Characterization of putative active factor in CM of CAG18439.

<p>Effects of heat exposure and protease treatment of CM of CAG18439 on cell-to-cell transformation and cell growth in co-culture of MG1655 harbouring pHSG299 and MC4100. Heat treatment of CM was performed at 121°C for 20 min. Treatment of CM with trypsin (100 µg/mL) and proteinase K (200 µg/mL) was performed at 37°C for 120 min. CM was used for culture at 50% (v/v) in heat experiments and at 1% (v/v) in protease experiments. Data are presented as mean and S.D. (*: <i>t</i>-test: <i>P</i><0.05, <i>n</i> = 4; †: <i>t</i>-test: <i>P</i><0.005, <i>n</i> = 4).</p

Comparison of cell-to-cell transformation with artificial transformation.

<p>Strain names: CAG, CAG18439; HB, HB101; and MC, MC4100. Cell-to-cell transformation (A) was performed with three combinations including donor cells harbouring pHSG299. Artificial transformations by the CaCl₂ method (B) and PEG method (C) was performed as described in Methods. In artificial transformation, plasmid (pHSG299) was used in a semi-saturating amount (500 ng/sample) or the roughly estimated amount of leaked plasmid DNA in co-culture [CAG: 2×10^–10; HB: 1×10^–8; MC: 1×10^–9 ng per recipient ( = competent) cells]. The latter values were calculated on the assumptions that free plasmid DNA was supplied from dead donor cells (maximum 5% of total population) to co-existing recipient cells in each corresponding co-culture of cell-to-cell transformation, and that the pHSG299 copy number is ∼200 per cell <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016355#pone.0016355-Hong1" target="_blank">[38]</a>. Transformation frequency and transformation efficiency were calculated as described in Methods. Data are presented as mean and S.D. (<i>n</i> = 3).</p

Lateral plasmid transfer with various combinations of strains and plasmids in liquid culture.

<p>Frequency of plasmid transfer (mean, <i>n</i> = 3) in each combination is presented in decimal ranges as follows: ++++++, 1E–4 to 1E–5; +++++, 1E–5 to 1E–6; ++++, 1E–6 to 1E–7; +++, 1E–7 to 1E–8.</p

Lateral plasmid transfer through nylon membrane filter in colony biofilm culture.

<p>Frequency of plasmid transfer (mean, <i>n</i> = 3) in each combination is presented in decimal ranges as follows: ++++++++, 1E–2 to 1E–3; ++, 1E–8 o 1E–9; – : below detection limit.</p

Lateral plasmid transfer with various combinations of strains and plasmids in colony biofilm culture.

<p>Frequency of plasmid transfer (mean, <i>n</i> = 3) in each combination is presented in decimal ranges as follows: ++++++, 1E–4 to 1E–5; +++++, 1E–5 to 1E–6; +++, 1E–7 to 1E–8; ++, 1E–8 to 1E–9; +, 1E–9 to 1E–10; – : below detection limit; n.d., not determined or unable to examine because of coincidence of the same antibiotic resistance between strains and plasmids. Samples with plasmid transfer frequency >1E–6 are indicated by .</p

Effects of <i>Tn10</i> and <i>lacI</i> mutations on cell-to-cell transformation.

<p>DH5 harbouring pHSG299 (*) or DH5 harbouring pHSG299-cam (†) was used as the donor strain. Transformation frequency (mean, <i>n</i> = 3) in each combination is presented in decimal ranges as follows: +++++, 1E−5 to 1E−6; ++++, 1E−6 to 1E−7; +, 1E−9 to 1E−10; −, below detection limit.</p

Measurement of the dead cell ratio in liquid and colony biofilm cultures.

<p>*Measured at 24 h from culture start.</p

Effects of dilution of CM of CAG18439 on cell-to-cell transformation and cell growth.

<p>Effects of dilution of CM of CAG18439 on cell-to-cell transformation (A) and cell growth (B). Co-culture of MG1655 harbouring pHSG299 and MC4100 was performed in the presence of CM. Data are presented as mean and S.D. (<i>n</i> = 3).</p

Effect of DNase I activity on lateral plasmid transfer and detection of plasmid in culture medium.

<p>Effect of DNase I activity on lateral plasmid transfer in cell-mixed culture (A, B), and detection of pHSG299 in culture medium (C). (A) Frequency of plasmid transfer [mean and standard deviation (S.D.); *: <i>t</i>-test: <i>P</i><0.05, <i>n</i> = 5] in the absence (lane 1) and presence (lane 2) of DNase I (30 µg/mL) in a co-culture of MC4100 harbouring pHSG299 and CAG18439 in TSB. (B) Confirmation of workability of added DNase I in TSB culture. Plasmid pHSG299 DNA (10 µg/ml) and/or DNase I (30 µg/mL) was added to the co-culture of MC4100 and CAG18439 at culture start. After 16-hours culture, plasmid DNA in culture medium was isolated, digested with EcoRI and RNase A, and applied to 0.8% (w/v) agarose/Tris-borate-EDTA (TBE) gel. Lane M: size marker (lHind III); lane 1: control (no addition); lane 2: addition of purified pHSG299; lane 3: addition of purified pHSG299 and DNase I. The arrowhead shows the band of linear pHSG299 (2673 bp). (C) Detection of pHSG299 in liquid culture medium by PCR. Mixed culture medium of MC4100 harbouring pHSG299 and CAG18439 in TSB was prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016355#s4" target="_blank">Materials and methods</a> and this medium sample was directly subjected to 0.8% (w/v) agarose/TBE gel electrophoresis (lane 1) or used as PCR template (lanes 4 and 5). Lane M: size marker (pUC119 Hpa II); lane 1: medium sample (1 µL) of MC4100 harbouring pHSG299 and CAG18439 without PCR; lane 2: positive control (PCR product from purified pHSG299 DNA); lane 3: negative control (PCR product from medium sample of plasmid-free MC4100 and CAG18439); lanes 4 and 5: PCR products from medium sample of MC4100 harbouring pHSG299 and CAG18439. The pHSG299-specific primers amplify a 229 bp fragment (arrowhead).</p