Comparisons of the transferability of plasmids pCAR1, pB10, R388, and NAH7 among Pseudomonas putida at different cell densities

Abstract

The transferability of plasmids pCAR1, pB10, R388, and NAH7 was compared using the same donor-recipient system at different cell density combinations in liquid or on a solid surface. pCAR1 was efficiently transferred in liquid, whereas the other plasmids were preferentially transferred on a solid surface. Difference of liquid or solid affected the transfer frequency especially at lower cell densities.

Key words:

• cell density
• conjugation
• horizontal gene transfer
• plasmid
• Pseudomonas putida

Conjugative transfer of DNA elements, including plasmids, is the most important mechanism of horizontal gene transfer due to its high frequency and ability to disseminate large-sized DNA. ^{1–3 )} For an in-depth understanding of plasmid transfer in nature, their transfer frequency must be compared under various conditions. Cell density is a key determinant of the behavior of bacterial populations. ^{4 )} Environmental factors are important factors for bacterial behavior, such as growth on the surface of solid substrate(s) or planktonic cells in liquids or at air–liquid interfaces. ^{5 )} This study assessed the effect of donor and recipient cell density and mating conditions (liquid or a solid surface) on plasmid transfer frequency.

Bacterial strains were grown overnight in Lysogeny Broth (LB) ^{6 )} containing appropriate antibiotics at 30 °C. Antibiotics were used at final concentrations of 50 μg mL⁻¹ for kanamycin (Km), 30 μg mL⁻¹ for gentamicin (Gm), 25 μg mL⁻¹ for rifampicin (Rif), 30 μg mL⁻¹ for chloramphenicol (Cm), and 12.5 μg mL⁻¹ for tetracycline (Tc). Solid media were prepared by the addition of 1.6% (w v⁻¹) agar to liquid LB medium. Derivative plasmids of pCAR1, ^{7–9 )} pB10, ^{10 )} R388, ^{11 )} and NAH7 ^{12 )} (pCAR1::rfp, ^{13 )} pB10::rfp, ^{14 )} R388::rfp, and NAH7K2, ^{15 )} respectively) were used in this study. R388::rfp was constructed using a method similar to that described previously for pSM1833, ^{14,16 )} and Pseudomonas resinovorans CA10dm4RG ^{17 )} (pCAR1-cured strain, spontaneous Rif^r and Gm^r gene inserted into chromosome). The rfp-Km^r gene cassette insertion site was determined as described previously, ^{13 )} and the gene cassette was inserted at position 19965nt of R388 (DDBJ/EMBL/GenBank Accession No. BR000038), an intergenic region between ORF23 (hypothetical protein) and ORF24 (hypothetical protein). The resultant plasmid was transferred from P. resinovorans CA10dm4RG(R388::rfp) into Pseudomonas putida SM1443 (P. putida KT2440-derivative strain) ^{16 )} and used as a donor of R388::rfp. Similarly, NAH7K2 was transferred from Escherichia coli MVK2 ^{18 )} to P. putida SM1443 and the resultant strain was used as a donor of NAH7K2. P. putida SM1443(pCAR1::rfp) ^{13 )} and P. putida SM1443(pB10::rfp) ^{14 )} were used as donors of pCAR1::rfp and pB10::rfp, respectively. P. putida KT2440RG ^{17 )} (derivative strain of KT2440, spontaneous Rif^r and Gm^r gene inserted into chromosome) was used as the recipient.

To evaluate the effect of cell density on plasmid transfer frequency, overnight cultures of donor and recipient cells were harvested and washed in fresh LB. The resultant cells were suspended in fresh LB to an optical density at 600 nm (OD₆₀₀) of 2 × 10⁰, 2 × 10⁻¹, 2 × 10⁻², 2 × 10⁻³, and 2 × 10⁻⁴ for the donor, and 2 × 10¹, 2 × 10⁰, 2 × 10⁻¹, 2 × 10⁻², and 2 × 10⁻³ for the recipient. The ratio of donor to recipient cells was 1:10. For liquid mating, equal volumes (200 μL) of donor and recipient cells were mixed in 2 mL microtubes from which the caps had been removed and the lids sealed with a gas-permeable adhesive seal (Thermo Fisher Scientific). For filter mating (mating on a solid surface), a mixture of donor and recipient cells was transferred onto a 0.22 μm membrane filter (GTBP, 2.5 cm²; Millipore) using Glass Microanalysis Filter Holders and Filtering Flasks (Millipore). The resultant filter was placed on an LB agar plate and incubated at 30 °C for 3 h. After incubation, triplicate aliquots (10 μL) of serially diluted mixtures from each tube were spotted on selective agar plates. Transfer frequencies were calculated by dividing the colony forming units (CFU) mL⁻¹ (liquid) or CFU (cm²)⁻¹ (solid surface) of transconjugants by those of donor cells. All experiments were performed at least twice.

The transfer frequency of plasmids in liquid is shown in Fig. 1(A). Notably, the transfer frequency of pCAR1::rfp and pB10::rfp between a donor OD₆₀₀ of 2 × 10⁻¹ and a recipient OD₆₀₀ of 2 × 10⁰ (#2 in Fig. 1(A)) was higher than that between a donor OD₆₀₀ of 2 × 10⁰ and a recipient OD₆₀₀ of 2 × 10¹ (#1 in Fig. 1(A)), despite the identical ratio of donor to recipient cells. The reason why the transfer frequency was low at high-donor density condition was unclear. There might be many donor cells being not paired with recipient cells. Interestingly, the transfer frequencies of R388::rfp and NAH7K2 were below the detection limits at lower cell densities (#5, recipient OD₆₀₀ = 2 × 10⁻³ for R388::rfp, #4, recipient OD₆₀₀ = 2 × 10⁻² and #5 for NAH7K2; Fig. 1(A)). The plasmid transfer frequencies on filters (solid surface) are shown in Fig. 1(B). pB10::rfp, R388::rfp, and NAH7K2 differed by less than 10-fold according to cell density (Fig. 1(B)). In comparison, the transfer frequency of pCAR1::rfp differed markedly (more than 100-fold) according to cell density (Fig. 1(B)). The transfer frequencies of pB10::rfp, R388::rfp, and NAH7K2 on the filters were higher than those in liquid medium irrespective of cell density. By contrast, the transfer frequency of pCAR1::rfp on the filter was lower than that in liquid medium at lower cell densities (#4 and 5, Fig. 1(A) and (B)). Note that the number of donor cells after mating differed between mating in liquid and on filters (open triangles in Fig. 1(A) and (B)). The number of donors was ~100-fold larger in liquid at a donor OD₆₀₀ of 2 × 10⁻³ or 2 × 10⁻⁴ (#4 and #5 in Fig. 1(A) and (B)). This may have been due to differences in the growth rates of the donor cells between the liquid and solid media. Cell motility on the solid surface was likely restricted in comparison to that in liquid, so cells on the solid surface might experience greater competition for nutrients. Nevertheless, the transfer frequencies of pB10::rfp, R388::rfp, and NAH7K2 were > 10-fold higher on filters than in liquid, while that of pCAR1::rfp was lower on filters (Fig. 1AB). This indicates that the mating conditions (i.e. liquid or solid surface) affected the transfer frequency of pB10, R388, and NAH7, particularly at lower cell densities. This result was probably because donor and recipient cells were more likely to come into contact on the solid surface than in liquid, although this was not the case for pCAR1. At first, we hypothesized that the pili encoded by the plasmids caused this difference. The pilus types of transmissible plasmids are classified into MPF_F, MPF_G, MPF_I, and MPF_T based on the proteins used for mating pair formation. ^{19,20 )} The plasmids pB10, R388 and NAH7 encode MPF_T, ^{19 )} which forms short, rigid pili, ^{21 )} whereas pCAR1 encodes MPF_F as well as F, ^{19 )} which forms long, flexible pili. ^{21 )} The transferability of plasmids by long, flexible pili is equivalent in liquids and on solid surfaces, whereas transfer of plasmids by short, rigid pili is more efficient on solid surfaces. ^{22 )} To assess whether the MPF class influenced plasmid transferability, pDK1K ^{23 )} and F′tet (Pasteur Institute), both of which have MPF_F-type pili, were subjected to mating assays using various cell densities. Although pCAR1, pDK1, and F possess the same MPF class pili, their transfer frequencies in the liquid and on the filters differed markedly (Supplementary Fig. S1). These results indicate that the MPF class might not be a major determinant of plasmid transfer frequency in a liquid or solid medium. The results obtained in this study are partly in contradiction with previous report. ^{22 )} It is possible that different mating conditions (e.g. donor and recipient strains or different cell densities) caused the different results.

Fig. 1. Transfer frequency of plasmids according to cell density.

Notes: P. putida SM1443 cells were used as donors and P. putida KT2440RG cells as recipients. The OD₆₀₀ values of donor and recipient cells were adjusted using LB [donor: recipient ratios, 2 × 10⁰: 2 × 10¹ (1), 2 × 10⁻¹: 2 × 10⁰ (2), 2 × 10⁻²: 2 × 10⁻¹ (3), 2 × 10⁻³: 2 × 10⁻² (4), 2 × 10⁻⁴: 2 × 10⁻³ (5)] before mixing; the identical ratio of donor to recipient cells (1:10) were used in each cell density. Mixtures were incubated in microtubes (liquid mating; A) or on a solid surface (filter mating; B) for 3 h at 30 °C. Bars show the mean transfer frequencies (transconjugants/donor) calculated from triplicate spots of serially diluted mixtures. Filled diamonds show the frequencies calculated from each spot. Open triangles indicate the numbers of donor cells after mating. All experiments were performed at least twice.

Direct comparisons of the transfer frequency of pCAR1-, pB10-, R388-, and NAH7-derivative plasmids showed that pCAR1 could be efficiently transferred in liquid regardless of donor and recipient cell density. By contrast, R388 and NAH7 showed high transfer frequency on the solid surface, even at lower cell densities. pB10 showed a constant transfer frequency (<100-fold difference) under any conditions. It could be concluded that the mating conditions (liquid or solid medium) affected the transfer frequency of plasmids, especially at lower cell densities. Determination of the effects of various environmental factors on the transferability of plasmids is important to understand not only the influence of the plasmids on the host cell but also the mechanisms by which the plasmids survive in various environments.

Authors contribution

Kosuke Yanagida and Ayako Sakuda carried out the experiments with assistance from Kazuhiro Matsui. Kosuke Yanagida, Chiho Suzuki-Minakuchi, and Masaki Shintani prepared the manuscript. Kazunori Okada contributed analysis and discussion. Hideaki Nojiri designed the project and reviewed the manuscript.

Funding

This work was supported by JSPS KAKENHI [grant number 15H05618] (to M. S.) [grant number 15K14686] (to H. N.).

Supplemental materials

The supplemental material for this paper is available at http://dx.doi.org/10.1080/09168451.2015.1127131.

Acknowledgments

We are grateful to Prof. Eva Top of University of Idaho for providing pB10::rfp. We also thank Prof. Masataka Tsuda of Tohoku University for generously providing NAH7K2, pDK1, P. fluorescens Pf-5S, and P. fluorescens Pf-5G.

Disclosure statement

No potential conflict of interest was reported by the authors.

Notes

Abbreviations: Cm, chloramphenicol; Gm, gentamicin; Km, kanamycin; LB, Lysogeny Broth; OD₆₀₀, optical density at 600 nm; Rif, rifampicin; Tc, tetracycline.