Lb agar

Mixed bacterial suspensions in a final volume of 20 mL were incubated aerobically at 37 °C with vigorous shaking (200 rpm). At given time points, each planktonic culture was serially diluted in 1X PBS and plated on LB agar (Scharlab, S.L.) to count P. aeruginosa CFUs, and tryptic soy agar (TSA; Scharlab, S.L.) supplemented with 7.5% (w/v) NaCl to selectively count S. aureus CFUs^{73 (link)}.

The expression vector of pET17b was purchased from Novagen (Madison, Wisconsin, USA). Cloning E. coli strain TOP10 was from Invitrogen (Carlsbad, Canada). Purified BSA100X (10 mg/mL), restriction endonucleases AgeI HF and SacI HF, the expression host of SHuffle T7 Express Competent E. coli and one of the PNGase F enzymes were purchased from New England Biolabs (Ipswich, Massachusetts, USA). CarboClip PNGase F was ordered from Asparia Glycomics (San Sebastián, Gipuzkoa, Spain). T4 DNA ligase and PageRuler Prestained Protein Ladder were from Thermo Scientific (Waltham, Massachusetts, USA). Cells were cultured in LB Broth medium and LB Agar (Scharlau, Barcelona, Spain). Isopropyl β-d-1-thiogalactopyranoside (IPTG) was from BioChemica (Billingham, UK). EDTA-free Mini Complete Protease Inhibitor was purchased from Hoffman-La Roche (Basel, Switzerland). Template DNA (pGEX-3x-PNGase F) containing the coding sequence for PNGase F was kindly provided by Professor Miklos Guttman (University of Washington, Seattle, Washington, USA). DNA sequencing was performed by Macrogen Europe (Amsterdam, the Netherlands). The HiTrap Chelating Ni-affinity column was from GE Healthcare (Chicago, Illinois, USA). The human IgG (hIgG1) sample and all other chemicals were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

In this study, we used the Pseudomonas aeruginosa PAO1 (CECT 4122/ATCC 15,692) and PA14 [13 (link)] reference laboratory wild-type strains and different cystic fibrosis isolates, PAET1, PAET2, and PAET4, which were isolated from recurrent infections of three different chronic-infected patients [14 (link)]. The three different clinical isolates differed in the nature of infection they were isolated from: while PAET1 and PAET2 strains were isolated from patients that were not previously colonized, the PAET4 strain was isolated from a CF-patient that suffered from recurrent infections. In this sense, PAET1 and PAET2 could be treated as early isolates whereas PAET4 as a late P. aeruginosa clinical isolate. The isogenic nrdJ and nrdD mutants in the PAO1 strain were also used [15 (link)]. Overnight (O/N; ~16 h) cultures of P. aeruginosa were routinely grown in Luria-Bertani medium (LB; Scharlab) at 37ºC with vigorous shaking. LB-agar (Scharlab) was used to count the respective bacterial colony forming units (CFU) of the P. aeruginosa strains.