Pcold tf

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.

Escherichia coli strains DH5α and BL21 (DE3) (Invitrogen Corporation, Carlsbad, CA, USA) were grown at 37 °C in Lennox broth (LB) or on solid medium containing 1.5% agar at 37 °C and used for the cloning and expression of recombinant genes. When necessary, LB medium was supplemented with an appropriate dosage of ampicillin (100 μg/ml) or kanamycin (100 μg/ml).
The expression vector pCold-TF was purchased from Takara Bio, Inc. (Shiga, Japan). Restriction enzymes were purchased from MBI Fermentas, Inc. (Waltham, MA, USA). V. harveyi strains BB170 (sensor¹⁻ sensor²⁺) (ATCC BAA-1117)and BB152 (ATCC BAA-1119)were purchased from the American type culture collection (Manassas, VA, USA) and cultivated in modified autoinducer bioassay (AB) medium (Bassler et al. 1993 (link)). BB170 was used as the AI-2 biosensor strain and BB152 as a positive control for AI-2 production. All chemicals used were of analytical grade and purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA).

The methods to culture and extract the recombinant LLL proteins followed our previous works (Liu et al. 2019 (link); He et al. 2021 (link)). Briefly, the amplified products (EssLuc and EssLuc-LL2) were inserted into pEASY-T5 (Transgen) to produce the plasmids, from which the gene fragments were digested with NdeI and XhoI enzymes and then inserted into pCold-TF (Takara, Japan) to obtain the expression plasmid. Other genes synthesized by the company of Genecreate (Wuhan, China) were cloned into a pCold-I plasmid for expression. These expression plasmids were transformed into Escherichia coli BL21 (DE3; Tsingke, China) and expressed at 15 °C. Then, the proteins were purified using a nickel-nitrilotriacetic acid (Ni-NTA) column (Qiagen, Germany). The obtained proteins were stored at −80 °C, and the concentration was measured using an Enhanced BCA Protein Assay Kit (Beyotime, China).

The full‐length FoTIP4 coding sequence was inserted in the vector pCold TF (Takara) and expressed in Escherichia coli BL21 (DE3) by incubation with 0.5 mM IPTG for 5 hr at 16 °C. The recombinant protein was purified with a His60 Ni Gravity Column (Takara) according to the manufacturer’s instructions. The probes containing an RRPE or PAC box derived from FoSIK1 (FOXG_12883), pectate lyase gene (FOXG_13331), and β‐glucosidase gene (FOXG_01365) promoters were labelled with biotin using the EMSA Probe Biotin Labeling Kit (Beyotime). The same unlabelled DNA fragment was used as a competitor, while the RRPE or PAC box within a probe changed into AAAAAAAA was used as a negative control. The EMSA was performed using the electrophoretic mobility shift assay kit (Beyotime) according to the manufacturer’s instructions.

The EtCHP559 open reading frame (ORF) was amplified with HD1 and HD2 primers, with BamHI(GCGGATCC) and XhoI (CGCTCGAG) restriction sites at the 5' and 3' ends of the fragment, respectively. The products were digested with BamHI and XhoI, and purified with a gel extraction kit (Tiangen, Beijing, China). The purified fragments were ligated overnight into the expression vector pCold-TF (Takara), digested by the same restriction enzymes at 4°C, and transformed into competent Escherichia coli BL21(DE3) (Tiangen) for protein expression. The recombinant protein was harvested after induction with 1mM isopropylthio-α-D-galactoside (IPTG) (Sigma, St Louis, MO, USA) for 24 h at 16°C. The cell pellet was lysed by sonication and analyzed by 12% SDS-PAGE to confirm the distribution of expressed recombinant protein. rEtCHP559 protein was purified from lysate supernatants using His Bind Resin (Merck, Darmstadt, Germany). The purity of the protein was verified by 12% SDS-PAGE and the concentration of purified protein was determined by the BCA protein assay kit (Beyotime, Haimen, China), using bovine serum albumin (BSA) as a standard. The purified protein was stored in aliquots at −20°Cuntil further use. The TF protein was purified from the lysate supernatants of E. coli BL21 cells transformed with pCold-TF plasmid as same as the methods to rEtCHP559 protein.

The coding regions of hcp (VC1415) and hlyA (VCA0223) were amplified and cloned into the plasmids pET28a and pCold-TF (Takara), respectively. These recombinant plasmids were transferred into E. coli BL21(DE3) strain for protein expression. The His-tagged recombinant proteins were purified using Ni²⁺-NTA affinity chromatography, as was previously described.^{66 (link)} To prepare anti-Hcp and anti-HlyA antiserum for Western blotting analysis, 6-week-old Kunming female mice (Experimental Animal Center, Zunyi Medical University) were randomly assigned to each group (n = 10) and raised in a specific pathogen-free (SPF) environment. Mice were immunized subcutaneously with 30 µg of recombinant Hcp and HlyA proteins with an equal volume of aluminum adjuvant on days 1, 14, and 28. Blood samples were collected from the hearts on day 42, and the serum titers were tested by ELISA.