**4. Conclusions**

Genes coding for subunits of the non-ribosomal peptide synthetase, in nostocyclopeptideproducing *N*. *edaphicum* CCNP1411, revealed differences in nucleotide compositions, compared to the previously described *ncp* cluster of *Nostoc* sp. ATCC53789. Although the analysis of fragments of genes coding for active sites and ligand binding sites of the conserved protein domains derived from *N*. *edaphicum* CCNP1411 and *Nostoc* sp. ATCC53789 indicated identical amino-acid compositions, residues within adenylation domains and substrate binding sites were different between compared sequences. This finding may highlight sites prone to mutations within regions accounted for structure and substrate stability. Analysis of *ncp* gene products in *N*. *edaphicum* CCNP1411 led to the detection of new nostocyclopeptide analogues. However, modifications in their structure were minor and limited to three positions of the heptapeptides. Although the naturally produced nostocyclopeptides were previously described as cyclic structures, in *N*. *edaphicum* CCNP1411 they are mainly present as linear peptide aldehydes, indicating a slow cyclization process.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/18/9/442/ s1. Figure S1: Structure and enhanced product ion mass spectrum of the cyclic nostocyclopeptide Ncp-A1 cyclo [Tyr+Gly+Gln+Ile+Ser+MePro+Leu] identified based on the following fragment ions: *m*/*z* 757 [M+H]; 739 [M+H−H2O]; 729 [M+H−CO]; 721 [M+H−2H2O]; 628 [M+H−MePro−H2O], 626 [M+H−Ile−H2O]; 594 [M+H−Tyr]; 549 [Tyr+Gly+Gln+Ile+Ser+H]; 541 [M+H−(Ser+MePro)−H2O], 446 [M+H−(Ile+Ser+MePro)]; 428 [M+H−(Ile+Ser+MePro)−H2O]; 386 [Gly+Gln+Ile+Ser+H]; 372 [MePro+Leu+Tyr+H]; 300 [Leu+Tyr+Gly+H−H2O]; 209 [MePro+Leu+H]; 181 [MePro+Leu+H−CO]; 86−Ile/Leu immonium; 84 MePro immonium, Figure S2: Structure and enhanced product ion mass spectrum of the linear peptide aldehyde Ncp-A1-L (linear aldehyde of Ncp-A1) Tyr+Gly+Gln+Ile+Ser+MePro+Leu identified based on the following fragment ions: *m*/*z* 775 [M+H]; 757 [M+H–H2O]; 739 [M+H−2H2O]; 660 [M+H−Leu]; 549 [Tyr+Gly+Gln+Ile+Ser+H]; 531 [Tyr+Gly+Gln+Ile+Ser+H−H2O]; 521 [Tyr+Gly+Gln+Ile+Ser+H−CO]; 532 [M+H−(MePro+Leu)−H2O]; 462 [Tyr+Gly+Gln+Ile+H]; 434 [Tyr+Gly+Gln+Ile+H−CO]; 386 [Gly+Gln+Ile+Ser+H]; 349 [Tyr+Gly+Gln+H]; 301 [Gln+Ile+Ser+H−CO]; 227 [MePro+Leu+H]; 221 [Tyr+Gly+H]; 209 [MePro+Leu+H−H2O]; 181 [MePro+Ile+H−H2O−CO]; 148 [Tyr−NH2]; 136 Tyr immonium; 86−Ile/Leu immonium; 84, 101 (immonium),129 Gln; 84 MePro immonium, Figure S3: Structure and enhanced product ion mass spectrum of the cyclic nostocyclopeptide Ncp-A2 cyclo[Tyr+Gly+Gln+Ile+Ser+MePro+Phe] identified based on the following fragment ions: *m*/*z* 791 [M+H]; 773 [M+H−H2O]; 763 [M+H−CO]; 755 [M+H−2H2O]; 745 [M+H−CO−H2O]; 678 [M+H−Ile]; 628 [M+H−Tyr]; 593 [M+H−(Ser+MePro)]; 549 [Tyr+Gly+Gln+Ile+Ser+H]; 531 [Tyr+Gly+Gln+Ile+Ser+H−H2O]; 480 [M+H−(Ile+Ser+MePro)]; 462 [Tyr+Gly+Gln+Ile+H]; 406 [MePro+Phe+Tyr+H]; 379 [MePro+Phe+Tyr+H−CO]; 349 [Tyr+Gly+Gln+H]; 335 [Phe+Tyr+Gly+H−H2O]; 312 [Ile+Ser+MePro+H]; 307 [Phe+Tyr+Gly+H−H2O−CO]; 243 [MePro+Phe+H]; 215 [MePro+Phe+H−CO]; 158 [Gly+Gln+H−CO]; 132 Phe; 84 MePro immonium, Figure S4: Structure and enhanced product ion mass spectrum of the linear nostocyclopeptide aldehyde Ncp-A2-L (linear aldehyde of Ncp-A2) Tyr+Gly+Gln+Ile+Ser+MePro+Phe identified based on the following fragment ions: *m*/*z* 809 [M+H]; 791 [M+H−H2O]; 773 [M+H−2H2O]; 763 [M+H−CO−H2O]; 660 [M+H−Phe]; 628 [M+H−Tyr−H2O]; 549 [Tyr+Gly+Gln+Ile+Ser+H]; 531 [M+H−(MePro+Phe)−H2O]; 462 [Tyr+Gly+Gln+Ile+H]; 434 [Tyr+Gly+Gln+Ile+H−CO]; 312 [Ile+Ser+MePro+H]; 261 [MePro+Phe+H]; 243 [MePro+Phe+H–H2O]; 221 [Tyr+Gly+H], 193 [Tyr+Gly+ H−CO]; 148 [Tyr−NH2]; 136 Tyr immonium; 84, 101 (immonium), 129 Gln; 84 MePro immonium, Figure S5: Proposed structure and enhanced product ion mass spectrum of cyclic nostocyclopeptide Ncp-E2 cyclo[Tyr+Gly+Gln+Ile+Ser+Pro+Leu] characterized based on the following fragment ions: *m*/*z* 743 [M+H]; 725 [M+H−H2O]; 715 [M+H−CO]; 707 [M+H−2H2O]; 697 [M+H–H2O−CO]; 656 [M+H−Ser]; 638 [M+H−Ser−H2O]; 628 [M+H−Ser−CO]; 612 [M+H−Ile−H2O]; 549 [Tyr+Gly+Gln+Ile+Ser+H]; 541 [M+H−(Ser+Pro)−H2O]; 531 [Tyr+Gly+Gln+Ile+Ser+H−H2O]; 428 [M+H−(Ile+Ser+Pro)−H2O]; 349 [Tyr+Gly+Gln+H]; 300 [Leu+Tyr+Gly+H−H2O]; 195 [Pro+Leu+H]; 167 [Pro+Leu+H−CO]; 84 Gln; 70 Pro immonium, Figure S6: Proposed structure and enhanced product ion mass spectrum of the linear nostocyclopeptide aldehyde Ncp-E2-L (linear aldehyde of Ncp-E2) with general structure Tyr+Gly+Gln+Ile+Ser+Pro+Leu characterized based on the following fragment ions: *m*/*z* 761 [M+H]; 743 [M+H−H2O]; 725 [M+H−2H2O]; 549 [Tyr+Gly+Gln+Ile+Ser+H]; 532 [Tyr+Gly+Gln+Ile+Ser+H−H2O]; 462 [Tyr+Gly+Gln+Ile+H]; 349 [Tyr+Gly+Gln+H]; 434 [Tyr+Gly+Gln+Ile+H−CO]; 300 [Ser+Pro+Leu+H]; 221 [Tyr+Gly+H]; 213 [Pro+Leu+H]; 195 [Pro+Leu+H−H2O]; 148 [Tyr−NH2]; 136 Tyr immonium; 84, 101 (immonium), 129 Gln; 70 Pro immonium, Figure S7: Proposed structure and enhanced product ion mass spectrum of cyclic nostocyclopeptide Ncp-E3 cyclo[Tyr+Gly+Gln+Val+Ser+MePro+Leu] characterized based on the following fragment ions: *m*/*z* 743 [M+H]; 725 [M+H−H2O]; 715 [M+H−CO]; 707 [M+H – 2H2O]; 697 [M+H−H2O−CO]; 645 [M+H−Val]; 580 [M+H−Tyr]; 527 [M+H−(Ser+MePro)−H2O]; 428 [M+H−(Val+Ser+MePro)−H2O]; 410 [M+H−(Val+Ser+MePro)−2H2O]; 372 [Gly+Gln+Val+Ser+H]; 344 [Gly+Gln+Val+Ser+H−CO]; 300 [Leu+Tyr+Gly+H−H2O]; 233 [Leu+Tyr+H−CO]; 209 [MePro+Leu+H]; 181 [MePro−Leu+H−CO]; 84 MePro immonium; 72 Val immonium, Figure S8: Relative contents of nostocyclopeptides extracted from 10 mg of lyophilized biomass of N. edaphicum CCNP1411 with 20% MeOH of different pH (3.5, 6 and 8)

**Author Contributions:** Conceptualization, H.M.-M. and G.W.; methodology, H.M.-M. and G.W.; software, M.G., J.G. and R.G.; validation, M.G., J.G. and R.G.; formal analysis, A.F. and M.G.; investigation, A.F. and M.G.; resources, data curation, M.G., H.M.-M., J.G. and R.G.; writing—original draft preparation, A.F. and M.G.; writing—review and editing, A.F., M.G.; H.M.-M. and G.W.; visualization, A.F. and M.G.; supervision, H.M.-M. and G.W.; project administration, H.M.-M.; funding acquisition, H.M.-M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Science Centre in Poland 2016/21/B/NZ9/02304.

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