Gel purification kit

We have previously reported the isolation of 180 marine bacteria from 15 sponge samples that were collected from South China Sea [19 (link)]. We have extracted all strains’ genomic DNAs and discovered nonribosomal peptides (bacillibactin and bacillomycin D analogues) by using a PCR screening method [19 (link)]. In this study, the forward primer (5′-GGCAGCGGITTCGGCGGITTCCAG-3′) and the reverse primer (5′-CGITGTTIACIGCGTAGAACCAGGCG-3′), designed from the conserved sequences of KS_α and KS_β in the biosynthesis of tetracenomycin, daunorubicin, actinorhodin and fredericamycin [20 (link)], were used in the PCR screening for aromatic polyketide producers. A 20 μL PCR system consisting of 10 µL Easy Taq Polymerase (Beijing TransGen Biotech, Beijing, China), 2 µL of forward and reverse primer mixture (each for 10 µM), 1 µL genomic DNA (50–100 ng), and 7 µL sterilized water, were used. The PCR program was performed with an initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 1 min and extension at 72 °C for 1 min, followed by incubation at 72 °C for 10 min. In total, 167 strains were screened and the PCR products were analyzed by agarose gel electrophoresis, recovered with a gel purification kit (Beijing TransGen Biotech, Beijing, China) and sequenced to afford 12 “positive” strains.

The genomic DNAs of all 180 strains were extracted following standard protocols [48 ]. The forward primer (5′-GCSTACSYSATSTACACSTCSGG-3′) and the reverse primer (5′-SASGTCVCCSGTSCGGTAS-3′), designed from the conserved sequences of adenylation domains (A domains) in the biosynthesis of cephamycin, vancomycin, balhimycin, actinomycin, pristinamycin, and chloroeremomycin, were used in the screening [24 (link)]. A 20 μL PCR system consisting of 10 µL Easy Taq Polymerase (Beijing TransGen Biotech, Beijing, China), 2 µL of forward and reverse primer mixture (each for 10 µM), 1 µL genomic DNA, and 7 µL sterilized water, were used. The PCR program was performed with an initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C for 1 min and extension at 72 °C for 1 min, followed by incubation at 72 °C for 10 min. The PCR products were analyzed by agarose gel electrophoresis (Figure S2), recovered with a gel purification kit (Beijing TransGen Biotech, Beijing, China) and sequenced to afford 21 “positive” strains.

Molecular manipulation was performed according to a standard protocol for Gram-negative bacteria. E. coli APEC40 (WT) genomic DNA and plasmid DNA were extracted using a genome extraction kit (Sangon Biotech, Shanghai, China) and plasmid extraction kit (Transgen, Beijing, China), respectively. PCR amplification was conducted by using Taq or PrimeSTAR^® Max DNA Polymerase (Takara Bio Inc., Dalian, China). A gel purification kit was used to purify the PCR products and DNA restriction fragments (Transgen, Beijing, China). DNA restriction endonuclease (Thermo Fisher Scientific, Waltham, MA, USA) digestion and homologous recombination (Vazyme, Nanjing, China) were conducted using standard methods. The primer sequences used are listed in Table 2.