Bioscreen c

Bacterial growth experiments were performed in two biological replicates with two technical replicates for each strain on Bioscreen C (Thermo Labsystems, Helsinki, Finland) for 24 h at 37 °C. Briefly, 250 μL of MHB-II was inoculated with cells from LB agar plates to a final cell density of approximately 10⁶ CFU/mL, using a Sensititre™ Nephelometer (Thermo Fisher Scientific, Roskilde, Denmark) with a 0.5 McFarland standard (1–2 × 10⁸ CFU/mL). The cultures were then grown without antibiotic or supplemented with either 128 mg/L CTX, 0.5 mg/L GEN, or a combination of CTX and GEN of 1 + 0.25 mg/L. The OD₆₀₀ was measured every 20 min with continuous shaking, and the growth curves were obtained using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA).

The reduction of Cu²⁺ was measured indirectly in L. cremoris. When Cu²⁺ is reduced to the toxic species Cu⁺, growth of L. lactis was shown to be inhibited (Abicht et al., 2013 (link)). Therefore, inhibition of growth of L. cremoris was used as an indication of Cu²⁺ reduction.
Strains were cultivated statically in GM17 overnight, and then the cultures were diluted to an OD₆₀₀ value of 0.1 in fresh GM17. For Cu²⁺ reduction tests, 300 nM CuCl₂ was added to the media, and for growth controls CuCl₂ was not added. For anaerobic cultivation, aliquots of 450 μL were brought into 100-well Honeycomb microplates (Thermo Fisher Scientific) and incubated at 30°C for 48 h in Bioscreen C (Thermo Fisher Scientific), where the OD₆₀₀ was measured at 1-h intervals. For aerobic cultivation, aliquots of 350 μL were transferred to microplates which were subsequently incubated with continuous shaking at medium intensity. For respiration-permissive conditions, cultures were incubated the same way as for aerobic conditions, albeit with the addition of 2 μg/mL heme (hemin, Sigma).
The time to reach (TTR) OD₆₀₀ value 0.4 was used as a measurement of growth; the factor difference between TTRs in the test of 300 nM CuCl₂ and in the growth control without CuCl₂ was eventually used as the indicator of Cu²⁺ reduction-induced growth inhibition/toxicity.

The basal growth medium had the following composition: 20 mM sodium fumarate, 20 mM NaNO₃, 4.7 mM NH₄Cl, 1.3 mM KCl, 2 mM MgSO₄, 0.2 mM NaCl, 1.2 mM NaHCO₃, 5 mM NaH₂PO₄, 0.1 mM CaCl₂ with sterile vitamins and trace elements prepared as described by Widdel and Bak (1992 ). Initial cultures were grown aerobically. These were then diluted 20-fold into the experimental growth medium. For each experiment, where applicable, the indicated amounts of exogenous sulfur sources, uranyl acetate (U[VI]) and K₂Cr₂O₇ (Cr[VI]) were added to the basal growth medium. Cultures were grown in a 100 well Bioscreen plate with each well containing 400 μL of diluted preculture. The Bioscreen plate was incubated anaerobically or aerobically as indicated at 30°C with continuous shaking in a Bioscreen C (Thermo Labsystems, Milford, MA). In the case of anaerobic growths, the Bioscreen C was placed within an anaerobic chamber (Plas Labs, Lansing, MI) in an atmospheric composition of 95% Ar and 5% H₂. Growth was monitored at an absorbance of 600 nm. All experiments were performed in biological duplicate or triplicate, errors bars represent standard deviations.

To validate the predictions revealed by the TraDIS analysis, the WT strain (MG1655/pTF2) and its mutant derivatives (ΔacrZ, Δcls, ΔmdfA, ΔarcA, Δhfq, and ΔnlpI) were compared in terms of their ability to grow without antibiotics and in the presence of CHL using Bioscreen C (Thermo Labsystems, Helsinki, Finland) as previously described with slight modifications [19 (link)]. Briefly, a 10⁻² dilution of 0.5 MacFarland (1–2 × 10⁸ CFU/mL) was prepared in MHB-II for each strain, resulting in a final cell density of approximately 10⁶ CFU/mL. Then, 250 μL of the bacterial suspension was inoculated in each well. Next, the cultures were grown without antibiotics and in the presence of 2 mg/L CHL. The OD₆₀₀ was measured every 30 min with continuous shaking for 24 h at 37 °C. This experiment was performed with two biological replicates using two technical replicates, and the growth curves were generated using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA).

We analyzed the growth of the wild type, ∆hemK, and C-∆hemK using the Bioscreen C automated microbiology growth curve analysis system (Thermo Lab Systems, Helsinki, Finland) by adapting the method described by Medina et al. [47 (link)]. Briefly, a single colony of each strain was grown overnight at 28 °C in LB broth with an antibiotic (25 µg/mL gentamicin). The overnight cultures were adjusted to the same concentration by measuring the OD₆₀₀ values and were then diluted 100-fold in fresh YEB medium. An aliquot of 200 µL of this diluted solution was added into the wells of a 100-well microplate, with the fresh YEB used as a negative control. The OD at 600 nm was measured every 4 h at 28 °C with shaking. Four biological replicates were performed for each strain.

Kinetics of AcLys influence on viable bacterial cells was determined turbidimetrically using a 96-well plate reader Bioscreen C (Thermo Scientific, LabsystemsOy). Different concentrations of AcLys (1, 10, 50, 100, 200, 400 µg/mL)were dosed at time zero into 200 µL of microbial suspensionsin LB media collected at mid-log growth phase (OD₆₀₀ = 0.4) andnormalized to approximately 5 × 10⁸ CFU/mL with 20 mM Bis-Tris (pH 6.0). Optical density was monitored for 30 min with periodic shaking at room temperature. To reveal the influence of the incubation conditions on bacteriolytic activity the collected cells were centrifuged at 1500× g, washed with deionized water, and resuspended to the corresponding optical density with 20 mM Bis-Tris (pH 6.0). To calculate the minimum inhibitory concentration (MIC) the incubation of the reaction mixture was continued further, and the number of live cells was determined by removing aliquots from the microbial suspension at 15, 30, 60, 120, 240 min of incubation with the enzyme, seriallydiluting themand platingdilutions on nutrient agar. All measurements were performed in triplicates. The MIC of AcLys was defined as a concentration of an enzyme that reduces the bacterial population at least 10-fold, and inhibits the growth of the bacteria within a 2 h interval.

The 5 isolated variants were inoculated overnight in 5 ml NB before diluting 20x in micro titre plates. The range of hydrogen peroxide concentrations tested was between 5 and 100 mM. Plates were incubated at 30°C and read every 15 min at 600 nm for 48 h in Bioscreen C (Thermo Fisher Scientific).