Luria bertani agar

Restriction enzymes, DNA polymerases, the Gibson Assembly Cloning Kit, dNTPs and SOC (Super Optimal broth with Catabolite repression) were purchased from New England Biolabs and were used following the manufacturer’s instructions. Luria-Bertani (LB) agar, ampicillin, spectinomycin and other chemicals were obtained from Sigma-Aldrich. ampicillin and spectinomycin were used at 150 and 100 μg/ml, respectively. LB broth and agarose were purchased from Thermo Fisher Scientific and Melford, respectively. GelRed DNA dye was from Biotium Inc.

Surface sterilized roots were macerated with 0.8% saline solution, the liquid homogenate was diluted in 0.8% saline solution and 10⁻⁴ and 10⁻⁵ dilutions were used as inoculum for bacterial isolation. Four main culture media were used for the purification of bacterial isolates, namely Luria-Bertani (LB) agar (Sigma Aldrich, Germany), BD Difco R2A (R2A) agar (BD Diagnostics, Sparks, MD, USA) with and without 1.5 or 3% of added sodium chloride (NaCl) and Tryptone Soya Agar (TSA) agar (g/L: tryptone-15; soytone-5; NaCl-5; agar-15). 100 μl of diluted root extract was spread on different agar plate’s media. Inoculated plates were incubated at 28°C for 3–4 days and isolated colonies were purified by re-streaking until pure culture was achieved. Purified bacterial isolates were stored in 20% glycerol at -80°C. These isolates were then used for 16S classification, screening of biochemical traits and growth promotion of Arabidopsis.

Luria-Bertani (LB) agar, LB broth, chloramphenicol, ampicillin and CAZ were purchased from Sigma-Aldrich. Primers (P) used for this study are presented in Supplementary Table 7. All cloning enzymes were purchased from Thermo Fisher Scientific, if not stated otherwise. The E. coli E. cloni 10G (MP21-5) was obtained from Lucigen. All strains used and constructed in this study are presented in Supplementary Table 8. Kinetic data were fitted using Prism v. 9.0 (GraphPad Software).

P. aeruginosa FRD1 (CF isolate, mucA, Ohman and Chakrabarty, 1981) was used in this study. Bacteria from frozen stocks were plated on Luria-Bertani (LB) agar (Sigma, St. Louis, MO) and then inoculated into LB liquid medium, which was incubated at 37°C with agitation (200 rpm). FRD1 biofilms were fostered in Jensen's chemically defined medium at 37°C [15 (link)].

C57Bl/6N (Charles River) mice were kept under a constant light/dark cycle on a standard diet (Ssniff) and had access to food and water ad libitum. Male mice at the age of 10 weeks received either 3 mg/kg-bodyweight of LDN-193189 dissolved in 200 µl of 2% (2-Hydroxypropyl)-β-cyclodextrin intraperitoneally (i.p.) or only the solvent (vehicle) 2% (2-Hydroxypropyl)-β-cyclodextrin alone i.p. 1h prior to the infection and a second dose 11h after the infection. For the inhibition with oversulfated heparins, mice received 40 mg/kg-bodyweight of oversulfated heparins dissolved in 100 µl PBS subcutaneously or an equal volume of PBS alone 1h prior to the infection and a second dose 11h after the infection (Theurl et al., 2011b (link); Poli et al., 2014a (link)). Infection of mice was performed i.p. with either 1.1 x 10⁶ colony forming unites (CFU) of E. coli (O18:K1) or S.Tm (ATCC 14028), both suspended in 20 µl PBS, or PBS alone. Mice were terminated after 18h of infection and organ homogenates were plated in serial dilutions on Luria-Bertani (LB) agar (Sigma-Aldrich) plates to determine the bacterial load.
All animal experiments were performed in accordance with the Austrian Experimental Animal Welfare Act 2012 (Tierversuchsgesetz 2012) and were approved by the Federal Ministry of Science and Education (approval no. BMWFW-66.011/0115-V/3b/2019).

The PaD2HGDH pET20b(+) plasmid harboring the PA0317 gene was designed in-lab and purchased from GenScript. The plasmid was sequenced to verify the presence of the wildtype gene. E. coli strain Rosetta(DE3)pLysS was from Novagen. Bovine serum albumin was purchased from Promega. Luria–Bertani (LB) agar, LB broth, chloramphenicol, IPTG, lysozyme, sodium hydrosulfite (dithionite), PMS, and PMSF were obtained from Sigma–Aldrich. Ampicillin was purchased from ICN Biomedicals. d-2-hydroxyglutarate was purchased from MilliporeSigma. d-malate was purchased from Alfa Aesar. EDTA, glycerol, and all other reagents were of the highest purity commercially available.

Two strains of the Gram-negative bacillus K. pneumoniae of different virulence were used. K. pneumoniae KPC-Kp ST258 (L-78) is a clinical isolate derived from patients with bacteremia in Greek hospitals during 2009-2011 and its identity has been previously confirmed by standard techniques [13 (link)]. K. pneumoniae ATCC 43816 is a K2 strain used in animal models of infection. Both bacterial strains were stored at −80°C in medium containing 20% glycerol and were grown in freshly prepared Luria-Bertani (LB) agar (Sigma-Aldrich, St Louis, MO). Bacteria were incubated at 37°C in ambient air in order to reach the log phase of growth. Bacterial growth was spectrophotometrically monitored and adjusted to an initial optical density (OD_580nm) of 0.3. Prior animal infection, bacterial preparations were adjusted to 10⁹ CFU/mL for the L-78 strain and 5×10⁴ or 10⁷ CFU/mL for the ATCC 43816 strain.