Bacterial strains, plasmids and growth conditions
The bacterial strains and plasmids used in this study are described in Tables S1 and S2 in the supplementary information. All mutated B. subtilis strains were derivatives of B. subtilis strain 1A751. All B. subtilis strains were grown in super-rich medium containing 25 g Bacto tryptose, 20 g Bacto yeast extract and 3 g K2HPO4 per liter (pH 7.5) or agar plates with ampicillin (100 μg/ml), spectinomycin (100 μg/ml), zeocin (25 μg/ml) and kanamycin (25 μg/ml).
Construction of intracellular protease deletion mutants
The primers used in this study are summarized in Table S3 of the supplementary information. To create the gene deletion locifortepA, ymfH, yrrN and ywpE, the genes and flanking regions (about 2000 bp) were amplified from chromosomal DNA using their respective upstream and downstream primers. These PCR products were ligated into T-vectors to generate template vectors pT-tepA, pT-ymfH, pT-yrrN, and pT-ywpE, which were then used as templates to amplify the 5' and 3' flanks of these genes. pPIC9K was used as template to amplify the zeocin resistance gene. The zeocin resistance gene replaced the deletion gene and was inserted between the 5' flanks of the deletion gene and the 3' flanks of the deletion gene of template vectors to generate antibiotic selection marker knockout vectors pΔtepA, pΔymfH, pΔyrrN, and pΔywpE. Knockout vectors were transformed to B. subtilis 1A751. The suspect mutant cells resistant to zeocin were further identified by diagnostic PCR with the upstream forward primer /the downstream reverse primer of these deletion genes and the upstream forward primer of 5' flanks of these deletion genes/the downstream reverse primer of zeocin gene. The mutant was further confirmed by DNA sequencing.
Secretory expression of AiiO-AIO6
The AiiO-AIO6 expression plasmid pWB-AIO6BS was constructed following protocols as described previously (Pan et al. 2016). pWB-AIO6BS was transformed into the B. subtilis 1A751 and its four intracellular protease gene deletion derivatives. The secretion of AiiO-AIO6 from B. subtilis was studied using pWB-AIO6BS-harboring strains1A751, BSΔtepA, BSΔymfH, BSΔyrrN, and BSΔywpE. B. subtilis cells were cultured in SR medium with kanamycin (25 μg/ml) at 200 rpm for 24 h at 30°C. Bacterial growth was monitored by measuring optical density at 600 nm with the BioPhotometer plus of Eppendorf AG (Hamburg, Germany). Culture supernatant was separated from B. subtilis culture by centrifugation at 12, 000 g (10 min, 4°C) and subjected to AHL-lactonase activity bioassay. Proteins in the supernatants were precipitated with two volume of ice-cold acetone, and then acetone precipitations were separated on 12% polyacrylamide (TGX Stain-Free FastCast Acrylamide Kit, Bio-Rad) and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon; 0.45 μm pore size; Millipore). All stain-free gels were imaged with the Gel Doc XR+ documentation system (Bio-Rad). Western blot analysis was carried out using monoclonal mouse-anti-His-Tag antibody (TianGen, China) as the primary antibody and performed using One Step Western Kit HRP (mouse) according to the manufacturer's instructions (CW Biotech Company Beijing, China).
One unit of AHL lactonase activity was defined as the amount of enzyme that hydrolyzed 1 nmol 3-oxo-C8-HSL per minute. For the hydrolysis assay, the reaction mixture (500 μL) contained 50 μL AiiO-AIO6, 0.248 mM 3-oxo-C8-HSL, and 10 mM PBS (pH 7.0). The reaction was terminated at 70°C for 10 min after the mixture was incubated at 30°C for 30 min.The reaction mixtures were centrifuged at 10,000g for 10 min at 4°C and the supernatants after 0.22 μm filtration were used for HPLC analysis and quantification. The residual 3-oxo-C8-HSL was separated in a Shimadzu InertSustain C18 column at 30°C with a constant flow rate of 1 mL/min in isocratic elution with aqueous–organicmobilephase containing 0.375% triethylamine/acetonitrile (64:36, V/V), and then detected with an UV/visible light detector (Waters) at 201 nm. The remaining AHLs were quantified by calculating the peak areas for a given retention time compared to AHL solutions of known concentrations. Furthermore, for the control, AiiO-AIO6 was replaced with inactivated AiiO-AIO6 by heat treatment (100°C for 5 min).All determinations were performed in four replicates.
An enzyme assay was carried out using assay systems that consisted of mixtures of PBS (pH 7.0) and 0.249 to 0.870 nM 3-oxo-C8-HSL and an incubation time of 30 min at 30°C, and the corresponding reaction rate was calculated. The Michaelis-Menten equation in GraphPad Prism 8 was used to measure the Vmax, Km and Kcat of Michaelis-Menten kinetics.
LC-MS/MS analysis ofthe hydrolysis products of AHL by AiiO-AIO6
The above hydrolysis products of AHL by purified AiiO-AIO6 were extracted three times with ethyl acetate. The combined organic phase was then evaporated to dryness. The samples were dissolved in methanol and separated by a 50-min isocratic elution (mobile phase: methanol: water (60:40; v/v); flow rate: 0.25 ml/min) on a Thermo Scientific Dionex Ultimate 3000 UPLC system with C18 column. MS experiments were conducted on a Thermo Q Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) in a data-dependent acquisition mode with mass range 50 to 750 m/z using the Xcalibur 2.1.2 software followed by ten data-dependent MS/MS scans. The full scan and fragment spectra were collected with resolutions of 70,000 and 17,500 respectively.
For AHL lactonase AiiO-AIO6, homologous protein sequences were retrieved from NCBI after identification by BLAST. Multiple sequence alignment was constructed using DNAMAN. The phylogenetic tree was generated by the neighbor-joining method with the ClustalW (MEGA7).