Whole-genome sequencing and phylogenomic analyses of a novel zearalenone-degrading Bacillus subtilis B72

Abstract

Bacillus strain B72 was previously isolated as a novel zearalenone (ZEN) degradation strain from the oil field soil in Xinjiang, China. The genome of B72 was sequenced with a 400 bp paired-end using the Illumina HiSeq X Ten platform. De novo genome assembly was performed using SOAPdenovo2 assemblers. Phylogenetic analysis using 16S rRNA gene sequencing demonstrated that B72 is closely related to the novel Bacillus subtilis (B. subtilis) strain DSM 10. A phylogenetic tree based on 31 housekeeping genes, constructed with 19 strains closest at the species level, showed that B72 was closely related to B. subtilis 168, B. licheniformis PT-9, and B. tequilensis KCTC 13622. Detailed phylogenomic analysis using average nucleotide identity (ANI) and genome-to-genome distance calculator (GGDC) demonstrated that B72 might be classified as a novel B. subtilis strain. Our study demonstrated that B72 could degrade 100% of ZEN in minimal medium after 8 h of incubation, which makes it the fastest degrading strain to date. Moreover, we confirmed that ZEN degradation by B72 might involve degrading enzymes produced during the initial period of bacterial growth. Subsequently, functional genome annotation revealed that the laccase-encoding genes yfiH (gene 1743) and cotA (gene 2671) might be related to ZEN degradation in B72. The genome sequence of B. subtilis B72 reported here will provide a reference for genomic research on ZEN degradation in the field of food and feed.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-023-03517-y.

Keywords: Bacillus sp.; Degradation; Mycotoxin; ZEN-degrading enzyme; Zearalenone.

Fig. 1

Standard curve of ZEN

Fig. 2

B72 strain genome protein-coding gene function COG annotation by comparing with the six major databases

Fig. 3

a Neighbor-joining (NJ) phylogenetic tree based on 16S rRNA gene sequence shows the phylogenetic position of strain B72 among closely related taxa. Bootstrap values (expressed as percentages of 1000 replications) above 75% are shown at branch points. Bar, 0.01 nucleotide substitutions per position; b Neighbor-joining (NJ) phylogenetic tree based on comparing with 19 strains closest to each other at the species level with 31 housekeeping genes (dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB,rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC,rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB, tsf). Bootstrap values (expressed as percentages of 1000 replications) above 75% are shown at branch points. Bar, 0.05 nucleotide substitutions per position

Fig. 4

a ANIb analysis of strain B72 and 3 Bacillus (B. subtilis 168, B. licheniformis PT-9, and B. tequilensis KCTC 13622) reference genomes. The species cutoff value is 95%. b GGDC analysis of strain B72 and 3 Bacillus (B. subtilis 168, B. licheniformis PT-9, and B. tequilensis KCTC 13622) reference genomes. The species cutoff value is 70%

Fig. 5

a The growth curve of strain B72 was calculated by OD600nm at 0–24 h and measurements of ZEN degradation by B72 in minimal medium with 10 μg/mL ZEN at 0–24 h; b B72 was cultured in minimal medium with 10 μg/mL ZEN and incubated under different conditions for 24 h: PBS (control), protease K, cell extracts + 100 ℃, cell extracts + protease K, cell extracts + protease K + SDS and cell extracts. Data represent means ± SD of three independent replicates (b). Statistical significance was determined by Student’s t test (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001 (b)