*4.4. Antibacterial Testing*

Bacillimidazoles A–F (**1**–**6**) were tested for antibacterial activity against *E. coli* (ATCC #25922), *B. subtilis* strain NRS-231, and Methicillin-resistant *Staphylococcus aureus* (MRSA) (ATCC #33591), and MICs were determined using a dilution antimicrobial susceptibility test for aerobic bacteria. Compounds **1**–**6** were dissolved in DMSO and serially diluted to 10 concentrations (0.25–128 μg/mL) in 96-well plates. Vancomycin was used as a positive control against *B. subtilis* and MRSA, and exhibited MIC values of 0.25 μg/mL. Gentamicin was used as a positive control against *E. coli*, and exhibited an MIC of 4 μg/mL. Bacillimidazoles, vancomycin, and gentamicin were tested in triplicate. On each plate, there were six untreated media controls. The plates were incubated at 37 ◦C for 18 h. The MICs were determined as the lowest concentration that inhibited visible growth of bacteria.

#### *4.5. Sequencing and Identification of Candidate Bacillimidazole Biosynthetic Genes*

16S rDNA sequencing was conducted as previously described [27]. WMMC1349 was identified as a *Bacillus* sp. The 16S sequence for WMMC1349 was deposited in GenBank (accession number MK892477). PacBio sequencing data were converted from BAM to FASTQ format using bedtools [28], and this fastq file was then corrected, trimmed, and assembled using Canu v1.8 [29], with an estimated genome size of 4.5 megabases (Mb). The resulting assembly was 4.677Mb over 10 contigs. It has an N50 of 4.03Mb and an L50 of 1.

Genome sequence (accession number JABJUQ000000000) was subjected to antiSMASH 5.0 analysis [30]. Results were analyzed by BLAST analysis. Acetoin and amino acid biosynthetic genes (KEGG) were identified in the bacillimidazole producer and selected model *Bacillus* spp. By BLAST analysis using Geneious 11.1.3 [31], and verified using additional online platforms such as Phyre2 [32]. Genomes of the producer of **1**–**6** and selected model *Bacillus* spp. were aligned using the MAUVE algorithm [33]. Promoter regions were identified using the BPROM algorithm [34].

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/md20010043/s1. Detailed experimental discussions of isotopic enrichment studies and validation of bacillimidazoles as genuine natural products as well as Tables S1 & S2 summarizing all antibacterial data (Table S1), and bacillimidazole productivity of six *Bacilli* strains (Table S2). Also: Figure S1. Analytical HPLC analysis and 1H NMR analysis of active subfractions. Figure S2. LC-MS analysis (EIC *m/z* 383) of 3. Figure S3. LC-MS analysis (EIC *m/z* 397) of 4. Figure S4. LC-MS analysis of chemical reaction of tryptamine (10.0 mg/mL) and 2,3 butanedione (0.5 equivalent) in MeOH solution without HP-20 resin for 2 h. Figure S5. LC-MS analysis of chemical reaction of tryptamine (10.0 mg/mL) and 2,3 butanedione (0.5 equivalent) in MeOH solution with HP-20 resin for 2 h. Figure S6. LC-MS analysis of chemical reaction of tryptamine (10.0 mg/mL) and 2,3 butanedione (0.5 equivalent) in CHCl3:MeOH = 1:1 solution for 24 h. Figure S7. 1H NMR Spectrum of Bacillimidazole A (1, 600 MHz, CD3OD). Figure S8. 13C NMR Spectrum of Bacillimidazole A (1, 125 MHz, CD3OD). Figure S9. gCOSY Spectrum of Bacillimidazole A (1, 600 MHz, CD3OD). Figure S10. gHSQC Spectrum of Bacillimidazole A (1, 600 MHz, CD3OD). Figure S11. gHMBC Spectrum of Bacillimidazole A (1, 600 MHz, CD3OD). Figure S12. 1H NMR Spectrum of Bacillimidazole B (2, 600 MHz, CD3OD). Figure S13. 13C NMR Spectrum of Bacillimidazole B (2, 125 MHz, CD3OD). Figure S14. gCOSY Spectrum of Bacillimidazole B (2, 600 MHz, CD3OD). Figure S15. gHSQC Spectrum of Bacillimidazole B (2, 600 MHz, CD3OD). Figure S16. gHMBC Spectrum of Bacillimidazole B (2, 600 MHz, CD3OD). Figure S17. 1H NMR Spectrum of Bacillimidazole C (3, 600 MHz, CD3OD). Figure S18. 13C NMR Spectrum of Bacillimidazole C (3, 125 MHz, CD3OD). Figure S19. gCOSY Spectrum of Bacillimidazole C (3, 600 MHz, CD3OD). Figure S20. gHSQC Spectrum of Bacillimidazole C (3, 600 MHz, CD3OD). Figure S21. gHMBC Spectrum of Bacillimidazole C (3, 600 MHz, CD3OD). Figure S22. 1H NMR Spectrum of Bacillimidazole D (4, 500 MHz, CD3OD). Figure S23. 13C NMR Spectrum of Bacillimidazole D (4, 125 MHz, CD3OD). Figure S24. gCOSY Spectrum of Bacillimidazole D (4, 500 MHz, CD3OD). Figure S25. gHSQC Spectrum of Bacillimidazole D (4, 500 MHz, CD3OD). Figure S26. gHMBC Spectrum of Bacillimidazole D (4, 500 MHz, CD3OD). Figure S27. 1H NMR Spectrum of Bacillimidazole E (5, 600 MHz, CD3OD). Figure S28. 13C NMR Spectrum of Bacillimidazole E (5, 125 MHz, CD3OD). Figure S29. gCOSY Spectrum of Bacillimidazole E (5, 600 MHz, CD3OD). Figure S30. gHSQC Spectrum of Bacillimidazole E (5, 600 MHz, CD3OD). Figure S31. gHMBC Spectrum of Bacillimidazole E (5, 600 MHz, CD3OD). Figure S32. 1H NMR Spectrum of Bacillimidazole F (6, 500 MHz, CD3OD). Figure S33. 13C NMR Spectrum of Bacillimidazole F (6, 125 MHz, CD3OD). Figure S34. gCOSY Spectrum of Bacillimidazole F (6, 500 MHz, CD3OD). Figure S35. gHSQC Spectrum of Bacillimidazole F (6, 500 MHz, CD3OD). Figure S36. gHMBC Spectrum of Bacillimidazole F (6, 500 MHz, CD3OD). Figure S37. Positive Ion HRESIMS of Bacillimidazole A (1). Figure S38. Positive Ion HRESIMS of Bacillimidazole B (2). Figure S39. Positive Ion HRESIMS of Bacillimidazole C (3). Figure S40. Positive Ion HRESIMS of Bacillimidazole D (4). Figure S41. Positive Ion HRESIMS of Bacillimidazole E (5). Figure S42. Positive Ion HRESIMS of Bacillimidazole F (6). Figure S43. 1H NMR Spectrum of 13C Labeled Bacillimidazole C (3, 500 MHz, CD3OD). Figure S44. 13C NMR Spectrum of 13C Labeled Bacillimidazole C (3, 125 MHz, CD3OD). Figure S45. 1H NMR Spectrum of 13C Labeled Bacillimidazole E (5, 500 MHz, CD3OD). Figure S46. 13C NMR Spectrum of 13C Labeled Bacillimidazole E (5, 125 MHz, CD3OD). Figure S47. Positive Ion HRESIMS of 13 C Enriched Bacillimidazole C (3). Figure S48. Positive Ion HRESIMS of 13C Enriched Bacillimidazole E (5). Figure S49. LC-MS analysis of strain *Bacillus* sp. WMMC325. Figure S50. LC-MS analysis of strain *Bacillus* sp. WMMC331. Figure S51. LC-MS analysis of strain *Bacillus* sp. WMMC1349. Figure S52. LC-MS analysis of strain *Bacillus* sp. WMMC1350. Figure S53. LC-MS analysis of strain *Bacillus* sp. WMMC1351. Figure S54. LC-MS analysis of strain *Bacillus* sp. WMMC1352. Figure S55. Culture broth of six different marine *Bacillus* strains. Figure S56. Pictures for sponge hosts of six different marine *Bacillus* strains.

**Author Contributions:** Conceptualization, C.R.C., J.C. and T.S.B.; methodology, J.-X.Y., Q.W., J.C. and T.S.B.; formal analysis, J.-X.Y., Q.W., E.J.N.H., M.G.C., H.H.; investigation, J.-X.Y., Q.W., E.J.N.H., M.G.C., D.R.B., G.E.A.; resources, H.H. and T.S.B.; writing—original draft preparation, J.-X.Y., Q.W., E.J.N.H. and S.R.R.; writing—review and editing, Q.W., S.R.R., J.C., M.G.C. and T.S.B.; supervision, C.R.C., J.C. and T.S.B.; project administration, C.R.C., J.C. and T.S.B.; funding acquisition, C.R.C., J.C. and T.S.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by NIH Grants U19AI109673, U19AI142720, and R01AT009874 in addition to the University of Wisconsin-Madison School of Pharmacy and the Graduate School at the University of Wisconsin. E.J.N.H. gratefully acknowledges funding by the Swiss National Science Foundation (Postdoctoral Mobility Fellowship). We would like to thank the Analytical Instrumentation Center at the School of Pharmacy, University of Wisconsin-Madison for the facilities to acquire spectroscopic data. This study made use of the National Magnetic Resonance Facility at Madison, which is supported by NIH gran<sup>t</sup> P41GM103399 (NIGMS). Additional equipment was purchased with funds from the University of Wisconsin, the NIH (RR02781, RR08438), the NSF (DMB-8415048, OIA-9977486, BIR-9214394), and the USDA.

**Conflicts of Interest:** The authors declare no conflict of interest.
