Aestuaramides, a Natural Library of Cyanobactin Cyclic Peptides Resulting from Isoprene-Derived Claisen Rearrangements

We report 12 cyanobactin cyclic peptides, the aestuaramides, from the cultivated cyanobacterium <i>Lyngbya aestuarii</i>. We show that aestuaramides are synthesized enzymatically as reverse <i>O</i>-prenylated tyrosine ethers that subsequently undergo a Claisen rearrangement to produce forward <i>C</i>-prenylated tyrosine. These results reveal that a nonenzymatic Claisen rearrangement dictates isoprene regiochemistry in a natural system. They also reveal one of the mechanisms that organisms use to generate structurally diverse compound libraries starting from simple ribosomal peptide pathways (RiPPs)

Phylogenetic tree of mitochondrial cytochrome <i>c</i> oxidase 1 (COXI) protein sequences from Didemnidae animals not included in Figure S3.

<p>The COXI sequences for <i>L. patella</i> amimals L2, L5 and L6 used to make this tree were obtained from the respective mitochondrial genome assembly from Illumina sequencing data. The inset identity matrix shows the pairwise nucleotide identities of sequences in this clade, indicating that <i>L. patella</i> sequence AB602781.1 likely is a group B animal.</p

Primers and probes used in this study.

<p>Primers and probes used in this study.</p

Phylogenetic tree of mitochondrial cytochrome <i>c</i> oxidase 1 (COXI) protein sequences from our collected <i>L. patella</i> animals and other Didemnidae, with <i>Ciona savignyi</i> acting as the outgroup.

<p>Note: the <i>Didemnum vexillum</i> clade is collapsed for space. The Didemnidae COXI genes found in the NCBI database cover two non-overlapping regions of the gene (see Main Text), and therefore two separate trees were constructed (for the other tree, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095850#pone-0095850-g004" target="_blank">Figure 4</a>).</p

Phylogenetic tree of 18S rRNA nucleotide sequences from our collected <i>L. patella</i> animals and other Didemnidae, with <i>Ciona intestinalis</i> acting as an outgroup.

<p>Phylogenetic tree of 18S rRNA nucleotide sequences from our collected <i>L. patella</i> animals and other Didemnidae, with <i>Ciona intestinalis</i> acting as an outgroup.</p

Collection locations, phylogeny and divergence of <i>Lissoclinum patella</i> individuals collected across areas of the Southeastern Pacific between 2002 and 2011.

<p>(a) Collection sites, with a portion of the phylogenetic tree based on mitochondrial cytochrome c oxidase 1 (COXI) genes overlaid, with individuals colored by clades that diverge by 5% or more in their nucleotide sequence, as shown in (b).</p

Heatmap and hierarchical clustering of select secondary metabolite peak volumes in LCMS runs on <i>L. patella</i> extracts (top left).

<p>Clustering based on secondary metabolites that are produced by the symbiotic bacteria <i>Prochloron didemni</i> and <i>Ca.</i> Endolissoclinum faulkneri closely follows the hosts' phylogeny as determined by COXI sequences (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095850#pone-0095850-g002" target="_blank">Fig. 2</a>). The <i>P. didemni</i> compounds shown are all cyanobactins produced either by a patellamide-type pathway (red), or a trunkamide-type pathway (blue). These two types of are closely related ribosomal pathways that are highly tolerant to changes in the precursor peptide sequence. The patellazoles (magenta) are produced by another symbiont, <i>Candidatus</i> Endolissoclinum faulkneri, by a polyketide synthase pathway.</p

Schematic representation of the draft mitochondrial genome of <i>L. patella</i> animal L2 (top, to scale).

<p>The hive plots <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095850#pone.0095850-Krzywinski1" target="_blank">[61]</a> at the bottom of the figure show that the L2 assembly is syntenic with contigs assembled of the mitochondrial genomes of L5 and L6. In these plots, protein-coding genes are represented as circles, and contig boundaries are represented as zigzag lines. Homologous genes are joined by curved lines colored according to the sequence identity of the gene relevant gene pair. Abbreviations: COX, cytochrome <i>c</i> oxidase; NADH, nicotinamide adenine dinucleotide dehydrogenase.</p

Structure and Biosynthesis of the Antibiotic Bottromycin D

Drug resistant infectious diseases are quickly becoming a global health crisis. While <i>Streptomyces</i> spp. have been a major source of antibiotics over the past 50 years, efficient methods are needed to identify new antibiotics and greatly improve the rate of discovery. LCMS-based metabolomics were applied to analyze extracts of 50 <i>Streptomyes</i> spp. Using this methodology, we discovered bottromycin D and used whole genome sequencing to determine its biosynthesis by a ribosomal pathway