BUSCADOR RECOLECTA

Peer reviewed

Peer reviewed

Disc diffusion assay for testing fosfomycin resistance of WT AB5075, ΔcavA deleted mutant, vfr::Tn transposon mutant and their complemented derivative strains (ΔcavA+vfr and vfr::Tn+vfr respectively), as well as ΔcavA mutant overexpressing vfr (ΔcavA+vfr). CAMH agar inoculated with each corresponding strain and having a fosfomycin (50 μg) disc were assessed after 24 h at 37°C. Representative images (A) and the measured zone of inhibition in millimetres (B) are shown. Data represents the averages ± SD of biological triplicates. ns p>0.05, *p<0.05, ***p<0.001, ****p<0.0001 –One-Way ANOVA with Dunnet post-hoc test (B). All controls used are presented in S10B Fig, Peer reviewed

Cyclic di-GMP levels measured using CensYBL-Ab biosensor in ΔcavA mutant with low cAMP and ΔcavA+cavA complemented strain with high cAMP levels (A) as well as vfr::Tn mutant (B) compared to WT AB5075. Deletion of cavA reduced c-di-GMP levels by 66% (A) while disruption of vfr decreased them by 49% (B). Cultures were diluted in LB broth supplemented with apramycin (100 μg/ml) and biosensor expression was induced with anhydrotetracycline (50 ng/ml). Harvested cells were resuspended in sterile PBS and YFP and mCherry fluorescence signals were measured. The average normalised fluorescence (YFP/mCherry) ± SD from three independent biological repeats is presented. Data was analysed using One-Way ANOVA with Tukey post-hoc test (A) and Unpaired t-test (B) (* p<0.05, ** p<0.01). Strains harbouring empty miniTn7 were used as controls (S9B Fig)., Peer reviewed

Biofilm formation (A), EPS production (B) and twitching motility (C) of vfr::Tn transposon mutant AB07171, its complemented strain vfr::Tn+vfrWT, strains overexpressing vfr in the WT (WT+vfr) and ΔcavA backgrounds (ΔcavA+vfr) compared to AB5075 WT. In addition, vfr::Tn mutant was complemented with vfr variant harbouring two modified residues (T138A and T139W) in the cAMP binding site (vfr::Tn+vfrT138A,T139W). A—Biofilm formation microtiter assay was used as described above and biofilms were grown in LB broth for 24 h at 37°C shaking. Growth of the strains was assessed and strains harbouring empty miniTn7 were used as control. B—Images of colonies on Congo red agar for EPS production assessment were taken 5 days post-inoculation. C—Motility was assessed using soft agar at 48 h as described in the materials and methods section. Data including all controls is presented in S6 Fig. All experiments were repeated three independent times. Bar graphs present averages ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001—One-Way ANOVA with Dunnet (A) and Tukey (B & D) post-hoc tests, Peer reviewed

El trabajo realizado ha dado lugar al desarrollo y patente de un equipo basado en técnicas de confinamiento de campo que resuelve los problemas presentados por las generaciones anteriores de equipos basados en la misma familia de técnicas. Este equipo persigue poder realizar la monitorización continua de la corrosión en estructuras reales. El equipo es capaz de determinar la velocidad de corrosión de la armadura, además de la resistividad del hormigón y el potencial de corrosión de la armadura, tres parámetros clave a la hora de poder estimar el periodo de propagación dentro de la vida útil de las estructuras. La medida de la velocidad de corrosión y de la resistividad se realiza a través de dos nuevas técnicas orientadas a superar las limitaciones observadas en los corrosímetros actuales. La principal ventaja del sistema desarrollado es que evalúa directamente la propia armadura embebida sin necesidad de instalar o generar una sección de armadura como electrodo de trabajo. Esto permite una instalación no invasiva del sensor, ya que se coloca cerca de la armadura a evaluar sin perturbar sustancialmente las condiciones físico-químicas del entorno. De esta forma se garantiza una evaluación representativa y en condiciones reales de trabajo de los elementos de hormigón armado. Además, el sensor cuenta con diseño versátil que permite su instalación tanto en obra nueva (embebido) como en obra ejecutada (en superficie). Este sistema pretende ser una solución realmente pionera en el caso de la monitorización de estructuras existentes, y más en especial para la evaluación de armaduras activas en elementos pretensados o postesados donde no es posible alterar en absoluto el sistema acero-hormigón evaluado., Peer reviewed

Gene set enrichment analysis results of the significantly regulated genes in ΔcavA+cavA complemented vs ΔcavA EV strain in the dRNA-seq experiment., Peer reviewed

A—Cyclic di-GMP levels in WT AB5075, WT overexpressing constitutively active DGC gene pleD* (WT+pleD*) and WT with empty miniTn7 used for the strain construction. This demonstrates the activity of the modified CensYBL-Ab in detecting the elevated c-di-GMP levels in the WT+pleD* strain. The inactive CensYBL*-Ab biosensor was used to demonstrate that the increase in the signal was due to the changing c-di-GMP levels. B–Cyclic di-GMP levels in cavA related strains compared to the parental WT AB5075. The empty miniTn7 was used as control which demonstrates the empty vector did not have an effect on the c-di-GMP levels in the WT or the deleted ΔcavA mutant. ns p>0.05, ** p<0.01, **** p<0.0001—Two-Way ANOVA (A) and One-Way ANOVA (B) with Tukey post-hoc test., Peer reviewed

Expression of pilA gene (A) and pga operon (B) is regulated by CavA and Vfr. Promoter regions of pilA and pga fused with gfpmut3 fluorescent reporter (PpilA::gfpmut3 and Ppga::gfpmut3 respectively) were used to assess the effect of CavA and Vfr on their expression. Bacterial cells were harvested from diluted bacterial cultures grown for 4.5 h, after which were resuspended in sterile PBS. Fluorescence (A.U.) at 470-15/515-20 nm excitation/emission was measured to determine the expression of Gfp and thus the expression of each promoter. Optical density at 600 nm (OD600) was measured to determine growth. Data represents the average of three independent repeats ± SD. **p<0.01, ****p<0.0001 –One-Way ANOVA with Tukey post-hoc test., Peer reviewed

Growth (A), biofilm formation (B), EPS production (C) and motility (D) of cavA and vfr related strains demonstrating that the observed phenotypes were not attributed to growth alternations of the strains. Moreover, the empty miniTn7 system (EV) with Tetracycline (Tc) or Tellurate (Tel) resistance markers, used for the complementations and genes expression in different backgrounds, did not have an effect on any of the tested phenotypes. Growth and biofilm formation were assessed after 24 h at 37°C shaking. EPS production was assessed on Congo agar after 5 days incubation of the strains at 37°C and motility was tested after 48 h on soft agar at 37°C. ns p>0.05, *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001—One-Way ANOVA with Tukey post-hoc test., Peer reviewed