Article Figures & Data
Figures
- FIG 1
HPLC–HRMS-MS analysis of crude extracts from the strains investigated. Major puwainaphycin (PUW) and minutissamide (MIN) variants are highlighted. For variants without complete structural information, only m/z values are shown.
- FIG 2
Molecular network created using the Global Natural Products Social Molecular Networking (GNPS) web platform. Two separate networks were obtained during GNPS analysis: a group containing Cylindrospermum strains 1 to 3 and Anabaena strains 4 and 5 (a), and a group containing only variants detected in Symplocastrum muelleri strain 6 (b). The separate groups differ mainly in the peptide core of the molecule. For variants without complete structural information, only m/z values are shown. *, compound present in trace amounts; #, compound for which MS-MS data failed to resolve the structural information.
- FIG 3
Structural variability of the peptide cores of PUW/MIN variants. Examples of structural variants PUW F (a) and PUW A (b) with designated amino acid positions representing the two major peptide cores. (c) Table summarizing all types of the PUW/MIN peptide core found in known compounds reported in literature and compounds (Comp.) detected in studied strains. Columns shaded in gray highlight the conserved amino acid positions.
- FIG 4
Structural variability of the FA moiety of PUW/MIN variants. The relative proportions of variants with differences in FA lengths and substitutions are depicted using a color scale. For comparison, the peak area of a given variant was normalized against the peak area of the major variant present in the strain (MIN A for strains 1 to 5 and m/z 1,235.7 for strain 6).
- FIG 5
Structures of the puw gene cluster in the six cyanobacterial strains investigated. Gene arrangement and functional annotation of puwA to -L genes and selected PKS/NRPS tailoring domains are indicated by colored arrows. The distribution of the two types of putative starter modules (shaded boxes) observed is indicated by bars.
- FIG 6
Schematic view of the proposed biosynthesis assembly line of puwainaphycins and minutissamides. Variable amino acid positions and the ranges of fatty acyl lengths incorporated by the two putative alternative starter units are listed for individual strains. A, adenylation domain; ACP, acyl carrier protein; AmT, aminotransferase; AT, acyltransferase; C, condensation domain; DH, dehydratase; E, epimerase; ER, enoylreductase; FAAL, fatty acyl-AMP ligase; MT, methyltransferase; NRPS, nonribosomal peptide synthetase; KR, ketoreductase; KS, ketosynthetase; Ox, monooxygenase; PCP, peptidyl carrier protein; PKS, polyketide synthetase; TE, thioesterase.
- FIG 7
MS-MS fragmentation of MIN A (a, c, e) and the PUW variant at m/z 1,279 bearing an acetyl substitution of the fatty acid chain (b, d, f). (a, b) Base peak chromatograms. (c, d) Fragmentation of the protonated molecule at low fragmentation energy, yielding b series of ions, corresponding to the losses of particular amino acid residues. (e, f) Fragmentation of the protonated molecule at high energy (100 eV), yielding fragments characteristic for the β-amino fatty acid.
- FIG 8
Antifungal activities of PUW F against yeast strains Saccharomyces cerevisiae HAMBI 1164 (a) and Candida albicans HAMBI 261 (b). Discs were treated with a range of concentrations from 25.2 μg ml−1 to 0.0394 μg/ml to determine the MIC. Numbers and symbols represent concentrations and controls, as follows: 1, 25.2 μg ml−1; 2, 12.6 μg ml−1; 3, 6.3 μg ml−1; +, positive control (10 μg nystatin); −, negative control (10 μl methanol).
Tables
- TABLE 1
Strains analyzed for PUW/MIN production
Strain no. herein Strain Person who isolated strain Date Locality, habitat Reference 1 Cylindrospermum alatosporum CCALA 988 A. Lukešová 1989 Canada, Manitoba, Riding Mountain National Park, soil 55 2 Cylindrospermum moravicum CCALA 993 A. Lukešová 2008 Czech Republic, South Moravia, Moravian Karst, Amaterska Cave, cave sediment 55 3 Cylindrospermum alatosporum CCALA 994 A. Lukešová 2011 Czech Republic, Moravian Karst, earthworms collected from soil above Amaterska Cave, earthworm casings 55 4 Anabaena sp. UHCC-0399 M. Wahlsten N/A Finland, Jurmo, Southwestern Archipelago National Park, copepods 56 5 Anabaena minutissima UTEX B1613 T. Kantz 1967 South Texas, USA, soil 57 6 Symplocastrum muelleri NIVA-CYA 644 O. M. Skulberg 2009 Norway, Møre og Romsdal county, Halsa municipality, western slope of Slettfjellet mountain in semiterrestrial alpine habitat, biofilm on turf in ombrotrophic blanket bog 40 - TABLE 2
Deduced proteins encoded by the puw gene cluster in six cyanobacterial strains, including length and functional annotationc
Protein Length (aa) in strain no.: Predicted function(s)a 1 2 3 4 5 6 ORF1 659 664 664 643 643 647 ABC transporter PuwA 2,870 2,870 2,870 2,854 2,854 2,866 NRPS ORF2 1,116 1,499 1,875 643 670 376 Patatin-like phospholipase ORF3 696 696 Dynamin family protein PuwI 709 702 711 FAAL, ACP PuwJ 427 427 529 Cytochrome-like protein PuwB 2,534 2,592 2,592 2,549 2,537 2,555 Hybrid PKS/NRPS, aminotransferase, oxygenase PuwC 597 590 590 597 589 FAAL PuwD 101 104 96 93 92 ACP PuwK 465 Halogenase PuwE 3,077 3,121 3,121 3,099 3,112 3,113 NRPS PuwF 2,370 5,851b 5,851b 5,877b 5,871b 3,310 NRPS PuwG 3,492 2,620 NRPS PuwH 1,102 1,081 1,102 1,121 1,121 1,408 NRPS PuwL 217 O-Acetyltransferase - TABLE 3
Fragmentation and amino acid composition of PUW variants from Symplocastrum muelleri strain 6 bearing acetyl substitutions on the FA moietya
Level of fragmentation
energy, peptide sequence
or fragment obtainedValue when X, Y, and FA are as indicated X, Ala; Y, Thr; FA, C16 X, Gly; Y, Thr; FA, C18 X, Ala; Y, Thr; FA, C18 X, Gly; Y, Val; FA, C18 X, Ala; Y, Val; FA, C18 m/z Δ(ppm) Sum formula m/z Δ(ppm) Sum formula m/z Δ(ppm) Sum formula m/z Δ(ppm) Sum formula m/z Δ(ppm) Sum formula Low fragmentation energy (60 eV) [M+H]+ 1,265.7338 +0.7 C59H101N12O18 1,279.7496 +0.9 C60H103N12O18 1,293.7654 +0.8 C61H105N12O18 1,277.7695 +1.6 C61H105N12O17 1,291.7870 +0.1 C62H107N12O17 [M-CH3OH]+ 1,233.7170 −6.6 C58H97N12O17 1,247.7194 +4.1 C59H99N12O17 1,261.7494 −7.3 C60H101N12O17 Low int. C60H101N12O16 Low int. C61H103N12O16 [M-CH3OH-NMeAsn]+ 1,105.6558 −4.9 C53H89N10O15 1,119.6619 +3.7 C54H91N10O15 1,133.681 +0.6 C55H93N10O15 1,117.6924 −5.0 C55H93N10O14 1,131.7307 −25.0 C56H95N10O14 [M-CH3OH-NMeAsn-Dhb]+ 1,022.6180 −4.7 C49H84N9O14 1,036.6365 −7.3 C50H86N9O14 1050.6478 −3.1 C51H88N9O14 1,134.6603 −10.3 C51H88N9O13 1,048.6671 −1.7 C52H90N9O13 [M-CH3OH-NMeAsn-Dhb-X]+ 951.5785 −2.5 C48H83N8O13 979.589 +18.8 C48H83N8O13 979.6041 +3.4 C48H83N8O13 977.6481 −20.5 C49H85N8O12 977.6518 −24.1 C49H85N8O12 [M-CH3OH-NMeAsn-Dhb-X-Gln]+ 823.5253 −9.4 C41H71N6O11 851.5473 +1.8 C43H75N6O11 851.5478 +1.2 C43H75N6O11 849.5838 −16.7 C44H76N6O10 849.5589 +12.5 C44H77N6O10 [M-CH3OH-NMeAsn-Dhb-X-Gln-Y]+ 722.4729 −4.2 C37H64N5O9 750.5005 +0.9 C39H68N5O9 750.5147 −18.1 C39H68N5O9 Low int. C40H72N5O8 Low int. C40H72N5O8 [M-CH3OH-NMeAsn-Dhb-X-Gln-Y-Thr]+ 621.4223 −0.2 C33H57N4O7 649.4526 +1.4 C35H61N4O7 649.4539 −0.6 C35H61N4O7 649.465 −17.8 C35H61N4O7 649.4483 +8.0 C35H61N4O7 High fragmentation energy (100 eV) Fragment 1 411.3208 +2.2 C23H43N2O4 439.3559 −6.5 C25H47N2O4 439.3556 −5.8 C25H47N2O4 439.3556 −5.8 C25H47N2O4 439.3508 +5.1 C25H47N2O4 Fragment 1, C2H4O2 351.3006 +0.0 C21H39N2O2 379.3334 −4.0 C23H43N2O2 379.3329 −2.6 C23H43N2O2 379.3328 −2.4 C23H43N2O2 379.3360 −10.8 C23H43N2O2 Fragment 2 284.2583 +0.4 C17H34NO2 312.2919 −6.9 C19H38NO2 312.2892 +1.6 C19H38NO2 Low int. C19H38NO2 Low int. C19H38NO2 Fragment 2, C2H4O2 224.2367 +2.6 C15H30N 252.2686 0.0 C17H34N 252.2687 −0.5 C17H34N 252.2684 +0.7 C17H34N 252.2677 +3.5 C17H34N ↵a Low int., low intensity (defined here as a signal-to-noise ratio lower than 2). For detailed methods, see Materials and Methods.
- TABLE 4
Bacterial and yeast strains used for antimicrobial testing of PUW F and MIN A, C, and D
Test organism (HAMBI no.)a Mediumb Incubation temp (°C) Incubation time (h) Gram stain reactionc Pseudomonas sp. (2796) TGY 28 24 − Micrococcus luteus (2688) TGY 28 24 + Bacillus subtilis (251) TGY 28 24 + Pseudomonas aeruginosa (25) TGY 37 24 − Escherichia coli (396) TGY 37 24 − Bacillus cereus (1881) TSA 28 24 + Burkholderia cepacia (2487) TSA 37 24 − Staphylococcus aureus (11) TSA 37 24 + Xanthomonas campestris (104) NA 28 24 − Burkholderia pseudomallei (33) NA 37 24 − Salmonella enterica serovar Typhi (1306) NA 37 24 − Arthrobacter globiformis (1863) NA 28 24 − Kocuria varians (40) NA 28 24 + Candida albicans (261) YM agar 37 24 Yeast Cryptococcus albidus (264) YM agar 28 24 Yeast Saccharomyces cerevisiae (1164) YM agar 28 24 Yeast ↵a HAMBI, culture collection of University of Helsinki, Faculty of Agriculture and Forestry, Department of Microbiology.
↵b The compositions of all media were obtained from the American Type Culture Collection (ATCC). TGY, tryptone glucose yeast; TSA, tryptic soy agar; NA, nutrient agar; YM agar, yeast malt agar.
↵c −, negative; +, positive.
Supplemental material
- Supplemental file 1 -
List of PUW/MIN variants detected in the strains under study and their MS/MS fragmentation analysis data (Table S1); analysis of amino acid adenylation domains encoded in puw gene clusters of five newly sequenced cynobacterial strains (Table S2); NMR data for the fatty acid moiety of MIN C and D in DMSO-d6 (Table S3); formation of characteristic fragments in high-energy fragmentation in major MIN variants reported for Anabaena minutissima UTEX B1613 (Fig. S1); fatty acyl-AMP ligase alignments (Fig. S2); 1H NMR spectra of minutissamides C and D from Anabaena sp. UHCC-0399 (Fig. S3); 13C HSQC spectrum of minutissamide C from Anabaena sp. UHCC-0399 (Fig. S4).
PDF, 4.9M
- Supplemental file 1 -
