*4.3. Strain Identification*

DNA was extracted from cultures using the ZymoBIOMICS Fecal/Soil Kit (Zymo Research, Irvine, CA, USA) following manufacturer's instructions including a 3 min disruption of cells using ceramic beads. Concentrations were measured using Nanodrop and Qubit. Fragments of the 16S

rRNA-encoding gene were amplified by PCR for 35 cycles using two primer sets commonly used to specifically amplify cyanobacterial genes. Annealing temperature of 58 ◦C was used for primer set 8F (5'-AGAGTTTGATCCTGGCTCAG 3') and 920R (5--TTGTAAGGTTCTTCGCGTTG-3'), and annealing at 55 ◦C was used for primer set 861F (5--TAACGCGTTAAGTATCCC-3-) and 1380R (5--TAACGACTTCGGGCGTGACC-3-) [27,28]. For each strain, sequence chromatograms (Genoscreen, Lille, France) were examined, assembled using Geneious (https://www.geneious.com/), and compared to the GENBANK database using BLAST. Sequences are deposited in GENBANK under accession numbers MN823169 to MN823186; MN824246 and MN824247.

A dataset was built consisting of sequences from the 20 isolated strains, their best BLAST hits, and representatives of major cyanobacterial lineages. Sequences from genus *Gloeobacter* were used as an outgroup. Sequences were aligned using the secondary structure-aware Infernal Aligner v. 1.0 tool available on the Ribosomal Database Project website [29]. Alignment was controlled to remove ambiguously-aligned zones. Phylogenetic tree was reconstructed using the software MEGA7 [30]. Relationships were inferred using a Maximum Likelihood approach using a General Time Reversible model (5 categories and invariants), and 1280 nucleotide positions. Support values at nodes were obtained from 100 boostrap replicates analyzed using the same method. A pairwise p-distance matrix (Table S1) was built to support preliminary genus and species delimitation.

### *4.4. Preparation of Cyanobacteria Chemical Extracts*

An aliquot each culture was deposited in a 15 mL Falcon tube containing 10 mL of unsalted Z8 medium. After centrifugation at 4000 g and three successive washes with unsalted Z8 medium, the pellets were lyophilized under vacuum at −40 ◦C for 12 h. The dry extracts were weighed and then re-suspended in a MeOH/CH3CN/H2O mixture (40:40:20) for having a final concentration of 1 mg /100 μL of solvent mixture. After three successive sonications (6 min cycle: 1 min ON/30 s OFF) and centrifugations at 14,000 g for 5 min, the supernatants were evaporated and the dry extracts were prepared for having a final concentration of 1 mg/mL and were then filtered on a membrane of 13 mm in diameter and 0.2 μm in pores (VWR International). An aliquot of 30 μL was reserved for LC-MS<sup>2</sup> analysis. The remaining samples were evaporated and diluted in DMSO at a concentration of 10 μg/μ<sup>L</sup> for antibacterial activities evaluation.

### *4.5. LC-MS<sup>2</sup> Analyzes of Extracts*

The extracts were subjected to an Agilent 1260 HPLC (Agilent Technologies, Les Ulis, France) coupled to an Agilent 6530 Q-ToF-MS equipped with a Dual ESI source. The chromatographic separation was performed using an HPLC (C18 Sunfire ® Waters 150 × 2.1 mm, 3.5 μm column, 250 μL/min gradient elution (A: CH3CN, B: H2O + 0.1% formic acid) from 5% to 100% A, over 20 min). The divert valve was set to waste for the first 3 min. In positive ion mode, purine C5H4N4 [M + H]+ ion ( *m*/*z* 121.0509) and hexakis (1 *H*, 1 *H*, 3 *H*-tetrafluoropropoxy) phosphazine C18 H18F24 N3O6P3 [M + H]+ ion ( *m*/*z* 922.0098) (HP 0921) were used as internal lock masses. Source parameters were set as follow: capillary voltage at 3500 V, gas temperature at 320 ◦C, drying gas flow at 10 L/min, nebulizer pressure at 40 psi. Fragmentor was set at 175 V. Acquisition was performed in auto MS<sup>2</sup> mode on the range *m*/*z* 100–1200 with an MS rate of 1 spectra/sec and an MS/MS scan rate of 3 spectra/sec. Isolation MS/MS width was 2 *<sup>m</sup>*/*<sup>z</sup>*. Fixed collision energies 45 eV was used. MS/MS events were performed on the three most intense precursor ions per cycle with a minimum intensity of 5000 counts. Full scans were acquired at a resolution of 11,000 [FWHM] ( *m*/*z* 922).

### 4.5.1. MS/MS Data Pretreatment

The MS data were converted from RAW (Thermo) standard data format to mzXML format using the MSConvert software, part of the ProteoWizard package [31]. The converted files were treated using the MZmine software suite v. 2.38 [12].

The parameters were adjusted as following: the centroid mass detector was used for mass detection with the noise level set to 1.0E6 for MS level set to 1, and to 0 for MS level set to 2. The ADAP [32] chromatogram builder was used and set to a minimum group size of scans of 2, minimum group intensity threshold of 3.0E3, minimum highest intensity of 3.0E3 and *m*/*z* tolerance of 10.0 ppm. For chromatogram deconvolution, the algorithm used was the wavelets (ADAP). The intensity window S/N was used as S/N estimator with a signal to noise ratio set at 10, a minimum feature height at 1000, a coe fficient area threshold at 10, a peak duration range from 0.02 to 1.0 min and the RT wavelet range from 0.02 to 0.6 min. Isotopes were detected using the isotopes peaks grouper with a *m*/*z* tolerance of 10.0 ppm, a RT tolerance of 0.3 min (absolute), the maximum charge set at 1 and the representative isotope used was the most intense. Peak alignment was performed using the RANSAC alignment method ( *m*/*z* tolerance at 10 ppm), RT tolerance 0.3 min, RT tolerance after correction 0.5 min, RANSAC iterations 0, Minimum number of points: 80.0 %, Threshold value: 0.3, requiring the same charge state. The peak list was gap-filled with the same RT and *m*/*z* range gap filler ( *m*/*z* tolerance at 10 ppm). Eventually the resulting aligned peaklist was filtered using the peak-list rows filter option in order to keep only features associated with MS<sup>2</sup> scans.

### 4.5.2. Molecular Networks Generation

In order to keep the retention time, the exact mass information and to allow for the separation of isomers, a feature-based molecular network (https://ccms-ucsd.github.io/GNPSDocumentation/ featurebasedmolecularnetworking/) was created using the mgf file resulting from the MZmine pretreatment step detailed above. Spectral data was uploaded on the GNPS molecular networking platform. A network was then created where edges were filtered to have a cosine score above 0.7 and more than six matched peaks. Further edges between two nodes were kept in the network if and only if each of the nodes appeared in each other's respective top 10 most similar nodes. The spectra in the network were then searched against GNPS' spectral libraries. All matches kept between network spectra and library spectra were required to have a score above 0.7 and at least six matched peaks. The output was visualized using Cytoscape 3.6 software [33]. The GNPS job parameters and resulting data are available at the following address (https://gnps.ucsd. edu/ProteoSAFe/status.jsp?task=9581427a15b7422d8bd2b3b4b086189e). The DEREPLICATOR job resulting data is available at the following address (https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task= 0c058507ac774dd7b881c2ee36d57720).

### *4.6. Evaluation of the Antibacterial Activity of Cyanobacterial Strains*

The antibacterial activities of the chemical extracts of the various cyanobacterial strains were tested against six human pathogenic bacteria (*Escherichia coli* ATCC 8739, *Klebsiella pneumoniae* ATCC 11296, *Pseudomonas aeruginosa* ATCC 13388, *Enterococcus faecalis* ATCC 29212, *Staphylococcus aureus* ATCC 6538 and *Bacillus cereus* ATCC 14579) and four marine pathogenic bacteria (*Vibrio alginolyticus* ATCC 17749, *Vibrio anguillarum* ATCC 19264, *Pseudoalteromonas atlantica* ATCC 19262 and *Pseudoalteromonas distincta* ATCC 700518). The selected pathogenic human and marine bacteria were cultured in LB (Luria Bertoni) medium at 37 ◦C or in MB (Marine Broth) at 25 ◦C, respectively. The di fferent bacteria were isolated on LB or MB agar by incubation at 37 ◦C or 25 ◦C overnight. A pre-culture of 5 mL was prepared by inoculating a colony of each bacterial strain, and incubated at 37 ◦C or 25 ◦C and stirring overnight. The bacterial suspension was adjusted by dilution to obtain an optical density (OD) of 0.03 at 620 nm. The antibacterial assays were performed by a liquid method in 96-well microplates. Briefly, 100 μL of the bacterial suspension of di fferent bacteria strains were distributed in each well. The extracts, diluted in DMSO, were tested in triplicate at a concentration of 100 μg/mL. The 96-well microplates were incubated overnight at 37 ◦C or 25 ◦C and shaked at 450 rpm. The OD of each well was measured at 620 nm using an absorbance reader plate (Multiscan, Thermofisher, Saint-Herblain, France). The percentage of growth inhibition was calculated using the formula: % inhibition = 100 − [(ODS − ODB)/(ODT − ODB) × 100] where T = bacterial suspension without test sample, B = culture

medium without bacteria and S = bacterial suspension test sample. Standard antibiotics were used as positive controls (ampicillin against *E. faecalis*, *B. cereus, P. distincta*, *V. anguillarum*; chloramphenicol against *E. coli*, *P. aeruginosa*, *V. alginolyticus, P. atlantica*; gentamycin against *S. aureus*, *K. pneumoniae*).

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/18/1/16/s1, Table S1: Pairwise distance values among new cyanobacterial strains isolated during this study. Values below 0.05 (5% divergence, as congenerics) are in bold. Table S2. List of the 54 candidate structures, which are consistent with previously identified peptides using DEREPLICATOR algorithm. Figure S1. Global molecular network obtained from LC-MS/MS data of 20 cyanobacteria extracts (red ellipses are DEREPLICATOR peptide matches). Figure S2. A selection of clusters and self-loops annotated with putative cyanobacterial peptides and their origin.

**Author Contributions:** Conceptualization and supervision: S.D. (Sébastien Duperron) and M.-L.B.-K., Phylogenetic studies: C.D., S.D (Sylvain Durand) and S.D. (Sébastien Duperron), Chemical studies: S.D. (Sylvain Durand), A.L., and M.-L.B.-K., Molecular Network: M.A.B. and S.D. (Sylvain Durand). Antimicrobial assays: S.D. (Sylvain Durand), A.L. and M.-L.B.-K. All the authors contributed to the writing and editing the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** We obtained financial support from the CNRS MITI X-Life 2018-2019 program (CABMAN project) and the ATM CHEMCYANGROV from the 2019 MNHN gran<sup>t</sup> "biodiversity of microorganisms".

**Acknowledgments:** We acknowledge the financial support of the CNRS and the MNHN.

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