Fig 4. GTAs are released into the supernatant and can transfer genetic material.

(A) Micrographs of cells bearing P_xyl-gafYZ on a high-copy vector grown with 0.2% glucose or 0.3% xylose for 3 h. Samples were stained with SYBR-gold and imaged for fluorescence. Scale bar = 2 μm. (B) Transmission electron micrographs of filtered supernatant from wild-type or ΔrogA cells in stationary phase. Scale bar = 100 nm. (C) Representative images of gene transfer with induction of gafYZ at different optical densities. (D) Quantification of the gene transfer rate of the tet^R resistance marker from a donor strain bearing P_xyl-gafYZ on a high-copy plasmid (n = 3, error bars indicate SD). Transfers were carried out with induction of gafYZ at different culture densities as marked. Transfer rates are shown relative to transfer measured at cell density = 0.8. Data are available in S1 Data. (E) GTA-mediated gene transfer rates with or without addition of DNase. A total of 5 U/mL DNase I was added to donor culture for 1 h following induction, followed by 3 h co-culture with recipient strain (n = 3, error bars indicate SD). ns = not statistically significant via t test. Data are available in S1 Data. (F) GTA-mediated gene transfer rates with donor and recipient cells at different ratios (n = 3, error bars indicate SD). Recipient cells were also assayed when in exponential phase. * = p-value <0.05 comparing data to “9:1 stationary” sample. Data are available in S1 Data. (G) Transfer rate of the tet^R resistance marker from donors bearing P_xyl-gafYZ on a high-copy plasmid and a tet^R resistance marker either at a region of high packaging (1.0 Mb) or low packaging (2.0 Mb) (n = 3, error bars indicate SD). Each donor and recipient strain were grown either alone or together in the presence of 0.3% xylose. * = p-value <0.05 comparing data of same time point between the “donor (tet^R @ 1 Mb) + recipient” and “donor (tet^R @ 2 Mb) + recipient” samples. Data are available in S1 Data. GTA, gene transfer agent.