Fig 2: Absorbance spectra of acetone/hexane (2:3, v/v) extracts of different parts of tomatoes (blue, peel; orange, fruit; gray, pulp; yellow, seeds) in (a) Arsicolo, (b) Crovarese, (c) Datterino, and (d) San Marzano cultivars. The absorbance of the supernatants was recorded at 453, 505, 645, and 663 nm by SpectroStar Nano (BMG Labtech).

Fig 3: 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).

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: Comparison of the growth curves of some of the cold-tolerant genes characterized in this study and their homologous genes in E. coli. DH10B strain carrying an empty pBluescript vector was used as a positive control and the single cold-sensitive mutant (DH10B ?csdA) also carrying an empty pBluescript vector was used as a negative control. All clones were grown during 5 h at 37°C with a 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.