></tr><tr><td>Agar</td><td>Nakarai tesque</td><td>01162-15</td><td></td></tr><tr><td>rifampicin</td><td>Wako</td><td>185-01003</td><td></td></tr><tr><td>gentamicin</td><td>Wako</td><td>077-02974</td><td></td></tr><tr><td>kanamycin</td><td>Wako</td><td>115-00342</td><td></td></tr><tr><td>Petri dish</td><td>AXEL</td><td>3-1491-51</td><td>JPND90-15</td></tr><tr><td>microtubes</td><td>Fukaekasei</td><td>131-815C</td><td></td></tr><tr><td>500 mL disposable spinner flask</td><td>Corning</td><td>CLS3578</td><td></td></tr></tbody></table>

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.

Comparison of conjugation frequency by the MPN method
In our previous report, we compared the conjugation frequencies of pBP136::gfp and pCAR1::gfp in three-fold diluted LB (1/3 LB) liquid medium with different stirring rates after a 45 min mating using 125 mL spinner flasks10. We compared the conjugation frequencies of pBP136::gfp and pCAR1::gfp with 106 CFU/mL...

Watch this Scientific Journal Video about High-Resolution Comparison of Bacterial Conjugation Frequencies at JoVE.com

High-Resolution Comparison of Bacterial Conjugation Frequencies

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 ...

high-resolution-comparison-of-bacterial-conjugation-frequencies

Research

JoVE Journal

Genetics

10.7K Views.  Shizuoka University. 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.

Video: High-Resolution Comparison of Bacterial Conjugation Frequencies

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 ...

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Bioengineering

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.

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.

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

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 ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Biology

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...

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 ...

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