Re-analysis of HPLC peaks previously assigned as phenazines from original data collected on an older HPLC.

UV–vis chromatograms are shown on the left, and the full UV–vis spectra of the peaks indicated with an arrow are shown on the right. Where indicated on the y-axis, the data have been normalized to bring the data into the same scale for clarity. (A) Analysis of the PCA peak, comparing a pure PCA standard (chromatogram at 364 nm, orange) to a sputum sample (chromatogram at 398 nm, black). (B) Analysis of the pyocyanin peak, comparing pyocyanin in P. aeruginosa-free sputum (blue) to a P. aeruginosa-positive sputum sample (black) (both chromatograms at 387 nm).</p

Correlation between ferriprotoporphyrin IX concentration and disease status as measured by percent predicted FEV1 (Spearman’s ρ = −0.47, <i>p</i> = 3.6 × 10⁻⁵).

Each data point represents a single sputum sample from a unique patient. The dashed line illustrates a linear regression through the data.</p

Patient cohort characteristics.

Patient cohort characteristics.</p

Refinement of metabolite detection in cystic fibrosis sputum reveals heme correlates with lung function decline - Fig 1

Identification of heme in sputum samples by comparing sputum (black) to a hemin standard (green). (A) UV–vis chromatogram (top) and extracted ion chromatogram (616.1773 ± 0.01 Da) from the positive mass channel (bottom) demonstrating identical retention times. (B) The associated positive ions detected from the peak shown in A, comparing the sputum peak (top) to the hemin standard (bottom). (C) The associated negative ions detected from the peak shown in B, comparing the sputum peak (top) to the hemin standard (bottom). A collision-energy ramp of 10 to 14 eV was applied to generate a fragmentation pattern. (D) Comparison of the UV–vis spectra for the peaks in A (black and green) compared to the same peak identified in our prior study (gray, dashed). For clarity, the spectra are normalized to their maximum value. (E) The chemical structure assigned to the peak in A, ferriprotoporphyrin IX.</p

Additional file 1 of Distribution and diversity of dimetal-carboxylate halogenases in cyanobacteria

Additional file 1: Table S1. Accession numbers of cylC homologs and aurFgenes used for primer design. Figure S1. (a) Phylogenetic tree (FastTree GTR with a rate of 100) of cylC homologs highlighted according to the groups selected for degenerate primer design. (b) Schematic representation of the different pairs of degenerate primers. Figure S2. PCR-based detection of cylC homologs in the LEGEcc. Five pairs of primers were designed based on conserved regions identified in the cylC gene. Each primer pair was used in a PCR screen of the gDNA obtained from diverse strains (n = 326) of the LEGEcc. The resulting amplicons were cloned and sequenced. Sequences for each primer pair were aligned with the corresponding regions of cylC genes found in the NCBI reference genomes (cyanobacteria only) and those from LEGEcc strains’ genomes. Shown are the resulting cladograms (RaxML, 1000 replicates) for each primer pair used in the screening. Blue squares indicate sequences obtained from the PCR screen. Figure S3. RaxML cladogram (1000 replicates) of the 16S rRNA gene of LEGEcc strains (grey squares) and from cyanobacterial strains with NCBI-deposited reference genomes, screened in this study. Taxonomy is presented at the order level (colored ranges). Strains whose genomes encode CylC homologs are denoted by black squares. Green squares indicate that at least one CylC homolog was detected by PCR-screening and verified by retrieving the sequence of the corresponding amplicon through cloning followed by Sanger sequencing. The cladogram topology is the same as shown in Fig. 3 of the main manuscript, but here bootstrap values (equal or above 0.7) are shown. Table S2. GenBank or RefSeq assembly acession number and LEGEcc genome used for CORASON analysis. Table S3. BLASTp search of CylC homologs against Aliterella sp., Chroococcidiopsis sp. and Gloeobacter sp. Figure S4. Rieske-containing biosynthetic gene clusters encoding CylC homolog(s). Figure S5. PriA-containing biosynthetic gene clusters encoding CylC homolog(s). Figure S6. Cytochrome P450/sulfotransferase-containing biosynthetic gene cluster encoding a CylC homolog. Figure S7. Type I PKS (chlorosphaerolactylate/columbamide/microginin/puwainaphycin-like) biosynthetic gene clusters encoding CylC homolog(s). Figure S8. Dialkylresorcinol biosynthetic gene clusters encoding CylC homolog(s). Figure S9. Type III PKS biosynthetic gene clusters encoding CylC homolog(s). Figure S10. Nitronate monooxygenase-containing biosynthetic gene clusters encoding a CylC homolog. Figure S11. Unclassified (likely incomplete) biosynthetic gene clusters encoding a CylC homolog. Table S4. BLAST search of Rieske-containing BGCs genes from Calothrix brevissima NIES 22 against Synechocystis sp. PCC 6803. Figure S12. Phylogenetic tree of FAD-dependent halogenases based on CORASON outputs with illustrative BGC architectures. Figure S13. Phylogenetic tree of nonheme iron-dependent halogenases based on CORASON outputs with illustrative BGC architectures. Figure S14. Phylogenetic tree of dimetal-carboxylate halogenases based on CORASON outputs with illustrative BGC architectures

Biosynthesis of the Unusual Carbon Skeleton of Nocuolin A

Nocuolin A is a cytotoxic cyanobacterial metabolite that is proposed to be produced by enzymes of the noc biosynthetic gene cluster. Nocuolin A features a 1,2,3-oxadiazine moiety, a structural feature unique among natural products and, so far, inaccessible through organic synthesis, suggesting that novel enzymatic chemistry might be involved in its biosynthesis. This heterocycle is substituted with two alkyl chains and a 3-hydroxypropanoyl moiety. We report here our efforts to elucidate the origin of the carbon skeleton of nocuolin A. Supplementation of cyanobacterial cultures with stable isotope-labeled fatty acids revealed that the central C13 chain is assembled from two medium-chain fatty acids, hexanoic and octanoic acids. Using biochemical assays, we show that a fatty acyl-AMP ligase, NocH, activates both fatty acids as acyl adenylates, which are loaded onto an acyl carrier protein domain and undergo a nondecarboxylative Claisen condensation catalyzed by the ketosynthase NocG. This enzyme is part of a phylogenetically well-defined clade within similar genomic contexts. NocG presents a unique combination of characteristics found in other ketosynthases, namely in terms of substrate specificity and reactivity. Further supplementation experiments indicate that the 3-hydroxypropanoyl moiety of 1 originates from methionine, through an as-yet-uncharacterized mechanism. This work provides ample biochemical evidence connecting the putative noc biosynthetic gene cluster to nocuolin A and identifies the origin of all its carbon atoms, setting the stage for elucidation of its unusual biosynthetic chemistry