Bl21 de3

In this study, the E. coli strains DH5α and BL21 (DE3) (Takara, Dalian, China) were used for cloning and expressing the fusion endolysins, respectively. E. coli strains (ATCC25922, CVCC1418 and O78), Acinetobacter baumannii (A. baumannii) strain, and Streptococcus suis (S. suis) strain were stored in the lab. The restriction endonucleases NdeI and SalI, T4 DNA ligase, and pET-28a vector (Takara, Dalian, China) were used to construct the recombinant vectors. Genscript Ni–NTA affinity chromatography medium (GE Healthcare, Beijing, China) was used for purifying endolysins, and primers were synthesized by Jilin Ku Mei Biotechnology Co., Ltd.

Pseudoalteromonas carrageenovora ASY5 was preserved in the College of Food and Biological Engineering of Jimei University. E. coli DH5α and BL21 (DE3) were obtained from TAKARA (Beijing, China). The pET-28a(+) vector was obtained from Thermo Fisher Scientific (Shanghai, China). Taq DNA polyMerase and polyacrylamide were purchased from TransGen Biotech (Beijing, China). Sodium alginate, polyM, and polyG were obtained from Yuanye BioTechnology (Shanghai, China).

E. coli JM109 (Takara, Shiga, Japan) was used for sequencing analysis, and E. coli BL21(DE3) (Takara), pColdTF (Takara), and pColdI (Takara) were used to express BmeTC^X genes. NMR spectra were recorded using a Bruker DPX 400 spectrometer (Billerica, MA, USA) at 400 MHz for protons (¹H) and 100 MHz for carbon (¹³C). GC–MS was performed on a JMS-T100GCV spectrometer (JEOL, Tokyo, Japan) equipped with a DB-1 capillary column (30 m × 0.25 mm × 0.25 µm; J&W Scientific. Inc., Folsom, CA, USA), using the EI mode operated at 70 eV. GC analyses were performed using a Shimadzu GC-2014 chromatograph equipped with a flame ionization detector and using a DB-1 capillary column (30 m × 0.25 mm × 0.25 µm; J&W Scientific, Inc.). GC and GC–MS conditions for the BmeTC^X products were as follows: injection temperature = 300 °C, column temperature = 220–300 °C (1 °C min⁻¹). GC and GC–MS conditions for the volatile compounds were as follows: injection temperature = 200 °C, column temperature = 40–300 °C (5 °C min⁻¹) for GC and 30–300 °C (5 °C min^-1) for GC–MS. Compound 6 was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Two ambergris samples (NSMT M55020 and NSMT M55019; Supplementary Fig. 7) stored in the National Museum of Nature and Science (Japan) for more than 30 years were used for the analysis of volatile components.

Marine bacterium Thalassomonas sp. LD5 was stored in our laboratory and in the China Center for Type Culture Collection (CCTCC; AB2017194). E. coli strains DH5α and BL21(DE3) (TaKaRa, Dalian, China) were cultured at 37 °C in Luria-Bertani (LB) broth or on LB agar plates containing kanamycin (30 μg/mL) when relevant. Plasmid pET-28a (+) (Novagen, Beijing, China) was used to express the recombinant proteins. T4 DNA ligase and PrimeSTAR DNA polymerase were purchased from TaKaRa (Dalian, China). Genomic DNA was extracted from Thalassomonas sp. LD5 using a commercial DNA purification kit (Thermo Scientific, Beijing, China). Sodium alginate was from Bright Moon Seaweed Group (Qingdao, China). PolyM and polyG (purity: about 95%) were from Qingdao BZ Oligo Biotech Co., Ltd. (Qingdao, China).

Escherichia coli DH5α and BL21 (DE3) (TaKaRa, Dalian, China) were grown in liquid Luria-Bertani (LB) medium at 37 °C for genetic manipulations and protein expression, respectively. Brucella suis S2 (WT strain) (CVCC reference number CVCC70502) was obtained from the Shaanxi Provincial Institute for Veterinary Drug Control (Xi’an, Shaanxi, China). Antibiotics were used at the following concentrations (μg/mL) when required: kanamycin, 50; ampicillin, 50; and gentamicin, 25 or 50. The arsR6 and omp25D gene deletion strains (ΔarsR6 and Δomp25D, respectively) were acquired by allelic exchange, as described previously [39 (link)]. The ΔarsR6 and Δomp25D complementing strains (CΔarsR6 and CΔomp25D) were acquired, as described previously [39 (link)]. All specific primers are listed in Supplementary Table S1.

The E. coli strains, BL21 (DE3), and expression vector, pET28a(+), were purchased from Takara (Dalian, China). O-Nitrophenol (ONP) and o-nitrophenyl-β-D-galactopyranoside (ONPG) were purchased from Sangon Biotech (Shanghai, China). Lactose, glucose, and gaLactose used for this study were supplied by Solarbio (Beijing, China). The thin-layer chromatography (TLC) silica gel plates were purchased from Merck (Darmstadt, Germany). All other materials used were of the analytical degree.

Bacterial strains used in the turbidity reduction assays, minimum inhibitory concentration (MIC) assay, and minimum bactericidal concentration (MBC) assay, are summarized in Table 1. The staphylococcal and streptococcal strains were grown at 37 °C with aeration in lysogeny broth (LB) containing 2% tryptone (Difco, Detroit, MI, USA), 0.5% yeast extract (Difco) and Tod-Hewitt broth (THB) (Kanto Kagaku, Tokyo, Japan). Escherichia coli DH5α and BL21(DE3) (Takara, Otsu, Japan) were grown at 37 °C with aeration in LB medium for all subcloning. When needed, 100 µg/mL ampicillin was added to the LB medium (final concentration).