6 bromoindole

V. parahaemolyticus strain ATCC 17802 and V. harveyi ATCC 14126 (American Type Culture Collection, Manassas, United States) were used in this study. Marine Luria-Bertani (mLB) broth containing 3% (w/v) NaCl was purchased from Becton Dickinson (Franklin Lakes, NJ, United States). V. parahaemolyticus was streaked from a glycerol stock −80°C onto mLB agar plates and incubated overnight at 30°C. For biofilm and other assays, a single colony from each plate was inoculated into 2 mL of an mLB broth and incubated overnight at 30°C in a shaking incubator at 250 rpm (Ashrafudoulla et al., 2020 (link)). Sixteen halogenated indole derivatives (4-bromoindole, 5-bromoindole, 6-bromoindole, 7-bromoindole, 4-chloroindole, 5-chloroindole, 6-chloroindole, 7-chloroindole, 4-fluoroindole, 5-fluoroindole, 6-fluoroindole, 7-fluoroindole, 4-iodoindole, 5-iodoindole, 6-iodoindole, 7-iodoindole, and indole) and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, United States) and Combi-Blocks, Inc. (San Diego, CA, United States). The compounds were dissolved in dimethyl sulfoxide (DMSO). Kanamycin, tetracycline, and crystal violet were obtained from Sigma-Aldrich. Dimethyl sulfoxide (DMSO) was also used as a negative control in all experiments at a concentration of 0.1% (v/v) in media; it did not affect cell growth or the biofilm formation.

Escherichia coli DH5α and BL21 (DE3) strains were used as cloning and protein expression hosts, respectively. E. coli strains carrying plasmids were cultivated in a Luria-Bertani (LB) medium supplemented with antibiotic (50 μg/mL kanamycin). An LB medium was purchased from Huankai Microbial (China). M9 media salts were purchased from Sangon Biotech (China). Plasmid DNA was isolated using a Tiangen plasmid miniprep kit (TIANGEN, Ltd. China). 6-Bromo-Tryptophan and 6-chloro-Tryptophan were purchased from GL Biochem (China). Tryptophan and NADH were purchased from Sigma (United States). Indole, 6-bromo-Indole, indigo, FAD, and isatin were purchased from Sigma (United States), Bidepharm (China), and Yuanye Biotech (China), respectively. Indirubin was purchased from TCI (Japan). Chemically synthesized 6, 6′-dibromoindigo was synthesized from Abace Biotech (China). Gene and oligomer synthesis and sequencing were carried out in BGI (China), Sangon (China), and Rui Biotech (China), respectively. Enzymes involved in restriction reaction, ligation, and PCR were purchased from New England Biolabs (United States), Vazyme (China), and TAKARA (Japan), respectively. All other chemicals used in this study were of analytical grade. All media and reagent solutions were prepared with Milli-Q water (Merck Millipore).

Escherichia coli LPS (O128:B12, Sigma), sulfanilic acid, N-(1-Naphthyl) ethylenediamine (NED), 85% orthophosphoric acid sodium nitrite (NaNO₂), isatin (8, Table 1), 5-bromoisatin (9, Table 1), 6-bromoisatin (4, Table 1), 7-bromoisatin (10, Table 1), 6-bromoindole (11, Table 1) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tyrian purple (6,6 dibromoindigo (1, Table 1), indirubin (7, Table 1), and 6-bromoindirubin (6, Table 1) were obtained from A. L. Skaltsounis (University of Athens). Penicillin–streptomycin solution, Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), sodium pyruvate, and l-glutamine were from Life Technology Australia (Mulgrave, VIC, Australia). RAW264.7 mouse macrophages and 3T3 Swiss albino (ATCC^® CCL92™) cell lines were obtained from the American Type Culture Collection (ATCC^®, Manassas, VA, USA).