Bioactive secondary metabolites from tomato-derived bacterium Bacillus velezensis Hnu24

Abstract A series of secondary metabolites have been isolated from the genus of Bacillus velezensis, most of which show antibacterial and insecticidal activities. In order to find more bioactive secondary metabolites from B. velezensis, one new natural component aminoindole dimer baciindole A (1), together with seven known compounds (2–8) were isolated from the tomato-derived bacterium Bacillus velezensis Hnu24. The structure of compound 1 was elucidated by its HR-ESI-MS spectral data and 1 D/2D NMR spectroscopic analysis. Compound 3 showed antibacterial activity against Staphylococcus aureus, S. epidermidis and Ralstonia solanacearum with the MIC values of 3.125, 12.5 and 50 μg/mL, respectively. Compound 4 showed antibacterial activity against S. aureus with the MIC value of 12.5 μg/mL. Compound 3 showed cytotoxic activity for human colon cancer HTC116 cancer cells with the IC50 value of 8.42 ± 0.48 μM. Five compounds (1–4 and 8) were obtained from the strain of B. velezensis for the first time. These results indicated that 3 will be useful in developing antimicrobial and treatment of colon cancer agents. Graphical Abstract


Biological activity
Compounds 1-8 were evaluated for their antibacterial and cytotoxic activities.Compound 3 showed antibacterial activity against Staphylococcus aureus, S. epidermidis and Ralstonia solanacearum with the MIC values of 3.125, 12.5 and 50.0 lg/mL, respectively.Compound 4 showed antibacterial activity against S. aureus with the MIC value of 12.5 lg/mL (Table 1).Compound 3 also showed cytotoxic activity against HTC116 cell lines with the IC 50 value of 8.42 ± 0.48 lM (the positive control doxorubicin hydrochloride with IC 50 value of 0.30 ± 0.005 lM).
Combined with these experimental results, we found that B. velezensis can metabolize biological dimerize indole compounds with antibacterial and cytotoxic activities.Subsequently, the preliminary structure-activity relationship of the isolated dimeric indole alkaloids (1-4) were discussed, and the substitution of different groups at C-1 can affect their antibacterial and cytotoxic activities.Compound 3 possesses a benzene ring at C-1 and showed better antibacterial and cytotoxic activities than that of 1 (methylbenzene connected at C-1), 2 (methylbenzene connected at C-1) and 4 (methyl connected at C-1).When compared 2 with 1, 1 which possesses an amino group at C-2 000 did not enhance antibacterial and cytotoxic activities.

Fungal materials
Bacillus velezensis Hnu24 was isolated from the roots of healthy tomato plants in the occurrence area of bacterial wilt in Haikou, Hainan province.The Bacillus Hnu24 was identified by its morphological characteristics, and a molecular biological protocol by 16S DNA amplification and sequencing of the ITS region.The sequence data had been submitted to GenBank, with an accession number SUB10455276, and Hnu24 was identified as Bacillus velezensis.The strain had been stored in the Guangdong Microbial Culture Collection Center.Ciprofloxacin, streptomycin and doxorubicin hydrochloride were used as positive control.

Antibacterial activity
Compounds 1-8 were evaluated for their antimicrobial activity against eight pathogenic bacteria, Staphylococcus aureus, S. epidermidis, Escherichia coli, Vibrio harveyi, V. alginolyticus, V. parahemolyticus, Pseudomonas aeruginosa and Ralstonia solanacearum by the microplate assay method (Pierce et al. 2008), and the broth medium containing pathogenic bacteria was used as the blank group, DMSO as the negative control, ciprofloxacin and streptomycin were used as positive control.

Cytotoxic activity
All compounds were tested for their cytotoxic activity against six human cell lines (A549, Hela, HTC116, NCI-1650, RKO, MCF-7 and NCM460) by MTT method (Mosmann et al. 1983).Sample concentrations were configured in six gradients of 20, 10, 5, 2.5, 1.25 and 0.625 lg/mL, the experiment was repeated more than 3 times, the experimental data were tested with a full-wavelength microplate reader (test wavelength was 492 nm).DMSO was the negative control and doxorubicin hydrochloride was the positive control, the inhibition rate of each sample was calculated according to the following formula: inhibition rate ¼ [(OD compound À OD DMSO )/OD DMSO ] Â 100%, the GraphPad Prism software was used to calculate the IC 50 value.

Conclusions
In summary, one new natural products baciindole A (1) and 7 known compounds (2-8) were obtained from Bacillus velezensis Hnu24.The structure of 1 was determined by detailed 1 D/2D NMR, HR-ESI-MS spectroscopic data.Compounds 1-4 and 8 were separated from this Bacillus velezensis Hnu24 for the first time.All compounds were tested for their antibacterial and cytotoxic activities.Compounds 3 and 4 showed moderated antibacterial activity against S. aureus, S. epidermidis, R. solanacearum or S. aureus with the MIC values ranging from 3.125 to 50 lg/mL, respectively.Compound 3 also exhibited cytotoxicity against HTC116 cell lines with the IC 50 value of 8.42 ± 0.48 lM.The preliminary structure-activity relationship of the isolated dimeric indole alkaloids (1-4) was also discussed.These results indicated that dimerize-indole derivatives will be useful to developing antimicrobial and treatment of human colon cancer agents.