Kh2po4

The following growth media were used: 2xTY medium (20 g/L Tryptone (Casitose Type-I, HiMedia, Mumbai, India), 10 g/L Yeast Extract (HiMedia, Mumbai, India), 20 g/L NaCl (Fisher Scientific, Loughborough, UK), pH 7.2) containing 100 μg/mL Ampicillin Ampicillin (Fisher Bioreagents, Pittsburg, PA, USA) and supplemented with 1 mM MgSO₄ (Fisher Scientific, Loughborough, UK) and 1% Glucose (Merck KGaA, Gernsheim, Germany) (2xTY-Amp); LB medium (10 g/L Tryptone, 5 g/L Yeast Extract, 10 g/L NaCl, pH 7.2) containing 100 μg/mL Ampicillin and supplemented with 1 mM MgSO₄ and 1% Glucose (LB-Amp); ZYP-5052 medium for autoinduction (1% Tryptone, 0.5% Yeast Extract, 25 mM (NH₄)₂SO₄ (Chem-Lab NV, Zedelgem, Belgium₎, 50 mM KH₂PO₄ (Fisher Bioreagents, UK₎, 50 mM Na₂HPO₄ (Fisher Bioreagents, Loughborough, UK), 0.5% glycerol (Sigma, St. Louis, MO, USA), 0.05% Glucose, 0.2% α-lactose (Carl Roth GmbH, Karlsruhe, Germany), 1 mM MgSO₄), containing 100 μg/mL Ampicillin; TYE agar (10 g/l Tryptone, 5 g/l yeast, 8 g/l NaCl, 15 g/l agar), containing100 μg/mL Ampicillin (TYE-Amp).

All strains were grown overnight in lysogeny broth (LB) at 37°C with shaking at 250 rpm. Swarming media consisted of 0.5% agar (Bacto) supplemented with 5g/L casamino acid, 1 mM MgSO₄, 0.1 mM CaCl₂ and 1X buffer (12 g/L Na₂HPO₄ (Fisher Scientific), 15 g/L KH₂PO₄ (Fisher Scientific) and 2.5 g/L NaCl, pH6.7) [55 (link)]. Biofilm assays were carried out in 96-well plates in 1% trypton at 25°C for 24 hours and quantified by crystal violet staining [56 (link)]. c-di-GMP measurements were obtained from colony biofilms incubated on trypton plates with 1% agar.

Potato dextrose broth (BD Biosciences, Le Pont de Claix, France) was used as a subculture medium for yeasts while the milk ingredient-based medium was prepared and used for bacteria. A milk ingredient-based liquid medium containing 5 g casamino acids (BD Biosciences, France), 1 g yeast extract (BD Biosciences, France), 5 g NaCl (Fisher Scientific, Illkirch-Graffenstaden, France), 20 g D-glucose (Fisher Scientific, France), and 1 g KH₂PO₄ (Fisher Scientific, France), per liter of deionized water, was prepared. Before sterilizing at 121 °C for 15 min, the pH of the medium was adjusted to 7.0 ± 0.2.
To prepare a microbial suspension, each of D. hansenii 304 and K. marxianus 44 was inoculated into 250 mL Erlenmeyer flask containing 50 mL of PDB, whilst the milk ingredient-based liquid medium was used instead of PDB for A. arilaitensis Po102. These flasks were incubated at 25 °C with agitation at 150 rpm for 2 days. After the cultivation period, the cells were harvested by centrifugation at 6000× g for 10 min, and resuspended in peptone saline diluent (1 g casein peptone (Sigma-Aldrich, Darmstadt, Germany) and 8.5 g NaCl, pH adjusted to 7.0 ± 0.2 at 25 °C)). A suspension containing 10⁷ cells mL⁻¹ was prepared to be used in the next step.

M9 minimal medium was made as per the Cold Spring Harbor Laboratory protocol: 5X salt solution (per liter: 33.92 g Na₂HPO₄, 15 g KH₂PO₄, 5 g NH₄Cl, all from Fisher Scientific, Mumbai, India) 2.5 g NaCl (Merck), 2 mM MgSO₄ (Fisher Scientific), 100 μM CaCl₂ (Merck), 4 g Glucose (Himedia). To induce strings unless mentioned 200 μM of 2,2-Bipyridyl (Bipd) (Sigma) was added to M9 minimal medium. Strings were harvested using sterile cut-tips after 10 hours at 37 °C with constant shaking at 180 rpm and processed appropriately. Planktonic growth was measured as optical density at 600 nm.

B. thetaiotaomicron was routinely grown on brain- heart infusion (BHI, OXOID) supplemented with hemin (1 μg/ml, Sigma-Aldrich). Agar (Fisher bioreagents), erythromycin (25 μg/ml, Duchefa Biochemies), gentamicin (200 μg/ml, Formedium) and 5-fluoro-2′-deoxyuridine (FUdR, 200 μg/ml, Sigma-Aldrich) were added when required. Minimal medium (MM) contained 7.5 mM NH₄SO₄ (Thermo Scientific), 9.5 mM Na₂CO₃ (Melford), 4 mM L-Cysteine (Melford), 100 mM KH2PO4 (pH 7.2)(Fisher Chemical), 1 μg/ml menadione (Sigma-Aldrich), 1 μg/ml hemin and a mixture of mineral salts (15 mM NaCl (Duchefa Biochemies), 0.18 mM CaCl₂•2H₂0 (Fisher Chemical), 0.1 mM MgCl2•6H₂O (Fisher Chemical), 0.5 mM MnCl₂•4H₂O (Acros Organics) and 0.04 mM CoCl2•6H₂O (Sigma-Aldrich)). Vitamin B₁₂ (Sigma-Aldrich) and L-methionine (Formedium) were added as required and described in the text. 0.5% of fructose (Thermo scientific) was added as carbon source. Cells were grown in a Whitley A35 anaerobic workstation with a mixture of gas of 80% N₂, 10% CO₂ and 10% H₂.

For enrichment of bacteriophages, a volume of 5 ml of sterile 10 x NB containing peptone (20 g, Sigma Aldrich), yeast extract (10 g, Oxoid), NaCl (5 g, Fisher Scientific) and K₂HPO₄ (16 g, Fisher Scientific) per 200 ml was added to the Stericup^™ Millipore Express TM® Plus filter receiver flask (Merck) containing the phage-cell filtrate, followed by equal volumes (2.5 ml) of P. carotovorum and P. atrosepticum (S1 Table) of liquid bacterial cultures at a cell concentration of ca. 10⁸ cfu ml^-1 (OD_600nm = 0.2). The resulting solution (bacteriophage and bacteria) was incubated at 25°C with 200 rpm agitation for 12–24 h. Following that, an aliquot of 10 ml of the solution was transferred into a centrifuge tube and centrifuged at (2000 rpm, 5°C) for 5 min. The supernatant which contained bacteriophages was filtered using a 10 ml syringe barrel fitted with a 0.22 μm filter Millex® GV filter unit (Merck). This bacteriophage filtrate was stored at 4°C until use. The 100 μl of filtrate was added to 900 μl of sterile phosphate-buffered saline (PBS) buffer containing NaCl (1.6g, Sigma Aldrich), KCl (0.04g, Fisher Scientific), K₂HPO₄ (0.22g, Sigma Aldrich) and KH₂PO₄ (0.04g, Fisher Scientific) per 100 ml, pH 7.4. Five–fold serial dilutions were made in PBS buffer pH 7.4 (Neat, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴) and subjected to plaque formation using the double layer agar method.

Standard nicotine 98 % (Riedel–de Haen, Germany), methanol HPLC grade (Fisher Scientific, UK), phosphoric acid and triethylamine (Riedel–de Haen, Germany) were used. A 25 mM phosphate buffer (pH 7.8) was prepared from potassium dihydrogen orthophosphate, KH₂PO₄ (Fisher Scientific, UK) and disodium hydrogen orthophosphate, Na₂HPO₄ (BDH Chemicals, UK) according to the procedure in British Pharmacopoeia (2012 ). All the experiments were done in The Chemical Laboratory of Ethiopian Pharmaceuticals Manufacturing Sh. Co. (EPHARM), Addis Ababa, Ethiopia.