Carboxin

Potentiating antibacterial activity by enhancing microbial ROS production has been demonstrated previously (Brynildsen et al., 2013 (link)). To test the impact of ROS modulation (by sdhC or cyoA deletion) on bacterial viability in E. coli ATCC 25922, EC04 and EC59 strains, time-kill curves were assayed in MHB (Mueller-Hinton broth, Becton Dickinson, Le Pont-de-Claix, France) at ciprofloxacin and ofloxacin concentrations of 2xMIC (Table 1). Growth in drug-free broth was evaluated in parallel, as a control. Cultures were incubated at 37°C, and shaken at 180rpm. An initial inoculum of 10⁶ CFU/mL from a fresh overnight culture was used in all experiments. Bacterial concentrations were determined at 0, 1, 2, 3, 4, and 24 h by colony counting on drug-free agar.
In parallel, the time courses of E. coli ATCC 25922, EC04 and EC59 cells treated with Carboxin (500 μM), an inhibitor of succinate dehydrogenase (Brynildsen et al., 2013 (link)), and a fluoroquinolone (ciprofloxacin or ofloxacin at 2xMIC), were compared with treatment with fluoroquinolones alone. Carboxin (Sigma–Aldrich, Madrid, Spain) was dissolved in 100% ethanol (Brynildsen et al., 2013 (link)).

The U. maydis strains used in this work (Table S3) derive from the lab strains AB33 [14 (link)], SG200 [7 (link)], and SG200-pit1∆ [23 (link)]. The cells were grown in complete medium (CM: 0.25% w/v cas-amino acids (Difco), 0.1% w/v yeast extract (Difco), 1.0% v/v vitamin solution, and 6.25% v/v salt solution from Holliday [24 ], 0.05% w/v deoxyribonucleic acid from herring sperm, and 0.15% w/v NH₄NO₃ adjusted to pH 7.0 with NaOH (Sigma Aldrich, Darmstadt, Germany) and supplemented with 1% glucose. The transformation of U. maydis protoplasts followed the protocol from [25 (link)]. The constructs were integrated using homologous recombination into the pep4 or upp3 locus, and the transformants were selected on the corresponding antibiotics (200 μg/mL hygromycin, 150 μg/mL nourseothricin, 2 μg/mL carboxin (Sigma Aldrich, Darmstadt, Germany)).
For fluorescence analysis with plate readers, the strains were grown overnight in CM-glucose, before shifting the cultures to nitrate minimal medium (NM)-glucose for the induction of filamentous growth in AB33. Hyphal growth was induced by changing the media to nitrate minimal medium (NM) supplemented with 1% glucose or arabinose. All strains were confirmed using Southern blot analysis [8 (link)] or genotyping PCR.

A. tumefaciens-mediated transformation of A. flavus was performed as described previously (Han et al. 2018 (link)), with some modification. The A. tumefaciens AGL-1 strain containing the pFC-eGFP plasmid was grown in 10 ml DYT medium supplemented with rifampicin (20 mg/ml) and kanamycin (50 μg/ml) overnight at 28° with shaking at 200 rpm. The overnight culture was then diluted to an OD₆₀₀ of 0.15 with induction medium (IM) and grown at 28° with shaking at 200 rpm until reaching an OD₆₀₀ 0.35-0.4. The A. tumefaciens cultures were then mixed with an equal volume of A. flavus conidial suspensions (2×10⁶ spores/ml), and subsequently 200 μl of the mixed cultures were plated onto cellulose nitrate membranes (0.45 μm pore, Sartorius Biotech, Goettingen, Germany) placed on co-cultivation medium and grown at 22° for 48 h. The cellulose nitrate membranes were then transferred to selection medium containing 300 μg/ml cefotaxime (Sangon Biotech, China), 60 μg/ml streptomycin (Bomei, China) and 50 μg/ml carboxin (45371, Sigma-Aldrich, Germany) and incubated at 28° in the dark until colonies appeared. The individual colonies were transferred to selection medium with the appropriate antibiotics, as described above, and grown at 28° for 3-4 days.