Bacto casamino acids

Luria–Bertani (LB) medium was prepared with 10 g/L Bacto™ Tryptone (Becton, Dickinson and Company, Detroit, MI, USA), 5 g/L Bacto™ Yeast Extract (Becton) and 10 g/L NaCl (Wako chemicals, Osaka, Japan). M9-YE medium was prepared with 6 g/L K₂HPO₄, 3 g/L KH₂PO₄, 0.5 g/L NaCl, 7.25 g/L yeast extract, 0.1 mM CaCl₂, and 1 mM MgSO₄. In the experiment for the examination of effects of nitrogen sources on ammonia production, Bacto™ Tryptone (Becton), Bacto™ Peptone (Becton), or Bacto™ Casamino acids (Becton) were used instead of Bacto™ Yeast Extract in M9-YE medium. Ampicillin (Meiji Seika Pharma, Tokyo, Japan, 100 μg/mL) and kanamycin (Nacalai Tesque, Kyoto, Japan, 25 μg/mL) were added as appropriate.

Anti-penta His mAb, Ni-NTA superflow resin and Ni-NTA His-Sorb plates were procured from Qiagen GmbH, Germany. Anti-mouse IgG-H&L-chain-HRP-conjugate was from JIR, USA. Acid washed glass beads (425-600 microns), HRP substrate TMB were from Sigma-Aldrich, USA. Montanide ISA720 was from Seppic Inc., France. BCA protein assay reagent was from Thermo Scientific, Rockford, USA. Vero cell lines and Pan-DENV prM-specific 2H2 mAb were obtained from ATCC, USA. DENV EDIII-specific mAb 24A12 was generated in-house. Alexa Fluor 488 for labeling mAbs was from Life Technologies. N1-Europium chelate was synthesized in-house at University of Turku, Finland. DENV-3 EDIII gene, codon-optimized for P. pastoris expression, was obtained from GeneArt AG, Germany. Casamino Acids (Bacto Casamino Acids: acid-hydrolyzed casein, low sodium chloride and iron concentrations) Cat # 223050 from Becton, Dickinson and company, USA. CA are a mixture of amino acids and some very small peptides (<5 amino acids) obtained from acid hydrolysis of casein.

E. coli ATCC 25922, a clinical isolate of B. bronchiseptica from pig (BB-P19; Veterinary Microbiological Diagnostic Center [VMDC], Utrecht University) and the laboratory strain P. aeruginosa PAO1 were used throughout this study. Both E. coli and P. aeruginosa were grown on tryptone soy agar (TSA) plates (Oxoid Ltd., Basingstoke, Hampshire, UK). Liquid cultures were grown in lysogeny broth (LB) containing 1% yeast extract (Becton, Dickinson and Company, Sparks, USA), 1% NaCl (Merck, Darmstadt, Germany), and 0.5% tryptone (Becton, Dickinson and Company). B. bronchiseptica was grown on Difco Bordet-Gengou (BG) agar plates (Becton, Dickinson and Company) containing 1% glycerol (Merck) supplemented with 15% (vol/vol) defibrinated sheep blood (Oxoid Ltd.). Liquid cultures were grown in Verwey medium (pH 7.4) (43 (link)) containing 0.1% (wt/vol) starch from potato (S2004; Sigma-Aldrich, St. Louis, MO, USA), 0.02% (wt/vol) KCl, 0.05% (wt/vol) KH₂PO₄, 0.01% (wt/vol) MgCl₂·6 H₂O (all from Merck), 0.002% (wt/vol) nicotinic acid (Sigma-Aldrich), 1.4% (wt/vol) Bacto Casamino Acids (Becton, Dickinson and Company), and 0.001% (wt/vol) l-glutathione reduced (Sigma-Aldrich).

Growth experiments were performed in defined M9 minimal media (M9MM) (Fluka) to confirm the observed phenotype. Single isolated colonies of SMG 9 and EPI300::pCC1FOS were grown overnight in M9MM (containing final concentrations of; D-glucose (0.4%), Bacto™ casamino acids (w/v 0.2%) (Becton, Dickinson and Co, Sparks, MD, USA), magnesium sulfate (MgSO₄) (2 mM), calcium chloride (CaCl₂) (0.1 mM) and 12.5 μg/ml Cm). Reagents were purchased from Sigma Aldrich (St. Louis, MO, USA) unless otherwise stated. Cells were harvested by centrifugation, washed in ¼ strength Ringers solution and resuspended in fresh M9MM. A 2% v/v inoculum was sub-cultured in fresh M9MM containing various concentrations of sodium chloride (0–8% w/v NaCl) and 1 mM of L-carnitine when required. Triplicate wells of a 96-well micro-titre plate were inoculated with 200 μl of the appropriate cell suspension. Plates were incubated at 37°C for 24–48 h in an automated spectrophotometer (Tecan Genios) which recorded the optical density at 595 nm (OD_{595 nm}) every hour. After 48 h the data was retrieved and analyzed using the Magellan 3 software program and graphs were created with Sigma Plot 10.0 (Systat Software Inc, London, UK). Results are presented as the average of triplicate experiments, with error bars being representative of the standard error of the mean (SEM).

C. aggregans MD-66^T (=DSM 9485^T) was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). C. aggregans strain NBF was previously isolated from Nakabusa hot spring (21 ). These strains were maintained photoheterotrophically in modified PE medium (pH 7.5) containing (L⁻¹) 0.1 g sodium glutamate, 0.1 g disodium succinate, 0.1 g sodium acetate, 0.1 g yeast extract (Nihon Seiyaku, Tokyo), 0.1 g Bacto casamino acids (Difco Laboratories, Detroit, MI, USA), 0.1 g Na₂S₂O₃·5H₂O, 0.1 g (NH₄)₂SO₄, 75 mg KH₂PO₄, 78 mg K₂HPO₄, 0.2 mL of a vitamin mixture (11 (link)), and 1 mL of a basal salt solution (11 (link)). Cultures were kept in screw-capped tubes that were completely filled with the same medium and incubated at 55°C under incandescent illumination (approximately 230 W m⁻²).

We used the strain RP437 considered wild type for motility and chemotaxis. The strains were transformed by heat shock with PZE1R-GFP and PZE1R-mCherry plasmids. Cells were cultured in 3 mL LB medium (Sigma) with ampicillin at 33°C, with shaking, up to mid-exponential phase (Optical density OD₆₀₀ = 0.5), and re-suspended after centrifugation in the medium used for the experiments: M9 Minimal Salts, 5× supplemented with 1 gL1 Bacto Casamino Acids (both from Difco Laboratories, Sparks), 4 gL1 D-Glucose, and 1 mM MgSO4. The two types of bacteria were cultured independently before being mixed at the desired ratio at a final concentration corresponding to OD₆₀₀ = 0.5.