*3.7. Isolation of Micrococcin P1 (1) and Micrococcin P2 (2)*

The active fraction of *B. marisflavi* JC556 (LS974830.1) was cultivated overnight and fractionated according to the respective OD600 (0.01 for bacteria and OD548 0.1 for yeast and fungi) using maXis 2 G (BRUKER DALTONICS, Germany); the retention times of the entire 96-well plate were recorded. Fractions with the lowest absorbances were considered to be the most active. LCMS-MS fragmentation of the compounds with the retention times of 9.43 min and 9.67 min, respectively, was realized with maXis 4G (Bruker, Germany). Generally, the methods of Okanya et al. were adopted [41]. Accordingly, HREIMS data were recorded on a maXis spectrometer (BRUKER DALTONICS, Germany). Molecular formulae were identified by including the isotopic pattern in the calculation (SmartFormula algorithm). Analytical RP HPLC utilised an Agilent 1100 HPLC system (AGILENT TECHNOLOGIES, Waldbronn, Germany) equipped with a UV diode-array detector and an evaporative light-scattering (PL-ELS 1000) detector. HPLC conditions were column 125 × 2 mm, Nucleodur C-18, and 5 μm (MACHEREY-NAGEL, Düren, Germany); solvent A, 5% acetonitrile in water, 5 mmol of NH4Ac, and 0.04 mL of CH3COOH; solvent B, 95% acetonitrile, 5 mmol of NH4Ac, and 0.04 mL of CH3COOH; gradient system, 10% B increasing to 100% in 30 min, 100% B for 10 min, to 10% B post-run for 10 min; 40 ◦C; and flow rate of 0.3 mL/min. Dereplication of these compounds along with the Dictionary of Natural products were used to identify the known antibiotics micrococcin P1 (**1**) and micrococcin P2 (**2**) from a strain of *B. marisflavi*.

#### **4. Conclusions**

The Kenyan isolate of "*Lyngbya majuscula*", also known as *Moorea producens* and recently renamed Moorena, was shown to be phylogenetically distinct from *L. majuscula* CCAP 1446/4 and *L. majuscula* clones. Surprisingly, epibiotic bacteria growing on the surface of *M. producens*' filaments were often found to be human pathogens. A new method for the isolation of genomic DNA (gDNA) from non-axenic filamentous marine cyanobacteria based on the CuSO4·5H2O assisted differential isolation of bacteria gDNA from a cyanobacteria biomass was reported. Debromoaplysiatoxin (DAT), which is an indicator toxin found earlier in *M. producens*, was not detected in the Kenyan strain of *M. producens*. The phylogenetic divergence, absence of DAT, and the detection of unique cyclodepsipeptides in the Kenyan strain of *M. producens* reinforce a need for molecular identification of non-axenic *M. producens* species worldwide. The IC50 of micrococcin P1 against *S. aureus* was within the range of values reported in literature. Our results showed that the genetic basis for synthesizing micrococcin P1 (**1**), originally discovered in *Bacillus cereus* ATCC 14579, is often species or strain dependent and provides evidence for *M. producens*-associated bacteria as an underexplored source of bacterial strains for the discovery of new antibiotics to fight drug resistance.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/md20020128/s1, Figure S1: Denaturation of *Moorea producens* DNA; Figure S2: Blasted sequences; Figure S3: Micrococcin P1 and P2 chromatogram (MS and UV at 220 nm); Figure S4: Micrococcin P1 MS/MS spectrum; Figure S5: Micrococcin P1 annotated MS/MS spectrum; Figure S6: Micrococcin P2 MS/MS spectrum; Figure S7: Micrococcin P2 annotated MS/MS spectrum; Figure S8: Activity of micrococcin P1 against *S. aureus* Newman.

**Author Contributions:** T.D. designed and performed the experiments and wrote the manuscript. M.J.H. and J.G.B. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by an EU-FP7 Marie Sklowdowska Curie IIF award to T.D. through his project Lyngbya-KENYA 29950 carried out at Newcastle University, United Kingdom (2012–2014) grant number Lyngbya-KENYA 29950; and by a fellowship to T.D. at the Helmholtz Institute for Pharmaceutical Research (HIPS), Saarland, Germany (2018–19) from the Organisation for the Prohibition of Chemical Weapons (OPCW) of the Hague, The Netherlands, grant number L/ICA/ICB/216416/18. The APC was funded by the Western Indian Ocean Marine Sciences Association (WIOMSA).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** We acknowledge Rebecca Goss of the University of St Andrews, Scotland, UK, for the LCMS analyses, Rolf Mueller of HIPS for providing his lab and facilities to T.D and Brian Whitton of Durham University, UK for confirming the identity of *M. producens*' stalked diatoms.

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