Kanamycin sulfate

Bacterial strains and plasmids used in these studies are described in Table 1. Escherichia coli, used for vector propagation, was grown in Luria-Bertani (LB) broth (Difco) containing 50 μg/ml kanamycin sulfate (Amresco, Solon, OH). S. pyogenes strains were grown in Bacto Todd Hewitt Broth (Becton, Dickinson and Company, Sparks, MD) supplemented with 2% Bacto yeast extract (THY medium) at 37°C or Brain Heart Infusion media (BHI) (Himedia Laboratories, India) at 37°C with the addition of the appropriate antibiotics (Sigma); growth was determined by monitoring the absorbance at 600 nm. Strain SF370SmR is a streptomycin resistant derivative of strain SF370, containing a spontaneous mutation in the rpsL gene [19 ]. No differences between SF370 and the streptomycin resistant derivative were observed with respect to growth rate or in the various biological assays employed [19 ]. Chemically defined media (CDM) was prepared as described in the literature [20 (link)].

M13KO7 was obtained from New England Biolabs, 2YT culture media was obtained from Novagen, agar–agar, citric acid, copper(ii) sulfate (CuSO₄·5H₂O), manganese(ii) sulfate monohydrate (MnSO₄·H₂O), nickel(ii) sulfate hexahydrate (NiSO₄·6H₂O), polyethylene glycol 6000 (PEG 6000) and potassium dihydrogen phosphate (KH₂PO₄), were obtained from R&M Chemicals. TG1 Escherichia coli cell was purchased from Agilent. Kanamycin sulfate was obtained from Amresco. Aluminium chloride hexahydrate (AlCl₃·6H₂O), guanidine hydrochloride (GuHCl) and zinc sulfate heptahydrate (ZnSO₄·7H₂O) were bought from Sigma-Aldrich, sodium chloride (NaCl) was bought from Friedemann Schmidt, iron(ii) sulfate heptahydrate (FeSO₄·7H₂O), potassium chloride (KCl) and sodium dihydrogen phosphate (NaH₂PO₄) were bought from Merck. Disodium hydrogen phosphate (Na₂HPO₄), iron(iii) chloride hexahydrate (FeCl₃·6H₂O) was purchased from Bendosen, calcium chloride (CaCl₂) and magnesium chloride (MgCl₂) were purchased from Fisher Scientific, chromium(iii) potassium sulfate dodecahydrate (KCr(SO₄)₂·12H₂O) was purchased from QRec. All the salts were dissolved in ultrapure water purified through a Milli-Q system (18.2 MΩ cm, Millipore).

The eukaryotic freshwater microalgae species, Dictyosphaerium pulchellum and Micractinium pusillum, used in this study were obtained from the Korea Marine Microalgae Culture Center (KMMCC), Busan, South Korea. Stock cultures were stored on the modified AF6 agar slants [40 ]. The cultures were streak plated and purified by subculturing by at least 5-6 times before use. Both microalgae species were cultivated in 250 mL flasks with 150 mL, modified AF6 medium while incubating at 25 ± 2°C, 50 ± 2 μmol photons m⁻² s⁻¹ and 50% humidity. Antibiotics, kanamycin sulfate (Amresco), and tetracycline hydrochloride (Bio101) with different concentrations ranging from 0 to 30 mg L⁻¹ were used. Growth rates were calculated by measuring the absorbance at 750 nm (OD₇₅₀) on every alternating day [41 (link)]. Additionally, all the experiments were repeated three times.