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. 2020 Nov 5;21(1):767.
doi: 10.1186/s12864-020-07160-2.

Genome mining and UHPLC-QTOF-MS/MS to identify the potential antimicrobial compounds and determine the specificity of biosynthetic gene clusters in Bacillus subtilis NCD-2

Zhenhe Su  1 , Xiuye Chen  1 , Xiaomeng Liu  1 , Qinggang Guo  2 , Shezeng Li  1 , Xiuyun Lu  1 , Xiaoyun Zhang  1 , Peipei Wang  1 , Lihong Dong  1 , Weisong Zhao  1 , Ping Ma  3
Affiliations

Genome mining and UHPLC-QTOF-MS/MS to identify the potential antimicrobial compounds and determine the specificity of biosynthetic gene clusters in Bacillus subtilis NCD-2

Zhenhe Su et al. BMC Genomics. .

Abstract

Background: Bacillus subtilis strain NCD-2 is an excellent biocontrol agent against plant soil-borne diseases and shows broad-spectrum antifungal activities. This study aimed to explore some secondary metabolite biosynthetic gene clusters and related antimicrobial compounds in strain NCD-2. An integrative approach combining genome mining and structural identification technologies using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UHPLC-MS/MS), was adopted to interpret the chemical origins of metabolites with significant biological activities.

Results: Genome mining revealed nine gene clusters encoding secondary metabolites with predicted functions, including fengycin, surfactin, bacillaene, subtilosin, bacillibactin, bacilysin and three unknown products. Fengycin, surfactin, bacillaene and bacillibactin were successfully detected from the fermentation broth of strain NCD-2 by UHPLC-QTOF-MS/MS. The biosynthetic gene clusters of bacillaene, subtilosin, bacillibactin, and bacilysin showed 100% amino acid sequence identities with those in B. velezensis strain FZB42, whereas the identities of the surfactin and fengycin gene clusters were only 83 and 92%, respectively. Further comparison revealed that strain NCD-2 had lost the fenC and fenD genes in the fengycin biosynthetic operon. The biosynthetic enzyme-related gene srfAB for surfactin was divided into two parts. Bioinformatics analysis suggested that FenE in strain NCD-2 had a similar function to FenE and FenC in strain FZB42, and that FenA in strain NCD-2 had a similar function to FenA and FenD in strain FZB42. Five different kinds of fengycins, with 26 homologs, and surfactin, with 4 homologs, were detected from strain NCD-2. To the best of our knowledge, this is the first report of a non-typical gene cluster related to fengycin synthesis.

Conclusions: Our study revealed a number of gene clusters encoding antimicrobial compounds in the genome of strain NCD-2, including a fengycin synthetic gene cluster that might be unique by using genome mining and UHPLC-QTOF-MS/MS. The production of fengycin, surfactin, bacillaene and bacillibactin might explain the biological activities of strain NCD-2.

Keywords: Bacillus subtilis NCD-2; Fengycin; Genome mining; Secondary metabolites; UHPLC–QTOF–MS/MS.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Circular genome of strain NCD-2 with specific features. The circular genome map was created using Circos v0.66 with COG (Cluster of Orthologous Groups of proteins) function annotation. From outside to inside: circle 1, the size of the complete genome; circles 2 and 3, the predicted protein-coding genes on the + and - strands, respectively, where different colours represent different COG function classifications; circle 4, tRNA (green) and rRNA (red); circle 5, G + C content, where peaks outside/inside the circle indicate above or below average GC content, respectively; the inner circle, G + C skew, with G% < C% in purple and with G% > C% in blue. The potato dextrose agar plate inside the representation of the circular genome shows the antifungal activity of strain NCD-2 and its derived strain constructed by atmospheric and room temperature plasma (ARTP) against Botrytis cinerea. The black bars outside the circular genome indicate the secondary metabolite biosynthetic gene clusters
Fig. 2
Fig. 2
Phylogenetic tree of 113 B. subtilis strains based on whole genome alignments. The position of strain NCD-2 in the phylogenetic tree is indicated by a black square mark, and the position of the reference strain B. subtilis NBRC 13719 is indicated by a black circle mark. Single Nucleotide Polymorphisms (SNPs) and short insertions or deletions (indels) within the multiple sequence alignments constructed by the REALPHY pipeline were extracted for subsequent phylogeny reconstruction. The phylogenetic tree was constructed using MEGA 5.0 by the Neighbor-joining method, with a bootstrap of 1000 replications. Bootstrap confidence levels > 50% are indicated at the internodes
Fig. 3
Fig. 3
Schematic diagram of nine secondary metabolite biosynthetic gene clusters in B. subtilis strain NCD-2. antiSMASH was used to predict potential secondary metabolite biosynthetic gene clusters. Different colour blocks represent genes with different functions; the genes marked with dark red, light red, blue, green, and gray are core biosynthetic, additional biosynthetic, transport-related, regulatory, and other genes, respectively
Fig. 4
Fig. 4
Comparisons of functional domains of core genes involved in synthesizing surfactin and fengycin in NCD-2. The functional domains of the core genes of clusters 1 (a) and 3 (b) in B. subtilis NCD-2. (c) The abbreviations indicate the functions of the corresponding structural domains. (d) The conserved binding pockets for substrates formed by amino acids in different adenylation domains
Fig. 5
Fig. 5
PCR and sequence of the fragment between fenE and dacC. a Schematic diagram used to design primers according to conserved bases from NCD-2 and B. velezensis FZB42; b PCR of the fenE-dacC fragment using the genomic templates NCD-2 and FZB42, with 16S rDNA as an internal reference control; c Schematic diagram of the constructed sequencing vector by ligating the fenE-dacC fragment to the pEASY-Blunt Zero vector, d BLAST of the fenE-dacC fragments from NCD-2 and pEASY-Blunt Zero fenE-dacC, in which the two sequences of fenE-dacC were complete same
Fig. 6
Fig. 6
The role of gms1961 in synthesizing fengycin. a FPLC of the lipopeptides of strain NCD-2 and Δgms1961, b quantitative production of fengycin in strain NCD-2 and Δgms1961, where the error bars represent the standard deviation and asterisks depict significant differences as measured by the t-test (**p < 0.01), c Extract Ions Using Dialog (XIC) and UHPLC-QTOF-MS of fengycin from NCD-2 and Δgms1961. The lipopeptide fengycin exhibited a difference at 25–50 min between strains NCD-2 and Δgms1961, and only the precursor related to m/z 725.4 was same (the light purple line), but the fragments were absolutely different from those of fengycin from strain NCD-2
Fig. 7
Fig. 7
MS/MS spectra of protonated cyclic fengycin and surfactin ions. a m/z 732.4, b m/z 746.4, c m/z 725.4, d m/z 739.4, e m/z 767.4, and f m/z 1008.7
Fig. 8
Fig. 8
MS/MS spectra of protonated cyclic bacillaene and bacillibactin ions. a m/z 581.4, b m/z 883.3
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