pbr322 with psti  (New England Biolabs)

Journal: Journal of Extracellular Vesicles

Article Title: Host Factor Induced Bacterial Extracellular Vesicles Promote Horizontal Gene Transfer in Vibrio cholerae

doi: 10.1002/jev2.70301

Figure Lengend Snippet: Stress‐induced BEVs are internalised by recipient cells and mediate horizontal gene transfer (HGT). (a) HGT rate of the tetR ‐cassette using BEVs derived from VC1620/1:: tetR (BEVs VC1620/1:: tetR ) as donor. BEVs VC1620/1:: tetR were isolated from AKI cultures in presence of bile (17.25 mM) or MMC (60 ng mL −1 ) or without any stressor (control, co). In addition, trypsin digested BEVs VC1620/1:: tetR derived from AKI cultures in presence of bile (17.25 mM) were used (see methods for detail). The purified linearised plasmid pBR322 (lin PstI) served as an extracellular DNA control. V. cholerae WT grown in AKI or SOC: HEPES served as recipient and was incubated for 20 h at 30°C and 150 rpm with respective BEVs VC1620/1:: tetR (2.5 × 10 11 particles) or pBR322 linearised with PstI (100 ng) before total CFU and transformants were determined by plating on LB‐Sm and LB‐Tet plates. (b) HGT rate of the tetR ‐cassette using BEVs VC1620/1:: tetR derived from AKI cultures in presence of bile (17.25 mM) as donor and V. cholerae WT or diverse V. cholerae mutants as recipient. Recipients, grown in AKI or SOC: HEPES, were incubated for 20 h at 30°C and 150 rpm with respective BEVs VC1620/1:: tetR (2.5 × 10 11 particles) before total CFU and transformants were determined by plating on LB‐Sm and LB‐Tet plates. (c) Internalisation of BEVs VC1620/1:: tetR by V. cholerae WT. Rhodamine‐labelled BEVs VC1620/1:: tetR derived from AKI cultures in presence of bile (17.25 mM) or MMC (60 ng mL −1 ) or without any stressor (control, co) were incubated with V. cholerae WT for 20 h. In addition, trypsin digested BEVs VC1620/1:: tetR derived from AKI cultures in presence of bile (17.25 mM) were used (see methods for detail). (d) Internalisation of BEVs VC1620/1:: tetR derived from AKI cultures in presence of bile (17.25 mM) by V. cholerae WT or the deletion mutants ∆ pilA and ∆ comEA . (c, d) Uptake is detected by an increase in relative fluorescence units (RFU) measured every 30 min. Wells containing rhodamine‐labelled BEVs VC1620/1:: tetR without recipient cells served as a blank. Shown is the median ± IQR ( n = 13). The bar chart on the right shows the median area under the curve (AUC) ± IQR calculated from the internalisation assays. Asterisks highlight significant differences between respective data sets (* p < 0.05 Kruskal–Wallis test followed by post hoc Dunn's multiple comparisons). (e) Colonisation fitness of VC1620/1:: tetR and ∆ comEA . Results are shown as competitive index (CI) for competitions of VC1620/1:: tetR or ∆ comEA to a fully virulent LacZ − derivative of the WT ( lacZ − ) in LB (in vitro) and in adult mice (in vivo). Each circle represents the CI from a single assay. Horizontal bars and error bars indicate the median ± IQR. Asterisks highlight data sets with significantly different CI from the theoretical value of 1 [ p < 0.05, Wilcoxon test against a hypothetical value of 1, n = 14 for VC1620/21:: tetR /WT ( lacZ − ) in vitro; n = 12 for ∆ comEA /WT ( lacZ − ) in vitro; n = 8 for VC1620/21:: tetR /WT ( lacZ − ) in vivo; and n = 6 for ∆ comEA /WT ( lacZ − ) in vivo]. (f) In vivo HGT rate of the tetR ‐cassette using BEVs VC1620/1:: tetR derived from AKI cultures in presence of bile (17.25 mM) as donor and V. cholerae WT or ∆ comEA as recipient. Mice colonised with WT or ∆ comEA received BEVs VC1620/1:: tetR VC1620/1:: tetR by oral gavage twice a day for two days before mice were euthanised and total CFU and transformants were determined by plating the homogenised colon on LB‐Sm and LB‐Tet plates. (a, b, f) Data are presented as median ± IQR. Assays yielding in no transformants on LB‐Tet were set to limit of detection (LOD), which is indicated by a dotted line. The LOD was defined as 0.5 CFU detected in the highest concentration plated on LB‐Tet plates divided by the total CFU determined by plating on LB‐Sm plates. HGT rates significantly higher than the LOD were by evaluated by the Wilcoxon Signed Rank test against the hypothetical value of the LOD (* p < 0.05, n = 8 for Panels a and b, n = 10 for Panel f). Statistically significant differences between the bile‐induced BEVs VC1620/1:: tetR before and after trypsin digest ( n = 8) or between the WT and ∆ comEA colonised mice ( n = 10) were analysed via the Mann–Whitney U test (* p < 0.05). BEV, bacterial extracellular vesicle; IQR, interquartile range; MMC, mitomycin C.

Article Snippet: Linearised pBR322 serving as control for HGT assays was generated by digestion of purified pBR322 with PstI (20 U μL −1 , NEB) according to the manufacturer protocol and subsequently heat inactivated at 80°C for 25 min. A proportion of the digestion reaction was analysed by agarose gel electrophoresis alongside undigested purified plasmid as control to conform complete linearisation.

Techniques: Derivative Assay, Isolation, Control, Purification, Plasmid Preparation, Incubation, Fluorescence, In Vitro, In Vivo, Concentration Assay, MANN-WHITNEY