New Structural Variants of Aeruginosin Produced by the Toxic Bloom Forming Cyanobacterium Nodularia spumigena

Abstract

Nodularia spumigena is a filamentous diazotrophic cyanobacterium that forms blooms in brackish water bodies. This cyanobacterium produces linear and cyclic peptide protease inhibitors which are thought to be part of a chemical defense against grazers. Here we show that N. spumigena produces structurally novel members of the aeruginosin family of serine protease inhibitors. Extensive chemical analyses including NMR demonstrated that the aeruginosins are comprised of an N-terminal short fatty acid chain, L-Tyr, L-Choi and L-argininal and in some cases pentose sugar. The genome of N. spumigena CCY9414 contains a compact 18-kb aeruginosin gene cluster encoding a peptide synthetase with a reductive release mechanism which offloads the aeruginosins as reactive peptide aldehydes. Analysis of the aeruginosin and spumigin gene clusters revealed two different strategies for the incorporation of N-terminal protecting carboxylic acids. These results demonstrate that strains of N. spumigena produce aeruginosins and spumigins, two families of structurally similar linear peptide aldehydes using separate peptide synthetases. The aeruginosins were chemically diverse and we found 11 structural variants in 16 strains from the Baltic Sea and Australia. Our findings broaden the known structural diversity of the aeruginosin peptide family to include peptides with rare N-terminal short chain (C2–C10) fatty acid moieties.

Chemical and Genetic Diversity of Nodularia spumigena from the Baltic Sea

“…Spumigins (SPUs), nodularins (NPs) and APs were detected in all 25 N. spumigena strains, whereas aeruginosins were not found in seven of them (Figure S9 and Table S3). These findings agree with previous studies [21,22,23]. Production of pseudoaeruginosins NS1 ( m / z 531) and NS2 ( m / z 533) was confirmed only in six strains (CCNP1402, BY1, CCY9414, AV1, KAC71, and KAC87), indicating that in N. spumigena this class of peptides belongs to the most rare ones.…”

“…These include 14 spumigins, 5 aeruginosins, 2 pseudoaeruginosins, 2 nodularins, and 36 anabaenopeptins (Table S2). Many of these peptides have been previously reported [7,16,20,21,22,23,25]. However, new proposed structures of 7 anabaenopeptins (APs) and one cyanopeptolin-like peptide were characterized in this study (Table S2 and Figures S1–S8).…”

“…In N. spumigena , five classes of non-ribosomal peptides have been identified: linear spumigins [20,21], aeruginosins [7,22], pseudoaeruginosins [23], cyclic nodularins [24], and anabaenopeptins [16,20]. Spumigins (SPUs) are linear tetrapeptides (Figure 1a) with hydroxyphenyl lactic acid (Hpla 1 ) in N -terminal position, Arg 4 or Arg mimetics in C -terminus and (2 S , 4 S )-4-methylproline (MePro) or Pro in position 3.…”

Baltic cyanobacteria – a source of biologically active compounds

“…2S) et al 1990;Ohta et al 1994;Sano et al, 2001), serine proteases (aeruginosins, spumigins, anabaenopeptins, cyanopeptolins) (Sano & Kaya, 1995;Ishida et al, 1999;Itou et al, 1999;Murakami et al, 2000;Ploutno et al, 2002;Radau et al, 2003;Elert et al, 2005;Fewer et al, 2009Fewer et al, , 2013 and compounds cytotoxic against human cancer cell lines (nostocyclopeptides) (Golakoti et al, 2001) (Supplementary Fig. 3S).…”

4-Methylproline Guided Natural Product Discovery: Co-Occurrence of 4-Hydroxy- and 4-Methylprolines in Nostoweipeptins and Nostopeptolides

“…Acid hydrolysis and amino acid analysis of Nwp W1 produced N-CH 3 -L-Phe, L-Ser, D-Ser, (2S,4S)-4-CH 3 -Pro, D-Leu, L-Ile, L-Tyr, and (2S,4R)-4-OH-Hyp. 15 N labeling showed that Nwp's contained 10 nitrogen atoms, indicating a presence of 10 amino acids. All these results together with the elemental composition data presented in Table 1 and accurate product ion masses presented in Figure 3 made it possible to draw the structures of the nostoweipeptins W1−W7 ( Figure 4).…”