High-Resolution Comparison of Bacterial Conjugation Frequencies

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 genes^1,2. 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 type^3,4,5. It can also be affected by the donor and recipient pair⁶ and the growth conditions of the cells^{7,8,9,10,11,12} (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 microscopy^15,16; 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.

Protokół

1. Preparation of a Donor with Green Fluorescent Protein (GFP)- and Kanamycin Resistance Gene-Tagged Plasmids

1. 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.
   1. Grow cultures of Escherichia coli DH10B harboring pBP136¹⁷ in 5 mL of sterile Luria broth (LB) and E. coli S17-1λpir¹⁸ 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 μg/mL Km at 37 °C overnight (O/N, 16–24 h) with shaking at 200 revolutions per minute (rpm).
   2. Harvesting and washing
      1. Harvest 1 mL of each culture, place it into a 2 mL microtube, and centrifuge (10,000 × g, room temperature, 2 min). Then, discard the supernatant and resuspend the cell pellet in 2 mL of sterile phosphate buffered saline (PBS).
      2. Centrifuge again (10,000 × g, room temperature, 2 min), and resuspend in 500 μL of sterile PBS.
   3. Filter mating
      1. Prepare sterile LB plates (with 1.6% agar), and place a sterile 0.22 μm pore size membrane filter on it. Mix 500 μL of E. coli S17-1λpir 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 °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 Π protein, encoded by the pir gene¹⁸, and can be transferred from S17-1λpir to DH10B. pBP136 carries no marker gene¹⁷ and can be transferred from DH10B to S17-1λpir. Therefore, we could not distinguish S17-1λpir 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.–1.1.5.).
   4. Grow an O/N culture of the above mating mixture in sterile LB containing 50 μg/mL Km at 37 °C with shaking at 200 rpm and a culture of Pseudomonas putida KT2440 [Km-sensitive (Km^s), rifampicin-sensitive (Rif^s), gentamicin-sensitive (Gm^s), and tetracycline resistant (Tc^r)] in medium containing 12.5 μg/mL Tc at 30 °C with shaking at 200 rpm.
   5. 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 °C) as in step 1.1.3.
   6. Prepare sterile LB plates containing 50 μg/mL Km and 12.5 μg/mL Tc (LB + Km + Tc plates).
   7. Dilute the resuspended mixture on the membrane filter with sterile PBS (10¹–10⁵-fold), and then spread each dilution onto LB + Km + Tc plates and incubate the plates at 30 °C for 2–3 d.
   8. Pick colonies from the plates, grow an O/N culture in sterile LB containing Km and Tc as well as P. resinovorans CA10dm4RG (Rif^r and Gm^r)⁶ in sterile LB containing Rif (25 μg/mL) and Gm (30 μg/mL) at 30 °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 S17-1λpir 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.
   9. After harvesting and washing the cells as in step 1.1.2, use them for filter mating (O/N, 30 °C) as in step 1.1.3.
   10. Prepare sterile LB plates containing Rif, Gm, and Km (LB + Rif + Km + Gm plates).
   11. Resuspend the mixture on the filter and then dilute it 10¹–10⁵-fold, spread it onto LB + Rif + Km + Gm plates, and incubate the plates for 2–3 d at 30 °C.
   12. Pick the colonies and check if they harbor pBP136 by PCR using specific primers for the plasmid.
2. Introduction of a selective marker gene into the target plasmid pCAR1
   Note: The goal of this protocol is to generate pCAR1::gfp
   1. Grow an O/N culture of P. putida KT2440 harboring pCAR1 (Km^s, Gm^s, Rif^s, Tc^r)²⁰ at 200 rpm and 30 °C and E. coli S17-1λpir¹⁸ harboring pJBA28 in 5 mL of sterile LB containing 50 μg/mL Km at 200 rpm and 37 °C.
   2. After harvesting and washing the cells as in step 1.1.2, use them for filter mating (O/N, 30 °C) as in step 1.1.3.
   3. Remove the filter from the LB plate, place it into a sterile 50 mL plastic tube, and add 1 mL of sterile PBS.
   4. Dilute the resuspended mixture with sterile PBS (10¹–10⁵-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.
   5. 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 °C).
   6. After harvesting and washing as in step 1.1.2, use the cells for filter mating (O/N, 30 °C) as in step 1.1.3.
   7. Resuspend the mixture on the filter and then dilute it, spread onto LB + Rif + Km + Gm plates, and incubate the plates for 2–3 d at 30 °C.
   8. Pick the colonies and check if they harbor pCAR1 by PCR with specific primers for the plasmid.
3. 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.
   1. 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 [Km^s, Gm^s, Rif^r, Tc^r, lacI^q, in which P_A1/O4/O3-gfpmut3* is not expressed because of its chromosomal lacI^q gene]²¹ in 3 mL of LB containing Tc (200 rpm, 30 °C).
   2. After harvesting and washing the cells as in step 1.1.2, use them for filter mating (O/N, 30 °C) as in step 1.1.3.
   3. 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 (10¹–10⁵-fold), spread the diluted mixture onto sterile selective LB + Tc + Km plates.
   4. 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.

2. Calculation of Conjugation Frequency by the MPN Method

1. Prepare sterile LB + Km and LB + Gm plates.
2. 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 (Gm^r, Rif^r) in 3 mL of LB containing Gm (140 rpm, 30 °C).
3. After harvesting and washing the cells as in step 1.1.2, use them for filter mating at 30 °C for 45 min as in step 1.1.3.
4. Serially dilute the above donor and recipient culture (10¹ –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 °C for 2 d.
5. Resuspend the mixture on the filter in sterile LB containing Km and Gm, and serially dilute (from 2¹ to 2²⁴–10^7.2) using a 96-well cell culture plate (in quadruplicate).
6. Incubate the 96-well plate for the appropriate time (2 d at 30 °C).
7. 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.
8. 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.
   1. Enter the name of the experiment (ex., ‘test’), the date of the experiment (ex., 2018/4/9), the number of test series, and the max. no. of dilutions (enter ‘24’) in row #7 of the ‘Program’ sheet of the Excel file (‘MPN_ver5.xls’).
   2. Enter ‘2^-1 (= 0.5) to 2^-24 (= 5.96 × 10^-8)’ in the ‘dilution factor d’ column, ‘0.01’ in ‘volume in ml or g w’ column, and ‘4’ in ‘No. of tubes n’ in the automatically produced tables of ‘input data’.
   3. Enter the number of wells in which the transconjugants grow at each sample dilution (0–4).
   4. Push the upper right ‘Calculate Results’ button, and then obtain the results (in MPN/μL) and their 95% confidence limits (lower and upper).
9. 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).

3. Preparation for Estimation of the Probability of Donor-Initiated Conjugation

1. 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 (Gm^r, Rif^r) in 3 mL of sterile LB containing Gm using 300 mL flasks (140 rpm, 30 °C) as precultures.
2. Transfer 200 μL of the preculture into 200 mL of fresh sterile LB containing Km or Gm in 500 mL flasks and incubate at 30 °C with shaking at 140 rpm.
3. Measure the turbidity at 600 nm (OD₆₀₀) of the culture using a UV-VIS spectrophotometer and spot the culture, diluted in LB (10¹–10⁸-fold dilutions), onto an LB plate containing Km or Gm. Incubate these LB plates at 30 °C for 1–2 d, and determine the CFU.
4. Plot the OD₆₀₀ values and the CFU with growth time to generate growth curves of the donor and recipient.
5. Grow cultures of the donor and recipient strain to mid-log phase, based on the growth curve.
6. After harvesting and washing the cells, prepare 10¹–10³ CFU of the donor in 10 μL of LB and 10⁵–10⁷ CFU of the recipient in 100 μL of LB.
7. Mix 10 μL of the donor and 100 μL 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.
8. Incubate the mixture at 30 °C for 45 min, and then add high concentrations of antibiotics (100 μg/mL Km and 60 μg/mL Gm) to each well to inhibit further conjugation.
9. Incubate the plate at 30 °C for 2 d.
10. Count the number of wells in which transconjugants grow.
11. 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.

4. Estimation of the Probability of Donor-Initiated Conjugation

1. 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 3.6–3.7.
2. Place 10⁶ CFU of the recipient in 100 μL of LB in each well of a 96-well plate.
3. Set up the FACS system (flow cytometry and cell sorter with a robotic arm, a 488 nm argon laser, and a 70 μm 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.
4. Sort a single donor cell by FACS on an LB plate (384 different spots), incubate the plate at 30 °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 90–95%.
5. Sort a single donor cell by FACS into each well of a 96-well plate with the recipient (4.2).
6. Incubate the plate for 45 min at 30 °C, and then add high concentrations of antibiotics (100 μg/mL Km and 60 μg/mL Gm) to each well to prevent further conjugation.
7. Incubate the plate at 30 °C for 2 d.
8. Count the number of wells in which transconjugants grew as determined by visual inspection with the naked eye.
9. 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.

Wyniki

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...

Dyskusje

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...

Materiały

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