Front. Microbiol. Frontiers in Microbiology Front. Microbiol. 1664-302X Frontiers Media S.A. 10.3389/fmicb.2025.1515483 Microbiology Original Research Prospection for potential new non-ribosomal peptide gene clusters in Bacillus genus isolated from fermented foods and soil through genome mining Waongo Blaise 1 * Ndayishimiye Libère 2 Tapsoba François 1 † Zongo Wend-Soo Abel 3 Li Jinquan 2 Savadogo Aly 1 1Laboratory of Applied Biochemistry and Immunology, University Joseph KI-ZERBO, Ouagadougou, Burkina Faso 2National Key Laboratory of Agricultural Microbiology, Key Laboratory of Environment Correlative Dietology, College of Food Science and Technology, College of Biomedicine and Health, Huazhong Agricultural University, Wuhan, China 3College of Food Science and Technology, Zhejiang University of Technology, Hangzhou, China

Edited by: Jørgen J. Leisner, University of Copenhagen, Denmark

Reviewed by: Maria Carla Martini, Worcester Polytechnic Institute, United States

Sajid Iqbal, Oujiang Laboratory (Zhejiang Laboratory for Regenerative Medicine, Vision, and Brain Health), China

 *Correspondence: Blaise Waongo, wangblaise85@gmail.com

†ORCID: François Tapsoba, https://orcid.org/0000-0001-6964-0442

 16 06 2025 2025 16 1515483 23 10 2024 21 05 2025 Copyright © 2025 Waongo, Ndayishimiye, Tapsoba, Zongo, Li and Savadogo. 2025 Waongo, Ndayishimiye, Tapsoba, Zongo, Li and Savadogo

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Experimental studies, though often very costly, lead to the discovery of known antimicrobial products. Yet, pathogenic microorganisms are proving increasingly resistant to pre-existing antimicrobial molecules, and this is a cause for worldwide concern. Therefore, it is necessary to search for new molecules that could serve as alternatives in the food, medical and agricultural sectors. Thus, 123 complete genomes of Bacillus strains isolated from soil and fermented foods were analyzed and annotated using bioinformatics prediction and characterization tools. The view was to discover new gene clusters for the biosynthesis of non-ribosomal peptides (lipopeptides, siderophores, antibiotics). This study revealed that 83% of the genomes analyzed possess biosynthetic gene clusters for the production of siderophore bacillibactin, 61% for surfactins, 37% for fengycins, 23% for iturins, 15% for kurstakins and 3% for bacitracin. Besides, seven new biosynthetic gene clusters coding Non Ribosomal Peptide Synthetases (NRPS) have been identified in B. velezensis ATR2, B. velezensis DSYZ, B. velezensis CGMCC11640, B. amyloliquefaciens HM618, B. amyloliquefaciens WF02, B. cereus CMCC P0011, B. cereus CMCC P0021, B. subtilis SJ-10 and B. anthracis CMF9. The results of this study revealed a significant potential of the genus Bacillus to produce new non-ribosomally synthesized peptides. Now, these predicted new antimicrobial molecules can be easily studied experimentally as many new gene clusters have been identified.

 Bacillus nonribosomal peptide lipopeptide biosynthetic gene clusters genome mining section-at-acceptance Antimicrobials, Resistance and Chemotherapy Highlights

Seven novel biosynthetic gene clusters that encode Non-Ribosomal Peptide Synthetases (NRPS) were discovered.

A strain of Bacillus can potentially co-produce 4 families of lipopeptides.

Bacillus genus has a significant potential to develop new non-ribosomally produced peptides.

Genome mining strategy makes it possible to discover new metabolites.

 Introduction

Bacillus are Gram-positive bacteria belonging to the firmicute genus and Bacillaceae family. They are spore-forming, aerobic or facultative aero-anaerobic and get energy by respiration or fermentation (Tamang et al., 2016). Their sporulating ability allows them to resist adverse environmental conditions. They are found in water, soil, dust, plants, food and in animals digestive tract. Some species of the genus Bacillus have important roles as antibiotics or antifungals producers (Khurana et al., 2020). For example, locillomycins and surfactin from B. subtilis 916 show antibacterial activity against Xanthomonas oryzae and Fusarium oxysporum (Zhao et al., 2018). Nowadays, 788 species from the genus Bacillus are sequenced, leading to various studies and explorations (www.ncbi.nlm.nih.gov/genome/?term=Bacillus, consulted 05/07/2024).

Indeed, many Bacillus strains have the ability to produce a wide variety of extracellular enzymes and nonribosomal secondary metabolites such as lipopeptides, the bacillibactin siderophore or antimicrobial compounds such as bacitracin which is a semi-cyclic peptide antibiotic commercialized as a mixture of polypeptides for the treatment of Gram-positive bacterial infections (May et al., 2001; Tapi et al., 2010). Other peptides, such as the lipopeptides have antimicrobial properties and can be used in food or soil biopreservation (Zhao et al., 2018).

Bacillus lipopeptides are subdivided into five families (fengycin, iturin, surfactin, kurstakin, and locillomycin) in which various subfamilies and variants can be found (Lam et al., 2021; Waongo et al., 2023). They are produced by modular mega-enzymes or assembly lines referred to as NRPSs (Iqbal et al., 2021). The NRPSs are composed of modules responsible for the incorporation of amino acids in the final nonribosomal peptide (Théatre et al., 2021). The modules contain the following main catalytic domains: the adenylation domain (A), responsible for the amino acid selection and activation, the carrier protein (CP), responsible for tethering the amino acid to the enzyme, and the condensation domain (C), forming a peptide bond between two amino acids attached to two consecutive modules (Leclère et al., 2016). Some NRPSs also include optional domains that can modify the incorporated amino acids during the synthesis, as the Epimerisation domain (E) leading to the D-isomery (Duban et al., 2022). The enzymatic domains work step by step to assemble the monomers into the peptide, so that NRPSs can be compared to assembly lines. A particular trait of NRPSs synthesising Lipopeptides (LP), except those belonging to the iturin and locillomycin families, is the presence of a condensation domain starting the assembly line. The role of this so-called C-starter is to condense the fatty acid into the first amino acid of the peptide chain. The fengycin family includes decapeptides fengycin A, B, C, and plipastatins A, B and S. The iturin family includes bacillomycins (D, DC, F, L and Lc), mycosubtilin, mojavensin A, subtulene A, mixirin and iturins (A, AL and C), all containing 7 amino acids. Heptapeptides are also found in the surfactin family and variant forms of lichenysin, pumilacidin, esperin and kurstakin. The fifth family contains the nonapeptides locillomycins A, B, and C (Luo et al., 2015a; Waongo et al., 2023).

Many drugs remain effective; however, the emergence of resistant organisms has become a major concern, drawing global attention and promoting the “One Health” concept (Aslam et al., 2021). This concept highlights the close links between human, animal, and environmental health. In this context, nonribosomal secondary metabolites produced by the genus Bacillus can be a source of interesting active compounds. Indeed, some antimicrobial peptides have been shown to be antibiotics with a broad spectrum against pathogens and lipopeptides and siderophores, which are not antibiotics but represent an alternative for food preservation and plant protection against phytopathogenic fungi (Huan et al., 2020).

An urgent challenge today is to discover new natural drugs to tackle emerging human, animal and plant pathogens. The identification of these biomolecules is usually performed by experimental studies including isolation, characterization, purification and activity tests (Fanaei et al., 2021). With the development of bioinformatics tools, this approach can now be accelerated and complemented by sequencing and subsequent genome or metagenome mining to identify natural product biosynthetic pathways (Blin et al., 2021).

Regarding nonribosomal peptide synthesis, specific tools have been developed to identify biosynthetic gene clusters in the genome sequences, to decipher the organization of NRPS into modules and domains, and to predict the nature of the monomers activated by each Adenylation-domain and their isomery in regard to the presence of epimerization domains (Blin et al., 2023). Notably, bacterial genome mining via in silico analysis, using the program antiSMASH, offers an attractive opportunity to discover new secondary metabolites such as NRPs (Rahman et al., 2014; Aleti et al., 2015). AntiSMASH 7 allows better visualization of enzyme assembly chain and good structuring of predicted molecules. Predicted peptides are likely to be characterized by comparison with other peptides available in Norine database (Flissi et al., 2020). The questions addressed here are (i) to review the structural and functional information together with annotation of gene clusters for known lipopeptides (LPs), antibiotics and siderophores produced by the genus Bacillus and (ii) to elucidate through genome mining the potential products of yet uncharacterized nonribosomal gene clusters. In addition, a bibliographical search was carried out to better understand the lipopeptides produced by Bacillus. The objective of this study is to combine literature exploration and genome analysis to identify and characterize the diversity of nonribosomal peptides as antibiotics, siderophores, and lipopeptides, which are potentially produced by Bacillus strains isolated from fermented foods and soil.

 Materials and methods An overview of known structures of nonribosomal lipopeptides produced by <italic>Bacillus</italic> strains

An overview of known structures of nonribosomal lipopeptides generated by Bacillus strains was performed. In brief, Scopus, PubMed, Web of Science, and ResearchGate databases were queried on October 2nd, 2024 using the keywords “lipopeptides AND Bacillus, lipopeptides AND structure, surfactin AND Bacillus, surfactin AND structure, fengycin AND Bacillus, Fengycin AND structure, kurstakin AND Bacillus, Kurstakin AND structure, locillomycin AND Bacillus, locillomycin AND structure, iturin AND Bacillus, iturin AND structure, New lipopeptides AND Bacillus, New lipopeptides Bacillus AND structure, non-ribosomal peptide AND Bacillus.”

 Database search for genome sequences

Sequences (including chromosome and plasmid) of Bacillus isolated from soil and fermented foods were retrieved from the NCBI nucleotide database.1 A total of 123 complete genomes from Bacillus strains isolated from soil and fermented food samples, were selected according to the quality (Table 1). Thus, information on assembly, genome size, number of contigs/scaffolds and N50 were examined.

Genome sequences including chromosomes and plasmids for the different Bacillus strains.

Number Strains Molecules Accession number Isolation source Country Molecule size Contig N50 Completeness (%) Contamination (%)
1 B. aerophilus KJ82 Chromosome CP091093.1 Soil China 3,754,440 bp 3.8 Mb 98.92 0.59
2 B. albus B-9 Chromosome NZ_JAAAWQ000000000.1 Soil China 5,371,297 bp 200.4 kb 99.33 0.1
3 B. albus YK87 Chromosome NZ_CP142670.1 Soil China 5,347,990 bp 5.3 Mb 98.82 0.10
4 B. altitudinis B4133 Chromosome NZ_JXCN00000000.1 Fermented food Netherlands 3,718,745 bp 95.4 kb 99.12 0.29
5 B. altitudinis GR-8 Chromosome CP009108.1 Soil China 3,674,849 bp 3.7 Mb 99.12 0.00
6 B. altitudinis GR-8 Plasmid NZ_CP009109.1 Soil China 6,935 bp 3.7 Mb 99.12 0.00
7 B. altitudinis NJ-V2 Chromosome NZ_CP012482.1 Soil China 3,787,818 bp 3.8 Mb 99.12 0.29
8 B. altitudinis NJ-M2 Chromosome NZ_CP012329.1 Soil China 3,793,419 bp 3.8 Mb 99.12 0.29
9 B. altitudinis NJ-V Chromosome NZ_CP012330.1 Soil China 3,792,999 bp 3.8 Mb 99.12 0.29
10 B. altitudinis G6S2 Chromosome NZ_CP126096.1 Soil Unknown 3,797,173 bp 3.8 Mb 99.41 0.29
11 B. amyloliquefaciens S499 Chromosome CP014700.1 Soil Congo 3,927,922 bp 3.9 Mb 97.18 0.98
12 B. amyloliquefaciens S499 Plasmid NZ_CP014701.1 Soil Congo 8,008 bp 3.9 Mb 97.18 0.98
13 B. amyloliquefaciens 205 Chromosome CP054415.1 Soil China 4,006,790 bp 4 Mb 95.66 1.32
14 B. amyloliquefaciens WF02 Chromosome CP053376.1 Soil Taiwan 4,026,648 bp 4 Mb 98.24 1.46
15 B. amyloliquefaciens bm1 Chromosome CP088005.1 Soil China 3,929,792 bp 3.9 Mb 98.03 1.25
16 B. amyloliquefaciens LS1-002-014 s Chromosome NZ_CP089530.1 Fermented food China 4,214,996 bp 4.2 Mb 98.26 0.94
17 B. amyloliquefaciens HM618 Chromosome CP029466.1 Soil China 4,021,851 bp 4 Mb 97.71 0.78
18 B. amyloliquefaciens B3 Chromosome NZ_JAJNPN000000000.1 Fermented food China 4,001,777 bp 171.8 kb 97.25 2.09
19 B. anthracis HYU01 Chromosome CP008846.1 Soil South Korea 5,213,498 bp 5.2 Mb 98.09 0.48
20 B. anthracis HYU01 Plasmid CP008847.1 Soil South Korea 181,894 bp 5.2 Mb 98.09 0.48
21 B. anthracis HYU01 Plasmid CP008848.1 Soil South Korea 94,732 bp 5.2 Mb 98.09 0.48
22 B. anthracis 9080-G Chromosome CM002398.1 Soil Georgia 5,232,192 bp 195.9 kb 98.43 0.71
23 B. anthracis 9,080-G Plasmid CM002399.1 Soil Georgia 181,656 bp 195.9 kb 98.43 0.71
24 B. anthracis 9,080-G Plasmid NZ_CM002400.1 Soil Georgia 94,815 bp 195.9 kb 98.43 0.71
25 B. anthracis CMF9 Chromosome CP085402.1 Soil China 5,324,354 bp 5.3 Mb 96.09 3.04
26 B. arachidis YX15 Chromosome CP127376.1 Soil China 4,913,320 bp 4.9 Mb 98.53 0.00
27 B. atrophaeus PENSV20 Chromosome CP050705.1 Soil Canada 4,148,820 bp 4.1 Mb 99.12 0.00
28 B. badius NBPM-293 Chromosome CP082363.1 Soil Unknown 3,868,812 bp 3.9 Mb 98.88 1.16
29 B. bombysepticus F12 Chromosome CP085406.1 Soil China 5,244,749 bp 5.2 Mb 99.12 0.05
30 B. bombysepticus Cuernavaca_S2 Chromosome CP126590.1 Soil Mexico 5,288,525 bp 5.3 Mb 98.82 0.00
31 B. cellulasensis NJ-V2 Chromosome CP012482.1 Soil China 3,787,818 bp 3.8 Mb 99.12 0.29
32 B. cellulasensis NJ-M2 Chromosome CP012329.1 Soil China 3,793,419 bp 3.8 Mb 99.12 0.29
33 B. cereus CMCC P0011 Chromosome CP011153.1 Soil China 5,506,876 bp 5.5 Mb 98.97 0.06
34 B. cereus CMCC P0011 Plasmid NZ_CP011154.1 Soil China 591,112 bp 5.5 Mb 98.97 0.06
35 B. cereus M3 Chromosome CP016316.1 Fermented food South Korea 5,218,997 bp 5.2 Mb 98.43 0.22
36 B. cereus CMCC P0021 Chromosome CP011151.1 Soil China 5,521,782 bp 5.5 Mb 99.13 0.06
37 B. cereus CMCC P0021 Plasmid NZ_CP011152.1 Soil China 591,110 bp 5.5 Mb 99.13 0.06
38 B. cereus NJ-W Chromosome CP012483.1 Soil China 5,370,032 bp 5.4 Mb 99.22 0.06
39 B. cereus NJ-W Plasmid NZ_CP012485.1 Soil China 11,744 bp 5.4 Mb 99.22 0.06
40 B. cereus NJ-W Plasmid NZ_CP012484.1 Soil China 9,032 bp 5.4 Mb 99.22 0.06
41 B. cereus NJ-W Plasmid NZ_CP012486.1 Soil China 7,703 bp 5.4 Mb 99.22 0.06
42 B. cereus C-1 Chromosome CP089601.1 Soil China 5,268,934 bp 5.3 Mb 97.79 0.06
43 B. gobiensis FJAT-4402 Chromosome CP012600.1 Soil China 4,597,707 bp 2.8 Mb 98.77 1.69
44 B. haikouensis MNJ12 Chromosome CP076017.1 Soil China 4,454,189 bp 4.5 Mb 99.41 1.78
45 B. halotolerans ZB201702 Chromosome CP029364.1 Soil China 4,154,245 bp 4.2 Mb 99.41 0.59
46 B. inaquosorum DE111 Chromosome CP013984.1 Soil USA 4,143,890 bp 4.1 Mb 98.82 0.00
47 B. inaquosorum LBA001 Chromosome NZ_CP127095.1 Soil Mexico 4,200,707 bp 4.2 Mb 98.82 0.00
48 B. infantis 63–11 Chromosome NZ_JAJBAP020000001.1 Fermented food Thailand 4,841,671 bp 4.8 Mb 99.41 0.21
49 B. licheniformis SCDB 14 Chromosome CP014842.1 Fermented food South Korea 4,136,986 bp 4.1 Mb 97.23 1.98
50 B. licheniformis P8_B2 Chromosome CP045814.1 Soil Denmark 4,343,379 bp 4.3 Mb 97.89 2.24
51 B. licheniformis SCK B11 Chromosome CP014795.1 Soil South Korea 4,300,706 bp 4.3 Mb 98.33 1.24
52 B. licheniformis 14ADL4 Chromosome CP026673.1 Fermented food South Korea 4,332,232 bp 4.3 Mb 97.73 3.06
53 B. methanolicus MGA3 Chromosome CP007739.1 Soil USA 3,337,035 bp 3.3 Mb 97.88 2.66
54 B. mojavensis B-41341 Chromosome NZ_JARLZB000000000.1 Soil Israel 3,856,892 bp 148 kb 99.41 0.00
55 B. mojavensis B-41812 Chromosome NZ_JARSHG000000000.1 Soil Argentina 3,924,370 bp 428.5 kb 98.82 0.00
56 B. mycoides KBAB4 Chromosome NC_010184.1 Soil Unknown 5,262,775 bp 5.3 Mb 98.82 0.32
57 B. mycoides KBAB4 Plasmid NC_010183.1 Soil Unknown 52,830 bp 5.3 Mb 98.82 0.32
58 B. mycoides KBAB4 Plasmid NC_010182.1 Soil Unknown 64,977 bp 5.3 Mb 98.82 0.32
59 B. mycoides KBAB4 Plasmid NC_010181.1 Soil Unknown 75,107 bp 5.3 Mb 98.82 0.32
60 B. mycoides BGSC 4BQ1 Chromosome NZ_NFDI00000000.1 Soil Spain 5,673,703 bp 639 kb 99.41 0.15
61 B. mycoides Gnyt1 Chromosome CP020743.1 Soil China 5,597,907 bp 5.6 Mb 99.41 0.00
62 B. mycoides ATCC 6462 Chromosome CP009692.1 Soil Unknown 5,255,868 bp 5.3 Mb 99.41 0.00
63 B. pacificus AT31 Chromosome NZ_CP142003.1 Soil China 4,903,194 bp 4.9 Mb 99.41 0.00
64 B. pacificus MP6 Chromosome CP093424.1 Fermented food China 5,038,982 bp 5 Mb 99.41 0.00
65 B. pacificus MP6 Plasmid CP093425.1 Fermented food China 291,500 bp 5 Mb 99.41 0.00
66 B. paralicheniformis UBBLI-30 Chromosome NZ_SULF00000000.1 Fermented food India 4,404,722 bp 332.9 kb 99.41 0.06
67 B. paralicheniformis 14DA11 Chromosome NZ_CP023168.1 Fermented food South Korea 4,535,069 bp 4.5 Mb 95.35 0.05
68 B. paralicheniformis CP47 Chromosome NZ_CP133705.1 Fermented food South Korea 4,537,254 bp 4.5 Mb 98.19 0.05
69 B. paranthracis SL1 Chromosome CP093423.1 Fermented food China 5,243,627 bp 5.2 Mb 99.41 0.29
70 B. paranthracis SL1 Plasmid CP093422.1 Fermented food China 325,335 bp 5.2 Mb 99.41 0.29
71 B. paranthracis Gxun-30 Chromosome NZ_CP065149.1 Soil China 5,149,464 bp 5.1 Mb 99.12 0.00
72 B. paranthracis KF11 Chromosome CP085413.1 Soil China 5,276,246 bp 5.3 Mb 99.41 0.29
73 B. paranthracis Bt C4 Chromosome NZ_CP101135.1 Soil China 5,245,328 bp 5.2 Mb 98.82 0.29
74 B. pumilus NJ-V Chromosome CP012330.1 Soil China 3,792,999 bp 3.8 Mb 99.12 0.29
75 B. pumilus DSM 1794 Chromosome CP187664.1 Soil Unknown 3,786,030 bp 3.8 Mb 95.31 2.17
76 B. pumilus MS32 Chromosome NZ_CP092829.1 Soil Germany 3,824,664 bp 3.8 Mb 95.53 2.6
77 B. pumilus B4127 Chromosome NZ_JXCL00000000.1 Fermented food Netherlands 3,886,280 bp 142 kb 95.37 3.01
78 B. pumilus B4127 Plasmid MH581228.1 Fermented food Netherlands 6,580 bp 142 kb 95.37 3.01
79 B. safensis G6S3 Chromosome CP128114.1 Soil Germany 3,670,853 bp 3.7 Mb 99.41 0.29
80 B. spizizenii T30 Chromosome NZ_CP011051.1 Soil Russia 4,031,727 bp 4 Mb 95.76 0.00
81 B. spizizenii AS2 Chromosome NZ_MUXL00000000.1 Soil Oman 4,041,230 bp 573.3 kb 98.82 0.29
82 B. spizizenii HUK15 Chromosome NZ_LSMU00000000.1 Soil France 4,254,363 bp 322.1 kb 98.82 0.29
83 B. stratosphericus MRPD-01 Chromosome PPGB00000000.1 Soil India 3,687,760 bp 322 kb 99.22 0.29
84 B. subtilis SJ-10 Chromosome NZ_CP025258.1 Fermented food South Korea 4,041,647 bp 4 Mb 99.36 0.59
85 B. subtilis MEC_B298 Chromosome NZ_CP100436.1 Soil China 4,030,831 bp 4 Mb 97.41 0.67
86 B. subtilis s-16 Chromosome NZ_CP063150.1 Soil China 4,209,504 bp 4.2 Mb 97.67 1.35
87 B. subtilis BSP1 Chromosome NZ_CP160396.1 Soil Lebanon 4,043,759 bp 4 Mb 96.28 1.21
88 B. subtilis KH2 Chromosome CP018184.1 Soil China 4,138,265 bp 4.1 Mb 96.73 1.28
89 B. subtilis FUA2231 Chromosome NZ_CP154918.1 Fermented food Zimbabwe 4,475,230 bp 4.5 Mb 97.31 1.62
90 B. subtilis UD1022 Chromosome CP011534.1 Soil USA 4,025,326 bp 4 Mb 97.53 0.91
91 B. subtilis SFA-H43 Chromosome KZ836066.1 Soil China 4,018,162 bp 2.1 Mb 96.73 1.48
92 B. subtilis CGMCC 2108 Chromosome CP014471.1 Soil China 4,122,154 bp 4.1 Mb 96.73 1.08
93 B. subtilis CGMCC 2108 Plasmid NZ_CP014473.1 Soil China 65,774 bp 4.1 Mb 96.73 1.08
94 B. subtilis CGMCC 2108 Plasmid NZ_CP014472.1 Soil China 5,820 bp 4.1 Mb 96.73 1.08
95 B. subtilis T30 Chromosome CP011051.1 Soil Russia 4,031,727 bp 4 Mb 95.76 0.00
96 B. subtilis NCIB 3610 Chromosome NZ_CP094361.1 Soil China 4,210,909 bp 4.2 Mb 98.51 1.33
97 B. subtilis ZD01 Chromosome NZ_CP046448.1 Soil China 4,015,360 bp 4 Mb 97.5 0.67
98 B. subtilis 73 Chromosome CP045826.1 Soil Germany 4,166,516 bp 4.2 Mb 97.33 1.97
99 B. subtilis G8 Chromosome NZ_AP025224.1 Fermented food Indonesia 4,017,503 bp 4 Mb 96.73 1.08
100 B. subtilis FUA2232 Chromosome NZ_CP154920.1 Fermented food Zimbabwe 4,475,228 bp 4.5 Mb 97.31 1.61
101 B. subtilis SRCM103517 Chromosome NZ_CP035226.1 Fermented food South Korea 4,192,706 bp 4.2 Mb 96.85 1.87
102 B. subtilis S1 Chromosome NZ_JAGFPW000000000.1 Fermented food India 4,488,261 bp 153.3 kb 95.77 1.85
103 B. thuringiensis HD12 Chromosome CP014847.1 Soil China 5,776,895 bp 5.8 Mb 98.39 0.96
104 B. thuringiensis HD12 Plasmid NZ_CP014853.1 Soil China 345,196 bp 5.8 Mb 98.39 0.96
105 B. thuringiensis HD12 Plasmid NZ_CP014852.1 Soil China 161,353 bp 5.8 Mb 98.39 0.96
106 B. thuringiensis HD12 Plasmid NZ_CP014851.1 Soil China 112,429 bp 5.8 Mb 98.39 0.96
107 B. thuringiensis HD12 Plasmid NZ_CP014850.1 Soil China 39,023 bp 5.8 Mb 98.39 0.96
108 B. thuringiensis HD12 Plasmid NZ_CP014848.1 Soil China 17,228 bp 5.8 Mb 98.39 0.96
109 B. thuringiensis Bt185 Chromosome CP014282.1 Soil China 5,243,635 bp 5.2 Mb 98.26 1.05
110 B. thuringiensis Bt185 Plasmid NZ_CP014284.1 Soil China 293,705 bp 5.2 Mb 98.26 1.05
111 B. thuringiensis Bt185 Plasmid NZ_CP014285.1 Soil China 55,372 bp 5.2 Mb 98.26 1.05
112 B. thuringiensis Bt185 Plasmid NZ_CP014286.1 Soil China 54,205 bp 5.2 Mb 98.26 1.05
113 B. thuringiensis Bt185 Plasmid NZ_CP014287.1 Soil China 41,937 bp 5.2 Mb 98.26 1.05
114 B. thuringiensis Bt185 Plasmid NZ_CP014288.1 Soil China 12,487 bp 5.2 Mb 98.26 1.05
115 B. thuringiensis Bt185 Plasmid NZ_CP014290.1 Soil China 7,486 bp 5.2 Mb 98.26 1.05
116 B. thuringiensis c25 Chromosome NZ_CP022345.1 Soil South Korea 5,334,660 bp 5.3 Mb 98.91 1.07
117 B. thuringiensis 97–27 Chromosome NC_005957.1 Soil Unknown 5,237,682 bp 5.2 Mb 99.44 0.32
118 B. thuringiensis YWC2-8 Chromosome CP013055.1 Soil China 5,674,369 bp 5.7 Mb 98.38 1.01
119 B. thuringiensis YWC2-8 Plasmid NZ_CP013056.1 Soil China 250,706 bp 5.7 Mb 98.38 1.01
120 B. thuringiensis YWC2-8 Plasmid NZ_CP013057.1 Soil China 84,491 bp 5.7 Mb 98.38 1.01
121 B. thuringiensis YWC2-8 Plasmid NZ_CP013058.1 Soil China 82,531 bp 5.7 Mb 98.38 1.01
122 B. thuringiensis YWC2-8 Plasmid NZ_CP013059.1 Soil China 80,699 bp 5.7 Mb 98.38 1.01
123 B. thuringiensis YWC2-8 Plasmid NZ_CP013060.1 Soil China 46,634 bp 5.7 Mb 98.38 1.01
124 B. thuringiensis YWC2-8 Plasmid NZ_CP013061.1 Soil China 8,512 bp 5.7 Mb 98.38 1.01
125 B. thuringiensis BGSC 4AA1 Chromosome CP010577.1 Soil China 5,652,292 bp 5.7 Mb 97.24 3.40
126 B. thuringiensis BGSC 4AA1 Plasmid NZ_CP010578.1 Soil China 232,994 bp 5.7 Mb 97.24 3.40
127 B. thuringiensis BGSC 4AA1 Plasmid NZ_CP010579.1 Soil China 92,619 bp 5.7 Mb 97.24 3.40
128 B. thuringiensis BGSC 4AA1 Plasmid NZ_CP010580.1 Soil China 76,979 bp 5.7 Mb 97.24 3.40
129 B. thuringiensis BGSC 4AA1 Plasmid NZ_CP010581.1 Soil China 68,444 bp 5.7 Mb 97.24 3.40
130 B. thuringiensis BGSC 4AA1 Plasmid NZ_CP010582.1 Soil China 51,723 bp 5.7 Mb 97.24 3.40
131 B. thuringiensis BGSC 4AA1 Plasmid NZ_CP010583.1 Soil China 4,845 bp 5.7 Mb 97.24 3.40
132 B. thuringiensis CTC Chromosome CP013274.1 Soil China 5,327,397 bp 5.3 Mb 97.63 1.10
133 B. thuringiensis CTC Plasmid NZ_CP013273.1 Soil China 25,529 bp 5.3 Mb 97.63 1.10
134 B. thuringiensis HS18-1 Chromosome CP012099.1 Soil China 5,292,526 bp 5.3 Mb 98.68 0.55
135 B. thuringiensis HS18-1 Plasmid NZ_CP012101.1 Soil China 337,579 bp 5.3 Mb 98.68 0.55
136 B. thuringiensis HS18-1 Plasmid NZ_CP012102.1 Soil China 92,085 bp 5.3 Mb 98.68 0.55
137 B. thuringiensis HS18-1 Plasmid NZ_CP012103.1 Soil China 94,695 bp 5.3 Mb 98.68 0.55
138 B. thuringiensis HS18-1 Plasmid NZ_CP012104.1 Soil China 42,726 bp 5.3 Mb 98.68 0.55
139 B. thuringiensis HS18-1 Plasmid NZ_CP012105.1 Soil China 14,336 bp 5.3 Mb 98.68 0.55
140 B. thuringiensis HS18-1 Plasmid NZ_CP012106.1 Soil China 4,669 bp 5.3 Mb 98.68 0.55
141 B. thuringiensis HS18-1 Plasmid NZ_CP012107.1 Soil China 8,287 bp 5.3 Mb 98.68 0.55
142 B. thuringiensis HS18-1 Plasmid NZ_CP012108.1 Soil China 7,386 bp 5.3 Mb 98.68 0.55
143 B. thuringiensis JW-1 Chromosome NZ_CP045030.1 Soil China 5,500,376 bp 5.5 Mb 99.30 0.74
144 B. thuringiensis HD-1 Chromosome CP004870.1 Soil USA 5,631,672 bp 5.6 Mb 97.67 1.75
145 B. thuringiensis YBT-1518 Chromosome CP005935.1 Soil China 6,002,284 bp 6 Mb 98.60 3.18
146 B. toyonensis UTDF19-29B Chromosome CP081872.1 Soil USA 5,240,743 bp 5.2 Mb 99.41 0.10
147 B. toyonensis Monterrey_S3 Chromosome CP126524.1 Soil Mexico 5,309,620 bp 5.3 Mb 99.41 0.10
148 B. toyonensis Cuernavaca_S4 Chromosome CP126520.1 Soil Mexico 5,270,039 bp 5.3 Mb 98.82 0.10
149 B. tropicus CK18 Chromosome CP085399.1 Soil China 5,237,233 bp 5.2 Mb 99.41 0.00
150 B. tropicus T36S-23 Chromosome NZ_CP119875.1 Soil South Korea 5,262,398 bp 5.3 Mb 98.82 0.62
151 B. vallismortis NBIF-001 Chromosome CP020893.1 Soil China 3,929,787 bp 3.9 Mb 98.82 0.00
152 B. vallismortis DSM 11031 Chromosome CP026362.1 Soil USA 4,286,362 bp 4.3 Mb 99.22 0.00
153 B. velezensis ATR2 Chromosome CP018133.1 Soil China 4,006,746 bp 4 Mb 99.41 0.00
154 B. velezensis SYP-B637 Chromosome CP043546.1 Soil China 3,915,550 bp 3.9 Mb 99.41 0.00
155 B. velezensis CGMCC 11640 Chromosome CP026610.1 Soil China 4,322,979 bp 4.3 Mb 99.41 0.00
156 B. velezensis Lzh-a42 Chromosome CP025308.1 Soil China 4,246,605 bp 4.2 Mb 99.41 0.00
157 B. velezensis YJ0-1 Chromosome NZ_CP128184.1 Soil China 4,043,341 bp 4 Mb 99.41 1.24
158 B. velezensis L-S60 Chromosome CP011278.1 Soil China 3,903,017 bp 3.9 Mb 99.41 0.00
159 B. velezensis CBMB205 Chromosome CP014838.1 Soil South Korea 3,929,745 bp 3.9 Mb 99.41 0.00
160 B. velezensis SB1216 Chromosome CP015417.1 Soil USA 3,814,720 bp 3.8 Mb 97.56 0.00
161 B. velezensis DSYZ Chromosome CP030150.1 Soil China 4,258,978 bp 4.3 Mb 99.41 0.00
162 B. velezensis DR-08 Chromosome CP028437.1 Soil South Korea 3,929,794 bp 3.9 Mb 99.36 0.00
163 B. velezensis CMF18 Chromosome CP085388.1 Soil China 3,963,155 bp 4 Mb 99.41 0.00
164 B. velezensis CK17 Chromosome CP085706.1 Soil China 3,921,806 bp 3.9 Mb 99.34 0.00
165 B. velezensis DMB06 Chromosome NZ_CP083763.1 Fermented food South Korea 4,157,945 bp 4.2 Mb 99.4 0.00
166 B. velezensis UD6-2 Chromosome NZ_JAGFMB000000000.1 Fermented food Thailand 3,951,373 bp 1 Mb 99.41 0.00
167 B. velezensis DMB05 Chromosome NZ_CP083715.1 Fermented food South Korea 3,262,563 bp 3.3 Mb 99.12 0.00
168 B. velezensis DMB05 Plasmid NZ_CP083716.1 Fermented food South Korea 806,695 bp 3.3 Mb 99.12 0.00
169 B. velezensis DMB05 Plasmid NZ_CP083717.1 Fermented food South Korea 72,020 bp 3.3 Mb 99.12 0.00
170 B. wiedmannii LN15 Chromosome CP126099.1 Soil China 5,391,143 bp 5.4 Mb 98.82 0.10
171 B. wiedmannii JAS08/1 Chromosome NZ_CP036073.1 Soil Poland 5,513,922 bp 5.5 Mb 99.41 0.10
172 B. wiedmannii JAS07/5 Chromosome CP036070.1 Soil Poland 5,239,489 bp 5.2 Mb 99.41 1.76
173 B. wiedmannii JAS07/5 Plasmid NZ_CP036072.1 Soil Poland 536,465 bp 5.2 Mb 99.41 1.76
174 B. wiedmannii JAS07/5 Plasmid NZ_CP036071.1 Soil Poland 79,335 bp 5.2 Mb 99.41 1.76
175 B. wiedmannii PL1 Chromosome AP022643.1 Soil Japan 5,309,441 bp 5.3 Mb 97.22 0.00
176 B. safensis ZK-1 Chromosome NZ_CP095759.1 Soil China 3,735,476 bp 3.7 Mb 99.06 0.29
177 B. safensis SRCM125915 Chromosome NZ_CP116774.1 Soil South Korea 3,769,976 bp 3.8 Mb 98.92 0.59
178 B. safensis PLA 1006 Chromosome NZ_CP114177.1 Soil China 3,891,304 bp 3.9 Mb 98.92 0.59
Detection of nonribosomal peptide biosynthetic gene clusters (BGCs)

The search for nonribosomal peptide sequences from the genome sequences of 123 Bacillus strains was performed following a workflow previously described (Leclère et al., 2016). First, the prediction of biosynthetic gene clusters (BGCs) was performed using antiSMASH version 7 (Blin et al., 2023). The regions encoding nonribosomal synthetases were further analyzed. The monomer composition of the predicted peptide was compared to all known NRPs annotated in the Norine database2 (Flissi et al., 2020) in order to identify them or predict them as a new peptide or new variant. When it was incomplete with truncated or fragmented clusters, we further investigated antiSMASH results to reconstruct partial or complete BGCs by assembling cluster fragments scattered in the shotgun genome. The number of genes, the order of modules, and the domains in the NRPSs and the predictions of the A-domain specificity helped identify clusters of known NRP families, even when they were fragmented.

Since poor sequence assembly can affect the quality of predicted metabolites, only high-quality complete genomes were considered in this study.

 Results

Out of 123 Bacillus chromosomes selected, 115 were found to carry BGC NRPSs. Among these NRPSs, those responsible for the synthesis of a siderophore (102), antibiotics (4) or lipopeptides (93) were found.

Prediction of NRP siderophore

The screened sequences harbor genetic potential to produce the siderophore bacillibactin. The gene cluster is composed of three modules with A-domain specificity for 2,3-dihydroxybenzoate (Dhb or diOH-Bz in Norine), glycine and threonine. The corresponding NRPS is known to follow an iterative mode of biosynthesis, which could lead to the production of active iron-chelating compounds named bacillibactins. This small cluster was predicted in 83% of the studied genomes.

 Overview of lipopeptide BGCs produced by <italic>Bacillus</italic>

The literature search revealed diverse non-ribosomal lipopeptides synthesized by the genus Bacillus. The structures of the different molecules are shown in Table 2.

Structure of different lipopeptides classified by families.

Name of lipopeptides Monomer composition of the peptide moiety Main fatty acid chains References
Fengycin family Decapeptide with a lactone ring between carboxy-terminal group β-OH fatty acids
Fengycin A1 L-Glu,D-Orn,D-Tyr,D-aThr,L-Glu,D-Ala,L-Pro,L-Gln,L-Tyr,L-Ile C14-C19 Ait Kaki et al. (2020)
Fengycin A2 L-Glu,D-Orn,D-Tyr,D-aThr,L-Glu,D-Ala,L-Pro,L-Gln,L-Tyr,L-Val C14, C15, C17 Ongena and Jacques (2007)
Fengycin B1 L-Glu-D-Orn-D-Tyr-D-aThr,L-Glu,D-Val,L-Pro,L-Gln,L-Tyr,L-Ile C14-C17 Fanaei et al. (2021)
Fengycin B2 L-Glu,D-Orn,D-Tyr,D-aThr,L-Glu,D-Val,L-Pro,L-Gln,L-Tyr,L-Val C14-C21 Ongena and Jacques (2007)
Fengycin C1 L-Glu,D-Orn,D-Tyr,D-aThr,L-Glu,D-Leu/D-Ile,L-Pro,L-Gln,L-Tyr,L-Val C15-C19 Vater et al. (2002)
Fengycin C2 L-Glu,D-Orn,D-Tyr,D-aThr,L-Glu,D-Val,L-Pro,L-Gln,L-aThr,L-Ile C14-C18 Ongena and Jacques (2007)
Fengycin S L-Glu,D-Orn,D-Tyr,D-Ser,L-Glu,D-Val,L-Pro,L-Gln,L-Tyr,L-Ile C17 Sang-cheol et al. (2010)
Plipastatin A1, A2 L-Glu,D-Orn,D-Tyr,D-a-Thr,L-Glu,D-Ala,L-Pro,L-Gln,D-Tyr,L-Ile nC16, aiC17 Nishikiori et al. (1986)
Plipastatin B1, B2 L-Glu,D-Orn,D-Tyr,D-a-Thr,L-Glu,D-Val,L-Pro,L-Gln,L-Tyr,L-Ile nC16, aiC17 Nishikiori et al. (1986)
Iturin family Heptapeptide cyclised by a lactam ring with the β-NH2 group of the acid chain β-NH2 fatty acids
Iturin A, AL L-Asn,D-Tyr,D-Asn,L-Gln,L-Pro,D-Asn,L-Ser nC14, iC15, aiC15, nC16, iC16 Pathak and Keharia (2014)
Iturin C L-Asp,D-Tyr,D-Asn,L-Gln,L-Pro,D-Asn,L-Ser nC14, iC15, aiC15 Pathak and Keharia (2014)
Bacillomycin D L-Asn,D-Tyr,D-Asn,L-Pro,L-Gln,D-Ser,L-Thr nC14, iC15, aiC15 Jin et al. (2017)
Bacillomycin DC L-Asn,D-Tyr,D-Asn,L-Pro,L-Glu,D-Ser,L-Thr C13 Jin et al. (2017)
Bacillomycin F L-Asn,D-Tyr,D-Asn,L-Gln,L-Pro,D-Asn,L-Thr C16, iC17, aiC17 Peypoux et al. (1985)
Bacillomycin L L-Asp,D-Tyr,D-Asn,L-Ser,L-Glu,D-Ser,L-Thr nC14, iC15, aiC15 Volpon et al. (2007)
Bacillomycin Lca L-Asn,D-Tyr,D-Asn,L-Ser,L-Glu,D-Ser,L-Thr nC14, iC15, aiC15, iC16 Volpon et al. (2007)
Mycosubtilin L-Asn,D-Tyr,D-Asn,L-Gln,L-Pro,D-Asn/D-Ser,L-Ser/L-Asn nC16, iC16, aiC17 Peypoux et al. (1986)
Mojavensin A L-Asp,D-Tyr,D-Asn,L-Gln,L-Pro,D-Asn,L-Asn aiC12 Ma et al. (2012)
Subtulene A L-Asn,D-Tyr,D-Asn,L-Gln,L-Pro,D-Asn,L-Ser (CH2)4HC = CH(CH2)3CH(CH3)2 Thasana et al. (2010)
Mixirin L-Asn,D-Tyr,D-Asn,L-Gln,L-Ser,D-Asn,L-Pro (A) C11, (B) C8, (C) aiC10 Zhang et al. (2004)
Kurstakin family Heptapeptide with a lactone ring between carboxy-terminal group of Gln7 and OH group of Ser4
Kurstakinb D-Thr,L-Gly,D-Ala,L-Ser,L-His,D-Gln,L-Gln iC11, nC12, iC12, iC13 Hathout et al. (2000)
Surfactin family Heptapeptide closed by a lactone ring with the b-OH group of the fatty acid chain β-OH fatty acids
Bamylocin Ac Glu,Leu,Met,Leu,Pro,Leu,Leu,Leu C13 Lee et al. (2007)
Surfactin L-Glu,L-Leu,D-Leu,L-Val,L-Asp,D-Leu,L-Leu iC14, nC14, iC15, aC15 Bonmatin et al. (2003)
[Orn2] surfactin L-Glu,L-Orn,D-Leu,L-Val,L-Asp,D-Leu,L-Ile C13-C15 Bonmatin et al. (2003)
[Orn7] surfactin L-Glu,L-Leu,D-Leu,L-Val,L-Asp,D-Leu,L-Orn – Bonmatin et al. (2003)
[Phe7] surfactin L-Glu,L-Leu,D-Leu,L-Val,L-Asp,D-Leu,L-Phe – Bonmatin et al. (2003)
[Cys7] surfactin L-Glu,L-Leu,D-Leu,L-Val,L-Asp,D-Leu,L-Cys – Bonmatin et al. (2003)
[Ala4] surfactin L-Glu,L-Leu,D-Leu,L-Ala,L-Asp,D-Leu,L-Leu iC14, nC14, iC15, aiC15 Bonmatin et al. (2003)
[Ile4] surfactin L-Glu,L-Leu,D-Leu,L-Ile,L-Asp,D-Leu,L-Leu aiC15 Bonmatin et al. (2003)
[Ile2, 7] surfactin L-Glu,L-Ile,D-Leu,L-Val,L-Asp,D-Leu,L-Ile C13-C15 Bonmatin et al. (2003)
[Ile4, 7] surfactin L-Glu,L-Leu,D-Leu,L-Ile,L-Asp,D-Leu,L-Ile C13-C15 Bonmatin et al. (2003)
[Leu4] surfactin L-Glu,L-Leu,D-Leu,L-Leu,L-Asp,D-Leu,L-Leu iC15 Bonmatin et al. (2003)
[Val7] surfactin L-Glu,L-Leu,D-Leu,L-Val,L-Asp,D-Leu,L-Val iC14, nC14, iC15, aiC15 Bonmatin et al. (2003)
[Ile7] surfactin L-Glu,L-Leu,D-Leu,L-Val,L-Asp,D-Leu,L-Ile iC14, nC14, iC15, aiC15 Bonmatin et al. (2003)
[Ile2, 4] surfactin L-Glu,L-Ile,D-Leu,L-Ile,L-Asp,D-Leu,L-Leu aiC15 Bonmatin et al. (2003)
[Val2, 7] surfactin L-Glu,L-Val,D-Leu,L-Val,L-Asp,D-Leu,L-Val C13-C15 Bonmatin et al. (2003)
[Val2, Ile7] surfactin L-Glu,L-Val,D-Leu,L-Val,L-Asp,D-Leu,L-Ile C13-C15 Bonmatin et al. (2003)
[Ile2, Val7] surfactin L-Glu,L-Ile,D-Leu,L-Val,L-Asp,D-Leu,L-Val C13-C15 Bonmatin et al. (2003)
[Ile2, 4, 7] surfactin L-Glu,L-Ile,D-Leu,L-Ile,L-Asp,D-Leu,L-Ile aiC15 Bonmatin et al. (2003)
Lichenysine L-Gln,L-Leu,D-Leu,L-Val,L-Asp,D-Leu,L-Ile iC13, aC13, nC14 iC15, aC15 Bonmatin et al. (2003)
[Ile4] lichenysin L-Gln,L-Leu,D-Leu,L-Ile,L-Asp,D-Leu,L-Ile aiC15 Bonmatin et al. (2003)
[Val7] lichenysin L-Gln,L-Leu,D-Leu,L-Val,L-Asp,D-Leu,L-Val iC13, aiC13, nC14, iC15, aiC15 Bonmatin et al. (2003)
[Ile2, 4] lichenysin L-Gln,L-Ile,D-Leu,L-Ile,L-Asp,D-Leu,L-Ile aiC15 Bonmatin et al. (2003)
Esperind L-Glu,L-Leu,D-Leu,L-Val,L-Asp,D-Leu,L-XE7-COOH C13, C14, C15 Thomas and Ito (1969)
Pumilacidin L-Glu,L-Leu,D-Leu,L-Leu,L-Asp,D-Leu,L-XP7 aC15, iC15, nC16, iC16, aC17, iC17 Naruse et al. (1990)
Locillomycin family Partially cyclized nonapeptides
Locillomycin A, B, C L-Thr,D-Gln,L-Asp,L-Gly,L-Asn,L-Asp,L-Gly,L-Tyr,L-Val C13, C14, C15 Luo et al. (2015a, 2015b)
Cerexin family Linear decapeptides
Cerexin A D-Asp,D-Val,D-val,L-Asn,D-Asn,L-ϒHyl,D-Thr,L-Ser,D-Trp,D-Ile iC11h3 Shoji et al. (1976b)
Cerexin B D-Asn,D-val,D-Phe,L-Asn,D-Asn,L-ϒHyl,D-Thr,L-Gly,D-Trp,D-Ile iC10h3, nC10h3, iC11h3, aiC11h3 Shoji and Kato (1976)
Cerexin C D-Asp,D-Val,D-val,L-Asn,D-Asn,L-Lys,D-Thr,L-Ser,D-Trp,D-Ile iC11h3 Shoji et al. (1976a)
Cerexin D D-Asp,D-Val,D-Phe,L-Asn,D-Asn,L-Lys,D-Thr,L-Gly,D-Trp,D-Ile iC10h3, nC10h3, iC11h3, aiC11h3 Shoji et al. (1976a)
Marihysin A family Cyclic heptapeptide
Marihysin A Pro,Gln,Asn,Ser,Asn,Tyr,Asn C14 Liu et al. (2010)
Octapeptin family
Octapeptin A1, A2, A3, A4 D-Dab,L-Dab,L-Dab,L-Dab,L-Dab,D-Leu,L-Leu,L-Leu aiC11h3, iC10h3, nC10h3, iC11h3 Meyers et al. (1976)
Octapeptin B1, B2, B3, B4 D-Dab,L-Dab,L-Dab,L-Dab,L-Dab,D-Leu,L-Leu,L-Phe aiC11h3, iC10h3, nC10h3, iC9h3 Meyers et al. (1976)
Octapeptin C1, C2, C3, C4 D-Dab,L-Dab,L-Dab,L-Dab,L-Dab,L-Leu,L-Leu,D-Phe aiC9h3, aiC9h3, iC10h3, nC10h3 Meyers et al. (1976)
Octapeptin D1, D2, D3, D4 D-Ser-L-Dab-L-Dab-D-Leu-L-Leu-L-Dab-L-Dab-L-Leu aiC11h3, iC10h3, nC10h3, iC11h3 Kato and Shoji (1980)

aBacillomycin Lca; or bacillopeptin.

bD forms of amino acid residue are deduced from NRPS modular structure.

cL and D forms are not specified.

dThe b-carboxyl of Asp5 is engaged in the lactone.

eOr halobacillin With XE7 = Leu or Val; XP7 = Val or Ile.

ai, anteiso; i, iso; n, normal; h3, 3-hydroxy; L-Hyl, L-threo—hydroxylysine; Dab, 2,4-Diaminobutyric acid.

 Prediction of the lipopeptide structures

Within BGCs detected by antiSMASH, special attention was paid to traits specific to LP synthesis. Thus, a condensation domain allowing the incorporation of fatty acid (FA) (so-called C-starter) was sought. C-starter is usually present in LP NRPS except for LP belonging to iturin and locillomycin families (Figure 1). As up to now, no bioinformatics tool enables the prediction of fatty acid (FA) structure, the work was focused on peptide moieties (Table 3).

Organization of the known non-ribosomal peptide synthetases (NRPS) encoding lipopeptides, siderophore bacillibactin and antibiotic bacitracin in Bacillus. Iterative domains: A, adenylation domain; C, condensation domain; CP, Carrier Protein; E, epimerization domain; TE, thioesterase domain; mal, malonyl-CoA; CAL, Co-enzyme A ligase; KS, ketosynthetase domain; AmT, aminotransferase. Organization of the known NRPSs: (a) iturin A in B. amyloliquefaciens S499, (b) known kurstakin, (c) surfactin in B. velezensis UD6-2, (d) lichenysin A in B. paralicheniformis CP47, (e) bacillibactin in B. aerophilus KJ82, (f) fengycin B in B. paralicheniformis CP47, (g) bacitracin in B. paralicheniformis CP47, (h) bacillomycin D in B. velezensis DMB06, (i) mycosubtilin in B. subtilis T30, (j) locillomycin in B. subtilis 916.

Structures of predicted and identified lipopeptides among different genomic sequences of Bacillus strains.

Predicted LP Structure analysis Comments Strains
[Ile7] surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) Complete B. aerophilus KJ82
[Ile7] surfactin (Glu,Leu,D-Leu)(Val,Asp,Leu)(Ile) Complete B. altitudinis B4133
[Ile7] surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) Complete B. altitudinis GR-8
[Ile7] surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) Complete B. altitudinis NJ-V2
[Ile7] surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) Complete B. altitudinis NJ-M2
[Ile7] surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) Complete B. altitudinis NJ-V
[Ile7] surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) Complete B. altitudinis G6S2
Iturin A (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Asn,Ser) Complete B. amyloliquefaciens S499
Fengycin A/B (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-X)(Pro,Gln,D-Tyr)(Ile) Incomplete (X could be Val or Ala) B. amyloliquefaciens S499
Surfactin (Glu,Leu,D-Leu)(…)(Leu) Partial and fragmented B. amyloliquefaciens S499
[Ile7] surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) Complete B. amyloliquefaciens 205
Fengycin (……)(…)(…)(…Glu,D-Tyr)(Ile) Partial and fragmented (Glu instead of Gln) B. amyloliquefaciens 205
Iturin A (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Asn,Ser) Complete B. amyloliquefaciens 205
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. amyloliquefaciens WF02
Fengycin A/B (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-X)(Pro,Gln,D-Tyr)(Ile) Incomplete (X could be Val or Ala) B. amyloliquefaciens WF02
Iturin A (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Asn,Ser) Complete B. amyloliquefaciens WF02
Surfactin (Glu,Leu,D-Leu)(Val,Asp,Leu)(Leu) Complete B. amyloliquefaciens bm1
Fengycin A/B (Glu,D-Orn,Tyr,D-Thr)(Glu,D-X)(Pro,Gln,D-Tyr)(Ile) Incomplete (X could be Val or Ala) B. amyloliquefaciens bm1
Iturin A (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Asn,Ser) Complete B. amyloliquefaciens bm1
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. amyloliquefaciens LS1-002-014 s
Fengycin A/B (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-X)(Pro,Gln,D-Tyr)(Ile) Incomplete (X could be Val or Ala) B. amyloliquefaciens LS1-002-014 s
Iturin A (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Asn,Ser) Complete B. amyloliquefaciens LS1-002-014 s
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. amyloliquefaciens HM618
Fengycin (……)(Tyr,D-Thr)(Glu,D-X)(Pro,Gln,D-Tyr)(Ile) Partial (X could be Val or Ala) B. amyloliquefaciens HM618
Iturin A (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Asn,Ser) Complete B. amyloliquefaciens HM618
Surfactin (Glu,Leu,…)(Val,Asp,…)(Leu) Partial and fragmented B. amyloliquefaciens B3
Fengycin (……)(…)(…,D-X)(Pro,Gln,D-Tyr)(Ile) Partial B. amyloliquefaciens B3
[Ile7] surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) Complete B. atrophaeus PENSV20
Fengycin A/B (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-X)(Pro,Gln,D-Tyr)(Ile) Incomplete (X could be Val or Ala) B. atrophaeus PENSV20
Iturin A (Asn)(D-Tyr,D-Asn,Gln,Pro)(…,Ser) Partial B. atrophaeus PENSV20
Kurstakin (D-Thr)(X,Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. bombysepticus F12
Kurstakin (D-Thr)(X,Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. bombysepticus Cuernavaca_S2
[Ile7] surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) Complete B. cellulasensis NJ-V2
[Ile7] surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) complete B. cellulasensis NJ-M2
Kurstakin (D-Thr)(X,Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. cereus CMCC P0011
Kurstakin (D-Thr)(X,Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. cereus CMCC P0021
Kurstakin (D-Thr)(X,Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. cereus NJ-W
Kurstakin (D-Thr)(X,Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. cereus C-1
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. halotolerans ZB201702
Fengycin (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-X)(Pro,Glu,…)(…) Partial (Glu instead of Gln and X could be Val or Ala) B. halotolerans ZB201702
Fengycin (…,D-Orn)(Tyr,D-Thr)(…,…)(Pro,…,…)(Ile) Partial and fragmented B. inaquosorum DE111
Surfactin (Glu,Leu,D-Leu)(Val,Asp,…)(Leu) Partial and fragmented B. inaquosorum DE111
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. inaquosorum LBA001
Fengycin (…,D-Orn)(Tyr,D-Thr)(Glu,…)(Pro,Glu,…)(Ile) Partial and fragmented (Glu instead of Gln) B. inaquosorum LBA001
Bacillomycin F (Asn)(D-Tyr,D-Asn,Pro,Gln)(D-Asn,Thr) Complete B. inaquosorum LBA001
Lichenysin A (Gln,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) Complete B. licheniformis SCDB 14
Lichenysin A (Gln,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) Complete B. licheniformis P8_B2
Lichenysin A (Gln,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) Complete B. licheniformis SCK B11
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. licheniformis 14ADL4
Fengycin (…)(…)(…,Glu,D-Tyr)(Ile) Partial and fragmented B. licheniformis 14ADL4
Fengycin (…,D-Orn)(Tyr,D-Thr)(…,D-X)(Pro,Glu,D-Tyr)(Ile) Partial and fragmented (Glu instead of Gln and X could be Val) B. mojavensis B-41341
Surfactin (Glu,Leu,…)(Val,Asp,…)(Leu) Partial and fragmented B. mojavensis B-41341
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. mojavensis B-41812
Fengycin B variant (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-X)(Pro,Glu,D-Tyr)(Ile) Incomplete (Glu instead of Gln and X could be Val) B. mojavensis B-41812
Kurstakin variant (D-Thr)(X,Ala)(Ser,Leu,D-Thr,Glu) Incomplete (Leu instead of His; Thr instead of Gln; Glu instead of Gln; X could be Gly) B. mycoides BGSC 4BQ1
Kurstakin (D-Thr)(X,Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. mycoides Gnyt1
Lichenysin A (Gln,Leu,D-Leu)(Val,Asp,…)(Ile) Partial and fragmented B. paralicheniformis UBBLI-30
Fengycin B (…,D-Orn)(Tyr,D-Thr)(…,D-Val)(Pro,Gln,D-Tyr)(Ile) Partial and fragmented B. paralicheniformis UBBLI-30
Lichenysin A (Gln,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) Complete B. paralicheniformis 14DA11
Fengycin B (Glu,…)(Tyr,D-Thr)(Glu,Val)(Pro,Gln,D-Tyr)(Ile) Partial and fragmented B. paralicheniformis 14DA11
Lichenysin A (Gln,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) Complete B. paralicheniformis CP47
Fengycin B (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-Val)(Pro,Gln,D-Tyr)(Ile) Complete B. paralicheniformis CP47
[Ile7] surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) Complete B. pumilus NJ-V
[Ile7] surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) Complete B. pumilus DSM 1794
[Ile7] surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Ile) Complete B. pumilus MS32
[Ile7] surfactin (Glu,Leu,D-Leu)(Val,Asp,…)(Ile) Partial B. pumilus B4127
[Ile4,7] surfactin (Glu,Leu,D-Leu)(Ile,Asp,D-Leu)(Ile) Complete B. safensis G6S3
Surfactin (Glu,Leu,D-Leu)(Val,Asp,Leu)(Leu) Complete B. spizizenii T30
Mycosubtilin (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Ser,Asn) Complete B. spizizenii T30
Mycosubtilin (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Ser,Asn) Complete B. spizizenii AS2
Surfactin (Glu,Leu,…)(Val,Asp,…)(Leu) Partial and fragmented B. spizizenii AS2
Mycosubtilin (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Ser,Asn) Complete B. spizizenii HUK15
Surfactin (Glu,Leu,…)(Val,Asp,…)(Leu) Partial and fragmented B. spizizenii HUK15
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. subtilis SJ-10
Fengycin B (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-Val)(Pro,Glu,D-Tyr)(Ile) Complete B. subtilis SJ-10
Bacillomycin D (Asn)(D-Tyr,D-Asn,Pro,Glu)(D-Ser,Thr) Complete B. subtilis SJ-10
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. subtilis MEC_B298
Fengycin B variant (…,…)(Tyr,D-Thr)(Glu,D-Val)(Pro,Glu,D-Tyr)(Ile) Partial (Glu instead of Gln) B. subtilis MEC_B298
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. subtilis s-16
Fengycin B variant (Glu,…)(Tyr,D-Thr)(Glu,D-Val)(Pro,Glu,D-Tyr)(Ile) Partial and fragmented (Glu instead of Gln) B. subtilis s-16
Fengycin B variant (Glu,…)(Tyr,D-Thr)(Glu,D-Val)(Pro,Glu,D-Tyr)(Ile) Partial and fragmented (Glu instead of Gln) B. subtilis BSP1
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. subtilis BSP1
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. subtilis KH2
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. subtilis FUA2231
Fengycin B variant (Glu,D-Orn,Tyr,D-Thr)(Glu,D-Val)(Pro,Glu,D-Tyr)(Ile) Complete (Glu instead of Gln) B. subtilis FUA2231
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. subtilis UD1022
Fengycin B variant (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-Val)(Pro,Glu,D-Tyr)(Ile) Complete (Glu instead of Gln) B. subtilis UD1022
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. subtilis SFA-H43
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. subtilis CGMCC 2108
Mycosubtilin (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Ser,Asn) Complete B. subtilis T30
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. subtilis T30
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. subtilis NCIB 3610
Fengycin (Glu,D-Orn)(Tyr,D-Thr)(…,…)(Pro,Glu,D-Tyr)(Ile) Partial and fragmented (Glu instead of Gln) B. subtilis NCIB 3610
Fengycin B variant (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-Val)(Pro,Glu,D-Tyr)(Ile) Complete (Glu instead of Gln) B. subtilis ZD01
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. subtilis ZD01
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. subtilis 73
Fengycin (…)(Glu,D-Tyr)(Ile) Partial and fragmented B. subtilis 73
[Ile7] surfactin (Glu,Leu,…)(Val,Asp,…)(Ile) Partial and fragmented B. subtilis G8
Fengycin (…)(…)(…,Glu,D-Tyr)(…) Partial and fragmented B. subtilis G8
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. subtilis FUA2232
Fengycin B variant (Glu,D-Orn,Tyr,D-Thr)(Glu,D-Val)(Pro,Glu,D-Tyr)(Ile) Complete (Glu instead of Gln) B. subtilis FUA2232
Surfactin (Glu,Leu, D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. subtilis SRCM103517
Fengycin B variant (Glu,D-Orn,Tyr,D-Thr)(Glu,D-Val)(Pro,Glu,D-Tyr)(Ile) Complete (Glu instead of Gln) B. subtilis SRCM103517
Surfactin (Glu,Leu,…)(Val,Asp,…)(Leu) Partial and fragmented B. subtilis S1
Kurstakin (D-Thr)(X,Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. thuringiensis HD12
Kurstakin (D-Thr)(X,Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. thuringiensis Bt185
Kurstakin (D-Thr)(X,Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. thuringiensis YWC2-8
Kurstakin (D-Thr)(X,Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. thuringiensis BGSC 4AA1
Kurstakin (D-Thr)(X,Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. thuringiensis HS18-1
Kurstakin (D-Thr)(X-Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. thuringiensis c25
Kurstakin (D-Thr)(X-Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. thuringiensis JW-1
Kurstakin (D-Thr)(X,Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. thuringiensis HD-1
Kurstakin (D-Thr)(X,Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. thuringiensis YBT-1518
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. vallismortis NBIF-001
Fengycin A/B (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-X)(Pro,Gln,D-Tyr)(Ile) Incomplete (X Could be Val or Ala) B. vallismortis NBIF-001
Iturin A (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Asn,Ser) Complete B. vallismortis NBIF-001
Fengycin B variant (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-Val)(Pro,Glu,D-Tyr)(Ile) Complete (Glu instead of Gln) B. vallismortis DSM 11031
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. vallismortis DSM 11031
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. velezensis ATR2
Fengycin (Glu,D-Orn)(Tyr,D-Thr)(……)(Pro,Gln…)(Ile) Partial and fragmented B. velezensis ATR2
Bacillomycin D (Asn)(D-Tyr,D-Asn,Pro,Glu)(D-Ser,Thr) Complete B. velezensis ATR2
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. velezensis SYP-B637
Fengycin A/B (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-X)(Pro,Gln,D-Tyr)(Ile) Incomplete (X could be Val or Ala) B. velezensis SYP-B637
Bacillomycin D (Asn)(D-Tyr,D-Asn,Pro,Glu)(D-Ser,Thr) Complete B. velezensis SYP-B637
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. velezensis CGMCC 11640
Fengycin A/B (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-X)(Pro,Gln,D-Tyr)(Ile) Incomplete (X could be Val or Ala) B. velezensis CGMCC 11640
Iturin A (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Asn,Ser) Complete B. velezensis CGMCC 11640
Surfactin (Glu,Leu,D-Leu)(Val,Asp,…)(Leu) Partial and fragmented B. velezensis Lzh-a42
Fengycin A/B (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-X)(Pro,Gln,D-Tyr)(Ile) Incomplete (X could be Val or Ala) B. velezensis Lzh-a42
Bacillomycin D (Asn)(D-Tyr,D-Asn,Pro,Glu)(D-Ser,Thr) Complete B. velezensis Lzh-a42
Fengycin A/B (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-X)(Pro,Gln,D-Tyr)(Ile) Incomplete (X could be Val or Ala) B. velezensis YJ0-1
Bacillomycin D (Asn)(D-Tyr,D-Asn,Pro,Glu)(D-Ser,Thr) Complete B. velezensis YJ0-1
Surfactin (Glu,Leu,D-Leu)(Val)(Asp,D-Leu)(Leu) Complete B. velezensis YJ0-1
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. velezensis L-S60
Fengycin (Glu,D-Orn)(Tyr,D-Thr)(……)(Pro,Gln,D-Tyr)(Ile) Partial and fragmented B. velezensis L-S60
Iturin A (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Asn,Ser) Complete B. velezensis L-S60
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. velezensis CBMB205
Fengycin A/B (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-X)(Pro,Gln,D-Tyr)(Ile) Incomplete (X could be Val or Ala) B. velezensis CBMB205
Iturin A (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Asn,Ser) Complete B. velezensis CBMB205
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. velezensis SB1216
Bacillomycin L (Asn)(D-Tyr,D-Asn,Ser,Glu)(D-Ser,Thr) Complete B. velezensis SB1216
Fengycin (………)(Tyr,D-Thr)(……)(Pro,……)(Ile) Partial and fragmented B. velezensis SB1216
Surfactin (Glu,Leu,…)(Val,Asp,D-Leu)(Leu) Partial and fragmented B. velezensis DSYZ
Fengycin A/B (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-X)(Pro,Gln,D-Tyr)(Ile) Incomplete (X could be Val or Ala) B. velezensis DSYZ
Iturin A (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Asn,Ser) Complete B. velezensis DSYZ
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. velezensis DR-08
Fengycin A/B (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-X)(Pro,Gln,D-Tyr)(Ile) Incomplete (X could be Val or Ala) B. velezensis DR-08
Iturin A (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Asn,Ser) Complete B. velezensis DR-08
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. velezensis CMF18
Fengycin A/B (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-X)(Pro,Gln,D-Tyr)(Ile) Incomplete (X could be Val or Ala) B. velezensis CMF18
Iturin A (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Asn,Ser) Complete B. velezensis CMF18
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. velezensis CK17
Fengycin A/B (Glu,D-Orn)(Tyr,D-Thr)(Glu,D-X)(Pro,Gln,D-Tyr)(Ile) Incomplete (X could be Val or Ala) B. velezensis CK17
Iturin A (Asn)(D-Tyr,D-Asn,Gln,Pro)(D-Asn,Ser) Complete B. velezensis CK17
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. velezensis DMB06
Bacillomycin D (Asn)(D-Tyr,D-Asn,Pro,Glu)(D-Ser,Thr) Complete B. velezensis DMB06
Fengycin (………)(…)(…,D-X)(Pro,…)(Ile) Partial and fragmented B. velezensis DMB06
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. velezensis UD6-2
Fengycin (……)(…)(…,D-X)(Pro…)(Ile) Partial and fragmented B. velezensis UD6-2
Bacillomycin D (Asn)(D-Tyr,D-Asn,Pro,Glu)(D-Ser,Thr) Complete B. velezensis UD6-2
Surfactin (Glu,Leu,D-Leu)(Val,Asp,D-Leu)(Leu) Complete B. velezensis DMB05
Fengycin (……)(……)(…X)(Pro,…)(Ile) Partial and fragmented B. velezensis DMB05
Bacillomycin D (Asn)(D-Tyr,D-Asn,Pro,Glu)(D-Ser,Thr) Complete B. velezensis DMB05
Kurstakin (D-Thr)(X,Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. wiedmannii JAS07/5
Kurstakin (D-Thr)(X,Ala)(Ser,Leu,D-X,Glu) Incomplete (Leu instead of His; Glu instead of Gln; X, X could be Gly and Gln respectively) B. wiedmannii PL1
[Ile4,7] surfactin (Glu,Leu,D-Leu)(Ile,Asp,D-Leu)(Ile) Complete B. safensis ZK-1
[Ile4,7] surfactin (Glu,Leu,D-Leu)(Ile,Asp,D-Leu)(Ile) Complete B. safensis SRCM125915
[Ile4,7] surfactin (Glu,Leu,D-Leu)(Ile,Asp,D-Leu)(Ile) Complete B. safensis PLA 1006

The monomers in brackets are those synthesized by a single gene. Monomers in bold differ from the known variants described in the same position. When the gene cluster was incomplete because scattered on several contigs or located on a too small contig, it was specify in the comment column and symbolized the truncated genes by “…”.

In silico analysis of the genomic sequences revealed the potential production of four lipopeptide families (Table 2). These are the surfactin, iturin, fengycin and kurstakin families depending on the strains. Over the 123 chromosomes analyzed, 75 (61%) possessed gene clusters responsible for surfactin production, 29 (23%) for iturin, 46 (37%) for fengycin and 19 (15%) for kurstakin (Table 3). No locillomycin synthetic gene cluster was detected in the genomes explored in this study. All chromosomes carrying kurstakin BGCs came from species belonging to the Bacillus cereus group. When this cluster was present, the other clusters (surfactin, fengycin, iturin) were absent. A kurstakin variant was detected in Bacillus mycoîde BGSC4BQ1 (Table 3, Figure 2). A variation was found in monomer 6, which is a threonine instead of Gln or Glu, which are most often found in this position. In addition, 25 chromosomes contained only clusters of surfactin genes. No chromosomes contained only fengycin or iturin gene clusters. A co-existence of gene clusters was also observed. As far surfactin and fengycin gene clusters are concerned, they were found in 21 chromosomes, while surfactin and iturin gene clusters were found in 4 chromosomes. A total of 25 chromosomes contained surfactin, fengycin and iturin gene clusters (Figure 3). In addition, six chromosomes contained four gene clusters (surfactin, fengycin, iturin, and novel gene clusters). All B. subtilis, B. velezensis, B. licheniformis, B. paralicheniformis, B. amlyloliquefaciens, B. pumilus, B. cellulasensis, B. altitudinis, B. spizizenii, B. safensis, B. vallismortis, B. halotolerans, B.inaquosorum, B. stratosphericus, B. aerophilus and B. atrophaeus have gene clusters to produce a known surfactin or variant. In addition, all B. licheniformis and B. paralicheniformis have gene clusters which produce lichenysin A (Table 3). All 29 iturin-producing strains belong to the B. subtilis group (B. subtilis, B. velezensis, B. amyloliquefaciens, B. spizizenii, B. atrophaeus). Gene clusters for the biosynthesis of bacillomycin D and bacillomycin L, variants of iturin were detected in the chromosomes of B. subtilis, B. amyloliquefaciens and B. velezensis species. In species such as B. atrophaeus and B. spizizenii, chromosomes possess gene clusters producing iturin A and mycosubtilin, respectively. For the fengycin family, A or B production gene clusters were detected in 1/3 of the chromosomes of producing strains. In fact, the 6th monomer, which may be Val in the case of fengycin B or Ala in the case of fengycin A, was not predicted during the study. A fengycin B variant was detected in the genomes of 10 strains belonging to the B. subtilis species (Table 3). However, all the genomes of B. paralicheniformis strains contained clusters of fengycin B biosynthesis genes (Table 3). The D-allothreonine observed in fengycin B is predicted to be a D-threonine by antiSMASH because these two threonine isomers can be selected by the same A-domain. A small difference is noticed in the beta carbon. Beta carbon of D-allothreonine (CαD, CβD) belongs to the D series whereas the beta carbon of D-threonine (CαD, CβL) belongs to the L series.

Organization of the predicted novel NRPSs and NRPS/PKS. Iterative domains: A, adenylation domain; C, condensation domain; CP, Carrier Protein; E, epimerization domain; TE, thioesterase domain; AT, acetyltransferase domain; KS, ketosynthetase domain; KR, ketoreductase domain; CAL, Coenzyme A ligase; C surrounded in light blue, condensation domain allowing condensation between a PKS monomer and a NRPS monomer. Predicted amino acid specificity is shown under each A domain. (a) a tetrapeptide in B. cereus CMCCP0011 (plasmid) and B. cereus CMCCP021 (plasmid); (b) a hexapeptide in B. velezensis ATR2 (chromosome); (c) a heptapeptide in B. velezensis DSYZ (chromosome), B. subtilis SJ-10 (chromosome) and B. velezensis CGMCC 11640 (chromosome); (d) a heptapeptide in B. amyloliquefaciens HM618 (chromosome); (e) a octapeptide in B. cereus CMCC P0011 (plasmid) and B. cereus CMCC P0021 (plasmid); (f) a heptapeptide in B. anthracis CMF9 (chromosome); (g) a hexapeptide in B. amyloliquefaciens WF02 (chromosome), B. velezensis CGMCC 11640 (chromosome) and B. velezensis DSYZ (chromosome); (h) predicted variant of kurstakin in B. mycoide BGSC 4BQ1, the difference with the other kurstakin predicted concerning the composition amino acids is located at the level of monomer 6; (i) predicted variant of fengycin B in B. vallismortis DSM 11031, B. subtilis UD1022, B. subtilis FUA2231, B. subtilis FUA2232, B. subtilis SRCM103517 and B. subtilis ZD01 genomes. Modular architecture is similar to the known fengycin B, the difference with fengycin B regarding the composition in amino acids is located at the level monomer 8.

Prevalence and coexistence of lipopeptide biosynthesis gene clusters in 123 chromosomes analysis from Bacillus (Venn diagram, n = 75).

 Prevalence of BGC NRPSs in chromosomes of the different species

The 123 strains represent 33 species with distributions as follows: 19 species were found in the soil samples only, 12 species were found in both soil and fermented food isolation media and 2 species were found only in the fermented food. Bacillus species that were only found in soil samples included B. albus, B. arachidis, B. atrophaeus, B. badius, B. bombysepticus, B. cellulasensis, B. gobiensis, B. haikouensis, B. halotolerans, B. methanolicus, B. mycoides, B. inaquosorum, B. stratosphericus, B. thuringiensis, B. toyoninsis, B. tropicus, B. anthracis, B. vallismortis and B. aerophilus (Figure 4A). Species found in soil and fermented foods were: B. licheniformis, B. paralicheniformis, B. paranthracis, B. pacificus, B. pumilus, B. safensis, B. spizizenii, B. subtilis, B. velezensis, B. wiedmannii, B. amyloliquefaciens and B. cereus. Two species, B. altitudinis and B. infantis, were only found in fermented foods (Figure 4B). Among genomes of soil Bacillus species, only B. atrophaeus had a gene cluster for iturin biosynthesis. Furthermore, only the genome of B. thuringiensis species carried a gene clusters for the biosynthesis of the antibiotic bacitracin and a new gene cluster. Analysis of genomes of the 12 species found in isolation media (soil and fermented food) revealed the presence of new gene clusters in species such as B. velezensis, B. amyloliquefaciens, B. subtilis and B. cereus. Prevalence of new gene clusters was higher in B. velezensis and B. amyloliquefaciens, respectively (Figure 4B) and the genomes of these two strains carried 3 new BGC NRPSs. The genome of B. infantis, a species that was only present in fermented foods had no NRPSs gene cluster (Figure 4B). Moreover, bacitracin biosynthesis genes were carried only by the genomes of B. paralicheniformis and B. thuringiensis. Genomes of B. pacificus, B. cereus, B. wiedmannii and B. paranthracis lacked surfactin biosynthesis genes (Figure 4).

Production capacity of known BGC NRPSs and new gene clusters depending on the species of the genus Bacillus. Strains in (A) are only isolated from soil samples, but in (B), apart from B. infantis and B. altitudinis which are only present in fermented food samples, the other strains can be found in soil or fermented foods. These two, which are only present in fermented food samples, are shown with asterisk in (B).

 Bacitracin production

Among the 123 genomes screened, only 4 (B. paralicheniformis 14DA11, B. paralicheniformis CP47, B. paralicheniformis UBBLI-30, and B. thuringiensis Bt185) bear BGC corresponding to the synthetic pathway for antibiotic bacitracin. This antibiotic is a semi-cyclic peptide constituted of 12 amino acids. In B. paralicheniformis CP47 chromosome sequence, a BGC encoding an NRPS containing 12 modules was found. These 12 modules correspond exactly to the 12 modules of bacitracin synthetase. In the genomes of B. thuringiensis Bt185, B. paralicheniformis 14DA11, and B. paralicheniformis UBBLI-30, modular structuring and amino acid composition were similar to bacitracin A1 (Figure 1), similarities ranged from 85 to 100%.

 BGC potentially producing new metabolites

A total of 7 new molecules carried by 7 new gene clusters were detected in this study, based on the exploration of 123 complete genomes of strains belonging to the Bacillus genus and isolated either from fermented foods or from soil. These newly identified gene clusters would be capable of producing other new lipopeptide families and new antibiotics. All newly predicted molecules were first compared with those identified subsequently, and then with the non-ribosomal peptides available in the Norine database. Low similarities ranged from 28.6 to 50%. The first new gene cluster detected consisted of 3 genes and 4 modules incorporating Thr, X, Asn and Asn monomers, respectively (Figure 2). Amino acid incorporated by module 2 was not detected, hence the letter X at this position (Figure 2). The predicted tetrapeptide showed a low similarity of 39.4% to Cis-7-tetradecenoyl-D-Asparagine. This new gene cluster was carried by plasmids from B. cereus CMCC P0011 and B. cereus CMCC P0021 strains. Two new gene clusters consisting of 6 modules each were detected by exploring the genomes of B. velezensis ATR2, B. velezensis DSYZ, B. velezensis CGMCC 11640 and B. amyloliquefaciens WF02. The hexapeptide predicted in the B. velezensis ATR2 genome was structured as follows: D-Cys-Ser-Cys-Ala-X-D-Asn, and contained a CAL (Co-enzyme A ligase) domain. The hexapeptide predicted in the genomes of B. velezensis DSYZ, B. velezensis CGMCC 11640 and B. amyloliquefaciens WF02 was structured: Val-D-Phe-Asp-D-Asn-Gly-D-Glu. Formation is ensured by the combination of an NRPS system and a PKS (polyketide synthase) system (Figure 2). CAL and C-starter domains were not detected in this hexapeptide. Therefore, it is probably an antibiotic. Literature searches and structural analyses in Norine did not reveal any similar molecules.

In silico analyses also revealed the presence of 3 new NRPS clusters capable of producing 3 different heptapeptides depending on the modular organization and amino acid composition of each. The first predicted heptapeptide detected was carried by B. velezensis DSYZ and B. subtilis SJ-10 genomes and is structured as follows: D-Cys-Ser-Cys-Ala-X-Asn-D-Asn (Figure 2). Monomer 5 has not been predicted. A CAL domain was detected at the start of the peptide chain (Figure 2). Structural analysis of this heptapeptide in Norine showed little similarity to iturin A1 (32.3%). Furthermore, literature searches revealed no molecules with a similar structure. For the last two predicted heptapeptides, each gene cluster consists of 7 modules incorporating 7 amino acids. One had the following structure: D-Phe-D-Leu-Phe-D-Thr-Val-Ala-Thr and was carried by the B. amyloliquefaciens HM618 genome while the other had the following structural architecture: D-Phe-Tyr-Ile-X-D-Phe-Leu-Leu and was carried by the B. anthracis CMF9 genome (Figure 2). Their structures were similar to kahalalide A (39.4%) and axinastatin 5 (40%) respectively. The heptapeptide identified in the B. amyloliquefaciens HM618 genome contained a thioesterase (TE) domain marking the end of the peptide chain. The heptapeptide identified in the B. anthracis genome contained a CAL domain at the start of the peptide chain and a C condensation domain at the end of module 7 (Figure 2).

Exploration of the plasmid sequence of B. cereus CMCCPOO11 and B. cereus CMCCPOO21 revealed a BGC probably responsible for the synthesis of a new octapeptide nonribosomal peptide (Figure 2). The chemical structure of unpredictable monomers are represented by the letter X in the peptide chain. The structure search on Norine showed little similarity (50%) with cyanostatin B. No molecules with a similar structure were found in the literature.

 Discussion

Bacillus species are known to produce a large variety of secondary metabolites. This production is influenced by environmental conditions. Thus, this study explored 123 complete genomes of Bacillus isolated from soil and fermented foods. Thus, in silico analysis of chromosomal and plasmid sequences using bio-informatics tools specific to non-ribosomal peptides revealed a potential gene cluster responsible for the biosynthesis of lipopeptides (sufactin, fengycin, iturin, kurstakin), the antibiotic bacitracin and the siderophore bacillibactin. Only B. subtilis 916 (Luo et al., 2015a) produced locillomycin, which was not detected in this study. However, surfactins are lipoheptapeptides with variants such as esperin, lichenysin, pumilacidin and surfactin (Waongo et al., 2023). Two distinct amino acids were predicted to be incorporated in the first module: either a glutamate (Glu) or a glutamic acid (Gln) (Hu et al., 2019). As for iturins, they are lipoheptapeptides whose main variants are: iturins A and C, bacillomycins D, F and L, mycosubtilin and mojavensin (Waongo et al., 2023). Bacillibactin is a siderophore whose sequence is Dhb-Gly-Thr (May et al., 2001). As a result, the modular organization and monomer composition of predicted peptides from the surfactin and iturin groups as well as bacillibactin, were similar to literature data. Compared with fengycin, the lipodecapeptide predicted in genomes of B. subtilis UD1022, B. vallismortis DSM11031, B. subtilis ZD01, B. halotolerans ZB201702, B. mojavensis B-41812, B. mojavensis B-41341, B. subtilis MEC_B298, B. subtilis s-16, B. subtilis FUA2231, B. subtilis FUA2232, B. subtilis SRCM103517, and B. subtilis BSP1, contains at position 8 a glutamic acid (Glu). In contrast, various authors have reported that module 8 incorporates glutamine (Gln) during peptide chain formation (Ait Kaki et al., 2020; Hussein, 2019). This observed variability shows that certain Bacillus could produce a fengycin B variant. Also, structural analysis of predicted kurstakins shows the presence of a threonine (Thr) at position 6 in the B. mycoide BGSC 4BQ1 genome. Generally, the amino acid occupying this position in the case of kurstakin is glutamine (Gln) (Béchet et al., 2012). Although monomers 2 and 6 have not been predicted in the genomes of other B. cereus group species (B. thuringiensis, B. cereus, B. wiedmannii, B. bombysepticus), it is quite possible that B. mycoide BGSC 4BQ1 would be capable of producing a kurstakin variant. Nevertheless, the two putative variants identified through this in silico screening approach will need to be further investigated through in vitro experiments.

This study revealed the coexistence of lipopeptide biosynthesis genes. Indeed, Luo et al. (2015b) reported the coexistence of surfactin (sfr), bacillomycin (bmy), fengycin (fen) and locillomycin (Loc) gene clusters in B. subtilis 916 genome. In this study, B. amyloliquefaciens WF02, B. amyloliquefaciens HM618, B. velezensis ATR2, B. velezensis CGMCC 11640, B. velezensis Lzh-a42, B. subtilis SJ-10 and B. velezensis DSYZ genomes also contained three known lipopeptide biosynthesis gene clusters and novel gene cluster that could produce a new lipopeptide family. To our knowledge, no study has demonstrated the coexistence of four or five gene clusters of lipopeptide in genomes of species such as B. amyloliquefaciens and B. velezensis. According to Luo et al. (2015b), a co-production of surfactin, fengycin, iturin and locillomycin by B. subtilis 916 is at the origin of its inhibitory capacity against multi-resistant Staphylococcus aureus. Similarly, the co-production of lipopeptides would reduce hemolytic activity of the producing strains (Luo et al., 2019; Waongo et al., 2023).

The emergence of multi-resistant strains to commonly used antibiotics is a major challenge (Eduardo-Correia et al., 2020). The exploration of new molecules that could serve as alternatives remains a necessity. Thus, the study identified seven new gene clusters (NRPS and NRPS/PKS), two of which were carried by plasmid sequences. These new gene clusters were responsible for the biosynthesis of a tetrapeptide, two hexapeptides, three heptapeptides and octapeptide. The predicted tetrapeptide and octapeptide were synthesized by gene clusters carried by plasmids. The structural architecture and monomer composition of all predicted molecules differed from the peptides available in the database (Flissi et al., 2023). Furthermore, literature searches did not reveal any molecules with similar structure and monomer composition. Except for the hexapeptide predicted in the chromosomes of B. velezensis DSYZ and B. amyloliquefaciens WF02, all the others carried a CAL or C-starter domain in the first module. In fact, lipopeptides are made up of chains of amino acids and fatty acids. Their structure is characterized by the presence of C-starter and CAL domains, which play a role in fatty acid chain activation (Ongena and Jacques, 2007). Thus, their presence in the first module of the seven predicted new peptides means that they could belong to the lipopeptide family. However, certain antibiotics of purely peptidic clinical interest, such as vancomycin and bacitracin, are characterized by the presence of an adenylation domain (A) in the first module (Konz et al., 1997; Wageningen et al., 1998). This A domain is responsible for selecting and activating the first amino acid to be integrated into the growing peptide chain. Consequently, the new hexapeptide predicted in the chromosomes of B. velezensis DSYZ and B. amyloliquefaciens WF02 would be a peptide antibiotic. Esmaeel et al. (2016) reported the synthesis of a new lipopeptide by analyzing the genomes of genus Burkholderia. On the other hand, since lipopeptides are synthesized non-ribosomally, the presence of gene clusters responsible for their synthesis in plasmids is rare. According to the literature, no study has revealed the presence of NRPS lipopeptide synthase genes on a Bacillus plasmid, only on the Burkholderia plasmid (Esmaeel et al., 2016). As a result, the putative new BGCs would constitute new families of lipopeptides as well as new antibiotics, which could have unique and interesting biological properties (e.g., antifungal, antibacterial, antiviral). Biomolecules newly predicted in this study could be at the center of current research in the interest of their future use in several fields such as agri-food, cosmetics and medicine. The results of this study reveal that the genomes of Bacillus strains available in databases contain many unknown molecules that could play important roles in antimicrobial control. However, it would be interesting to elucidate the biological functions of all these new molecules. Although the bioinformatic discovery of novel molecules often captures attention, rigorous experimental validation remains the crucial step in translating promising ideas into tangible scientific findings and ensuring genuine progress.

 Conclusion

Exploration of microorganisms genomes allows for the rapid identification of gene clusters located on chromosomes coding for new beneficial molecules. Thus, in addition to chromosomes, it would be interesting to analyze plasmids that could have BGC clusters of NRPs or lipopeptides. Then, this study allowed us to identify seven new gene clusters synthesizing new non-ribosomal peptides. Our results suggest that several NRPs capable of being produced by Bacillus strains isolated from fermented foods and soil are still uncharacterized and their properties still unknown. Therefore, targeted research should be conducted experimentally to validate these different predicted molecules.

 Data availability statement

Genome mining work was done base on the whole genome sequences of 123 Bacillus strains is listed in Table 1, which can be obtained from NCBI nucleotide database (https://www.ncbi.nlm.nih.gov/nuccore). All the other data supporting the findings is contained within the manuscript.

 Ethics statement

The manuscript presents research on animals that do not require ethical approval for their study.

 Author contributions

BW: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing. LN: Investigation, Methodology, Writing – review & editing. FT: Methodology, Writing – review & editing. W-SAZ: Methodology, Writing – review & editing. JL: Supervision, Writing – review & editing. AS: Supervision, Writing – review & editing.

 Funding

The author(s) declare that no financial support was received for the research and/or publication of this article.

We would like to thank University Joseph KI-ZERBO of Burkina Faso and Huazhong Agricultural University of China for their financial support.

 Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Generative AI statement

The author(s) declare that no Gen AI was used in the creation of this manuscript.

 Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

1https://www.ncbi.nlm.nih.gov/nuccore

2https://bioinfo.cristal.univ-lille.fr/norine

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