Resultados de investigación

Conjugation parameters obtained by fitting Eq.3 to data shown in Fig 1., Peer reviewed

(A) Conjugation kinetics for different plasmids of the same PTU. The conjugation frequency, expressed as Tc/ D is indicated on the y axis. The conjugation time, in minutes, is indicated on the x axis. (B) Circles indicating conjugation frequencies (y axis) obtained at different cell densities (x axis) for different plasmids belonging to PTU-W. Black lines indicate the interpolated [T]/[D] values according to Eq.3. Grey areas indicate the predicted confidence interval at 95% for the adjusted Kon and τ values. The estimated engagement times (τ) and encounter rates (kon), with their respective 95% confidence intervals are as follows: pMBUI4 Kon = 274 [131–580] μm2, τ = 4 [3.2–5.1]´; R388 Kon = 76 [28–263] μm2, τ = 18 [15–24]´; pIE321 Kon = 10 [4–32] μm2, τ = 36 [28–51]´; R7K Kon = 1 [0.3–4] μm2, τ = 262 [185–364]´., Peer reviewed

a) Effect of temperature on the conjugation rate. Conjugation assays were performed as described in Materials and Methods. (1 h, 37 ºC, 1D:100R). Each point corresponds to an independent experiment b) Effect of media composition on the transfer rate for R388 conjugation and mobilization (W-wire) and pKM101 conjugation (LBagar: autoclaved LBagar, LBagar pH7.4: PBS-buffered at pH 7.4 and TB agar: Terrific Broth agar. c) Overexpression of tra genes does not increase the transfer rate. On the x axis, genes overexpressed in R388-carrying donors using a pBAD33 vector. On the y axis, the fold change between the conjugation frequency (as [T]/[D]) obtained overproducing each of the constructions shown in the figure, and the conjugation frequency obtained with an empty pBAD33 vector. Statistical significancy was determined using Welch’s corrected ANOVA test for samples with unequal variances., Peer reviewed

Plasmid invasion of a susceptible population along time (x axis), expressed as transconjugants per donor (Y axis) A) Invasion dynamics of wild-type plasmids pKM101 (red dots) and R388 (black dots) obtained at two different donor: recipient ratios (1:100 and 1:10,000). Asterisks over t = 60, t = 90 and t = 120 minutes indicate statistically significant differences between plasmids pKM101 and R388 with p < 0.05 (*) B) Mobilization dynamics obtained with TRA genes from PKM101 (traN, red) and tra genes from R388 (traW, black) integrated in the chromosome of E. coli. The mobilizable plasmid contained the MOB region of the corresponding plasmid. Differences between plasmids were statistically significant with p < 0.01(***) for all time points analysed C) Differences in the dynamics of conjugation (black) and mobilization (red) in plasmid pKM101. Mobilization assays were perform using a vector containing mobN harboured by a strain that contained the traN genes inserted in the chromosome. Conjugation dynamics were obtained using the wild type pKM101 plasmid. Statistical differences were observed at t = 90 (* p < 0.05) and t = 120 (** p < 0.01) D) Comparison of plasmid R388 conjugation dynamics (black dots) vs R388-derived constructions. Black dots correspond to the wild-type plasmid transferred to a recipient strain that contains an empty pBAD expression vector. Green dots represent values obtained in conjugations where donors contained wild-type R388, and recipients harboured a pBAD::pifC construction, able to block secondary transmission. White dots correspond to the mobilization of a plasmid containing the mob region of plasmid R388 from a strain harbouring a chromosomal integration of the TraW genes. Black lines indicate comparisons between groups (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). When not explicitly indicated, the comparison between measurements was non statistically significant (n.s.)., Peer reviewed

A) Scheme showing the conjugative transmission of a plasmid (red circle) in a population of susceptible cells under Density-Dependent Transmission (DDT). In this regime, conjugation (black arrows) occurs when a plasmid containing cell contacts a plasmid-free one. The number of transconjugant cells formed is thus equivalent to the number of Donor:Recipient encounters. The conjugation frequency thus increases linearly with increasingly available recipients. B) Scheme showing the conjugative transmission of a plasmid (red circle) in the same population of susceptible cells as in A), but under Frequency-Dependent Transmission (FDT). In this regime, conjugation (black arrows) is limited by the time required to complete the transfer event, thus not all Donor:Recipient contacts result in successful Transconjugant formation. The conjugation frequency is thus constant, regardless of the number of available recipients. C) Ideal number of transconjugants obtained per donor (y axis, T/D) as a function of the total cell concentration (x axis, N). In a Holling´s Type II functional response there is a density-dependent region where transconjugant formation increases proportionally with N (leftwards from the blue line). When N becomes saturating the formation of transconjugants does not scale any further with cell density and becomes frequency-limited (rightwards from the blue line). D, E, F) Transconjugants per donor ([T]/[D], y axis) obtained at different cell densities ([N], x axis) in conjugations using E. coli BW27783, on solid LB-agar surfaces matings and 1 h. conjugation time. Each graph corresponds to plasmids R388 (D), pKM101 (E) and RP4 (F). The black line represents the ideal DDT dynamics, while the red line represents the FDT., Peer reviewed

A detailed set of derivations leading to analytical solutions for: (1) plasmid mobilization, (2) plasmid conjugation using the Holling’s Type II model, (3) the contribution of vegetative growth to the conjugation frequency, and (4) testing the approximations using chemical master equations., Peer reviewed

Antimicrobial resistance gene markers are coloured in red, mobilization genes are coloured in green, oriT and oriV sequences are indicated in blue., Peer reviewed

The study that generated these data aimed to evaluate the effects of incorporating a novel type of olive pulp into the diet of Murciano-Granadina dairy goats, focusing on ruminal fermentation, nutrient digestibility, energy and nitrogen metabolism, blood biochemical parameters, as well as milk production and quality. To this end, two in vivo experiments were conducted, assessing the impact of including olive pulp at a 12% level in the concentrate. The aforementioned parameters for each experiment are presented on separate sheets within an Excel file. Analytical replicates ranged from two to three, depending on the technique, while the number of observations was 18 (9 animals × 2 treatments) for the in vivo digestibility trial and 64 (32 animals × 2 treatments) for the on-farm milk yield trial, respectively., Project: GOPG-MA-20-0001 (BIORUMIOLI, www.biorumioli.es) funded by EU (EAFRD) and the Consejería de Agricultura, Pesca, Agua y Desarrollo Rural (An-dalusian Govern), Peer reviewed

A) Sequence conservation among PTU-W plasmids. Alignment of sequences indicating the synteny on Replication genes (indicated in blue), STB (brown), MOB (light blue) and MPF (green) operons in the 4 plasmids used in Fig 4. Antimicrobial resistance genes are indicated in red, transposases in orange. B) Conservation of the adhesin among PTU-W plasmids. TrwH-TrwG(maroon)-TrwJ(light red) complex from R388 pilus tip, as taken from PDB (https://www.rcsb.org/structure/8RT9). Mutated residues in pIE321, R7K or both are indicated in green, blue and pink, respectively., Peer reviewed

A) Doubling times in LB broth for recipient strains (E. coli Bw27783) and donor cells (Bw27783 containing the plasmids indicated in the legend) for the model plasmids employed in this work. B) Doubling times in LB broth for recipient strains (E. coli Bw27783) and donor cells (Bw27783 containing the plasmids indicated in the legend) for the plasmids from PTU-W employed in this work., Peer reviewed