Fig 2: L-histidine effect on cell growth is FLO11-dependent, and related to pH and cell-surface charge.The histogram and the line graph illustrate the cell growth and the cell surface net charge, respectively, of the 3238-32 and 3238?flo11 strains. Cells (104 cells/mL) were incubated in SC minimal medium. The media were buffered at pH 3, 4, 5 and 6 using 0.1 M citric acid monohydrate and 0.2 M sodium phosphate. Cells were grown in 96-well microplates, statically at 30°C for 48 h. Growth was monitored measuring the OD600 in a SPECTROstar nano-microplate spectrophotometer (BMG Labtech, Germany). Cell surface net charge (Z-potential) was measured under the same conditions using the Zetasizer Ver. 6.20 (Malvern Instruments Ltd) after 48 h of treatment. Data are means ±SD of three replicates. A one-way analysis of varience (ANOVA) was performed and followed by Tukey honestly significant difference test (P<0.05). The analyses were performed independently for growth and cell surface charge data and values with the same letter/symbol are not statistically different.

Fig 3: Transfer of TIG-R to AB and other bacterial strains. (a) Minimum amount of TIG-R EV inducing TIG-R against TIG-S (ATCC 17978) AB strain. Aliquots (5 µL) treated with TIG-R EVs at indicated concentrations were spotted onto an LB-agar plate containing TIG at 0.5 µg·mL-1 and grown for 16 h. Over 5 µg·mL-1 of TIG-R EVs, bacterial cells survived on the LB-agar plate. Growth curve analysis of TIG-S AB (ATCC 17978) strain with (b, Top) or without (c, Top) TIG-R EVs (5 µg) under different concentrations of TIG (0–4 µg·mL-1) was performed to evaluate the transfer activity. (b,c, Bottom) Cell viability. Aliquots (5 µL) of cells from (b, Top) and (c, Bottom) were spotted onto LB-agar plates and grown for 16 h. LB-agar plates were imaged by ChemiDocTM MP Imaging System (Bio-Rad, Hercules, CA, USA) and Image Lab Software (ver.5.2.1, Bio-Rad, Hercules, CA, USA). Error bars in growth curves indicate statistical significance at p < 0.05 (n = 3). (d) Dose-dependency of TIG resistance. Growth curve of TIG-S AB strain was conducted under TIG at 0.5 µg·mL-1 with increasing amounts of TIG-R EVs. Cells were grown at 37 °C for 24 h with 500 rpm in 96-well microplates and growth was monitored by measuring the OD600 using a SPECTROStar® Nano (BMG LABTECH GmbH, Ortenberg, Germany, n = 3). (e) Transfer spectrum of TIG-R EVs. TIG MICs of E. coli (ATCC 25922), K. pneumoniae (NCCP 16285), P. mirabilis (ATCC 29906), S. aureus (ATCC 25923), and S. typhimurium (14028S) against TIG (0–5 µg·mL-1) without or with TIG-R EVs of 5 µg was determined. One of the representative data from n = 3 was shown. The 96-well plates were imaged with a digital camera (Samsung NX200, Suwon, Korea). Red circles indicate MICs for individual samples.

Fig 4: Growth curves of the 20 cold-resistant clones isolated from Antarctic metagenomic libraries. The DH10B strain carrying an empty pBluescript vector was used as a positive control and both cold-sensitive strains (DH10B ?csdA and DH10B ?csdA ?rnr) also carrying empty pBluescript vectors were used as negative controls. All the clones were grown during 5 h at 37°C with slight agitation and then their OD600 nm values were adjusted to 0.03. They were grown in 96-well microtiter plates during 140 h at 15°C with a slight agitation (80 rpm) and OD measures were taken in the SPECTROstar Nano (BMG Labtech) four times per day. Six replicates per clone were introduced in each assay and each experiment was repeated at least three times using independent cultures to corroborate the results. (A) Results obtained for 6 cold-resistant clones (pC1–pC6) isolated from library C (Colobanthus quitensis) in the screening performed with the single cold-sensitive mutant (DH10B ?csdA). (B) Results obtained from the remaining 7 cold-resistant clones isolated from library C (pC7–pC13) in the screening performed with the single cold-sensitive mutant. (C) Results obtained for the 6 cold-resistant clones (pD1–pD6) isolated from library D (Deschampsia antarctica) in the screening performed with the single cold-sensitive mutant. (D) Results obtained for the cold-resistant clone pD7 isolated from library D in the screening performed with the cold-sensitive double mutant (DH10B ?csdA ?rnr).

Fig 5: Identification of determinants for the TIG transfer activity. Degradation of DNA components by DNase or restriction enzyme. TIG-R EV components. TIG-R EVs were treated with (a) DNase I and (b) Sau3AI. TIG-R EVs added with DNase I, Sau3AI were observed by 1% agarose gel electrophoresis with 1 kb Plus DNA Ladder (Thermo Fisher Scientific, Waltham, MA, USA). Growth curve analysis of TIG-S AB (ATCC 17978) strain with TIG-R EVs (5 µg) pre-treated with (c) DNase I (Red, circle), (d) NFW (Green, up-triangle), and (g) proteinase K (Blue, square) under TIG (0.5 µg·mL-1). Cell growth (OD600) was monitored by SPECTROStar® Nano (BMG LABTECH GmbH, Ortenberg, Germany, n = 3). (e,f) Characterization of proteins from TIG-R EVs after proteinase K treatment. EV proteins and proteinase K were identified using 12% Mini-PROTEAN® TGXTM Stain-Free gels (Bio-Rad, Hercules, CA, USA) with Precision Plus ProteinTM Standards (Bio-Rad, Hercules, CA, USA). Images (a,b,e,f) were obtained by ChemiDocTM MP Imaging System (Bio-Rad, Hercules, CA, USA) and Image Lab Software (ver.5.2.1, Bio-Rad, Hercules, CA, USA). (h) Cell viability. Aliquots of cells from (c,d,g) were spotted after growth for 16 h. All images (a,b,e,f,h) were obtained by ChemiDocTM MP Imaging System (Bio-Rad, Hercules, CA, USA) and Image Lab Software (ver.5.2.1, Bio-Rad, Hercules, CA, USA).