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Genetics and Molecular Biology

Alternative Biosynthetic Starter Units Enhance the Structural Diversity of Cyanobacterial Lipopeptides

Jan Mareš, Jan Hájek, Petra Urajová, Andreja Kust, Jouni Jokela, Kumar Saurav, Tomáš Galica, Kateřina Čapková, Antti Mattila, Esa Haapaniemi, Perttu Permi, Ivar Mysterud, Olav M. Skulberg, Jan Karlsen, David P. Fewer, Kaarina Sivonen, Hanne Hjorth Tønnesen, Pavel Hrouzek
Marie A. Elliot, Editor
Jan Mareš
aThe Czech Academy of Sciences, Biology Centre, Institute of Hydrobiology, České Budějovice, Czech Republic
bThe Czech Academy of Sciences, Institute of Microbiology, Center Algatech, Třeboň, Czech Republic
cUniversity of South Bohemia, Faculty of Science, České Budějovice, Czech Republic
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Jan Hájek
bThe Czech Academy of Sciences, Institute of Microbiology, Center Algatech, Třeboň, Czech Republic
cUniversity of South Bohemia, Faculty of Science, České Budějovice, Czech Republic
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Petra Urajová
bThe Czech Academy of Sciences, Institute of Microbiology, Center Algatech, Třeboň, Czech Republic
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Andreja Kust
aThe Czech Academy of Sciences, Biology Centre, Institute of Hydrobiology, České Budějovice, Czech Republic
bThe Czech Academy of Sciences, Institute of Microbiology, Center Algatech, Třeboň, Czech Republic
cUniversity of South Bohemia, Faculty of Science, České Budějovice, Czech Republic
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Jouni Jokela
dDepartment of Microbiology, Viikki Biocenter, University of Helsinki, Helsinki, Finland
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Kumar Saurav
bThe Czech Academy of Sciences, Institute of Microbiology, Center Algatech, Třeboň, Czech Republic
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Tomáš Galica
bThe Czech Academy of Sciences, Institute of Microbiology, Center Algatech, Třeboň, Czech Republic
cUniversity of South Bohemia, Faculty of Science, České Budějovice, Czech Republic
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Kateřina Čapková
aThe Czech Academy of Sciences, Biology Centre, Institute of Hydrobiology, České Budějovice, Czech Republic
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Antti Mattila
dDepartment of Microbiology, Viikki Biocenter, University of Helsinki, Helsinki, Finland
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Esa Haapaniemi
eDepartment of Chemistry, University of Jyväskylä, Jyväskylä, Finland
fDepartment of Biological and Environmental Science, Nanoscience Center, University of Jyväskylä, Jyväskylä, Finland
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Perttu Permi
eDepartment of Chemistry, University of Jyväskylä, Jyväskylä, Finland
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Ivar Mysterud
gDepartment of Biosciences, University of Oslo, Oslo, Norway
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Olav M. Skulberg
hNorwegian Institute for Water Research (NIVA), Oslo, Norway
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Jan Karlsen
iSchool of Pharmacy, University of Oslo, Oslo, Norway
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David P. Fewer
dDepartment of Microbiology, Viikki Biocenter, University of Helsinki, Helsinki, Finland
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Kaarina Sivonen
dDepartment of Microbiology, Viikki Biocenter, University of Helsinki, Helsinki, Finland
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Hanne Hjorth Tønnesen
iSchool of Pharmacy, University of Oslo, Oslo, Norway
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Pavel Hrouzek
bThe Czech Academy of Sciences, Institute of Microbiology, Center Algatech, Třeboň, Czech Republic
cUniversity of South Bohemia, Faculty of Science, České Budějovice, Czech Republic
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Marie A. Elliot
McMaster University
Roles: Editor
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DOI: 10.1128/AEM.02675-18
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  • FIG 1
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    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
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    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
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    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
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    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
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    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
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    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
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    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
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    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

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  • TABLE 1

    Strains analyzed for PUW/MIN production

    Strain no. hereinStrainPerson who isolated strainDateLocality, habitatReference
    1Cylindrospermum alatosporum CCALA 988A. Lukešová1989Canada, Manitoba, Riding Mountain National Park, soil55
    2Cylindrospermum moravicum CCALA 993A. Lukešová2008Czech Republic, South Moravia, Moravian Karst, Amaterska Cave, cave sediment55
    3Cylindrospermum alatosporum CCALA 994A. Lukešová2011Czech Republic, Moravian Karst, earthworms collected from soil above Amaterska Cave, earthworm casings55
    4Anabaena sp. UHCC-0399M. WahlstenN/AFinland, Jurmo, Southwestern Archipelago National Park, copepods56
    5Anabaena minutissima UTEX B1613T. Kantz1967South Texas, USA, soil57
    6Symplocastrum muelleri NIVA-CYA 644O. M. Skulberg2009Norway, Møre og Romsdal county, Halsa municipality, western slope of Slettfjellet mountain in semiterrestrial alpine habitat, biofilm on turf in ombrotrophic blanket bog40
  • TABLE 2

    Deduced proteins encoded by the puw gene cluster in six cyanobacterial strains, including length and functional annotationc

    ProteinLength (aa) in strain no.:Predicted function(s)a
    123456
    ORF1659664664643643647ABC transporter
    PuwA2,8702,8702,8702,8542,8542,866NRPS
    ORF21,1161,4991,875643670376Patatin-like phospholipase
    ORF3696696Dynamin family protein
    PuwI709702711FAAL, ACP
    PuwJ427427529Cytochrome-like protein
    PuwB2,5342,5922,5922,5492,5372,555Hybrid PKS/NRPS, aminotransferase, oxygenase
    PuwC597590590597589FAAL
    PuwD101104969392ACP
    PuwK465Halogenase
    PuwE3,0773,1213,1213,0993,1123,113NRPS
    PuwF2,3705,851b5,851b5,877b5,871b3,310NRPS
    PuwG3,4922,620NRPS
    PuwH1,1021,0811,1021,1211,1211,408NRPS
    PuwL217O-Acetyltransferase
    • ↵a ACP, acyl carrier protein; FAAL, fatty acyl-AMP ligase; PKS, polyketide synthase; NRPS, nonribosomal peptide synthetase.

    • ↵b The proteins PuwF and PuwG are encoded in a single ORF in this strain.

    • ↵c The empty fields in the table indicate the absence of a particular protein.

  • 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 obtained
    Value when X, Y, and FA are as indicated
    X, Ala; Y, Thr; FA, C16X, Gly; Y, Thr; FA, C18X, Ala; Y, Thr; FA, C18X, Gly; Y, Val; FA, C18X, Ala; Y, Val; FA, C18
    m/zΔ(ppm)Sum formulam/zΔ(ppm)Sum formulam/zΔ(ppm)Sum formulam/zΔ(ppm)Sum formulam/zΔ(ppm)Sum formula
    Low fragmentation energy (60 eV)
        [M+H]+1,265.7338+0.7C59H101N12O181,279.7496+0.9C60H103N12O181,293.7654+0.8C61H105N12O181,277.7695+1.6C61H105N12O171,291.7870+0.1C62H107N12O17
        [M-CH3OH]+1,233.7170−6.6C58H97N12O171,247.7194+4.1C59H99N12O171,261.7494−7.3C60H101N12O17Low int.C60H101N12O16Low int.C61H103N12O16
        [M-CH3OH-NMeAsn]+1,105.6558−4.9C53H89N10O151,119.6619+3.7C54H91N10O151,133.681+0.6C55H93N10O151,117.6924−5.0C55H93N10O141,131.7307−25.0C56H95N10O14
        [M-CH3OH-NMeAsn-Dhb]+1,022.6180−4.7C49H84N9O141,036.6365−7.3C50H86N9O141050.6478−3.1C51H88N9O141,134.6603−10.3C51H88N9O131,048.6671−1.7C52H90N9O13
        [M-CH3OH-NMeAsn-Dhb-X]+951.5785−2.5C48H83N8O13979.589+18.8C48H83N8O13979.6041+3.4C48H83N8O13977.6481−20.5C49H85N8O12977.6518−24.1C49H85N8O12
        [M-CH3OH-NMeAsn-Dhb-X-Gln]+823.5253−9.4C41H71N6O11851.5473+1.8C43H75N6O11851.5478+1.2C43H75N6O11849.5838−16.7C44H76N6O10849.5589+12.5C44H77N6O10
        [M-CH3OH-NMeAsn-Dhb-X-Gln-Y]+722.4729−4.2C37H64N5O9750.5005+0.9C39H68N5O9750.5147−18.1C39H68N5O9Low int.C40H72N5O8Low int.C40H72N5O8
        [M-CH3OH-NMeAsn-Dhb-X-Gln-Y-Thr]+621.4223−0.2C33H57N4O7649.4526+1.4C35H61N4O7649.4539−0.6C35H61N4O7649.465−17.8C35H61N4O7649.4483+8.0C35H61N4O7
    High fragmentation energy (100 eV)
        Fragment 1411.3208+2.2C23H43N2O4439.3559−6.5C25H47N2O4439.3556−5.8C25H47N2O4439.3556−5.8C25H47N2O4439.3508+5.1C25H47N2O4
        Fragment 1, C2H4O2351.3006+0.0C21H39N2O2379.3334−4.0C23H43N2O2379.3329−2.6C23H43N2O2379.3328−2.4C23H43N2O2379.3360−10.8C23H43N2O2
        Fragment 2284.2583+0.4C17H34NO2312.2919−6.9C19H38NO2312.2892+1.6C19H38NO2Low int.C19H38NO2Low int.C19H38NO2
        Fragment 2, C2H4O2224.2367+2.6C15H30N252.26860.0C17H34N252.2687−0.5C17H34N252.2684+0.7C17H34N252.2677+3.5C17H34N
    • ↵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.)aMediumbIncubation temp (°C)Incubation time (h)Gram stain reactionc
    Pseudomonas sp. (2796)TGY2824−
    Micrococcus luteus (2688)TGY2824+
    Bacillus subtilis (251)TGY2824+
    Pseudomonas aeruginosa (25)TGY3724−
    Escherichia coli (396)TGY3724−
    Bacillus cereus (1881)TSA2824+
    Burkholderia cepacia (2487)TSA3724−
    Staphylococcus aureus (11)TSA3724+
    Xanthomonas campestris (104)NA2824−
    Burkholderia pseudomallei (33)NA3724−
    Salmonella enterica serovar Typhi (1306)NA3724−
    Arthrobacter globiformis (1863)NA2824−
    Kocuria varians (40)NA2824+
    Candida albicans (261)YM agar3724Yeast
    Cryptococcus albidus (264)YM agar2824Yeast
    Saccharomyces cerevisiae (1164)YM agar2824Yeast
    • ↵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.

Additional Files

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  • 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

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Alternative Biosynthetic Starter Units Enhance the Structural Diversity of Cyanobacterial Lipopeptides
Jan Mareš, Jan Hájek, Petra Urajová, Andreja Kust, Jouni Jokela, Kumar Saurav, Tomáš Galica, Kateřina Čapková, Antti Mattila, Esa Haapaniemi, Perttu Permi, Ivar Mysterud, Olav M. Skulberg, Jan Karlsen, David P. Fewer, Kaarina Sivonen, Hanne Hjorth Tønnesen, Pavel Hrouzek
Applied and Environmental Microbiology Feb 2019, 85 (4) e02675-18; DOI: 10.1128/AEM.02675-18

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Alternative Biosynthetic Starter Units Enhance the Structural Diversity of Cyanobacterial Lipopeptides
Jan Mareš, Jan Hájek, Petra Urajová, Andreja Kust, Jouni Jokela, Kumar Saurav, Tomáš Galica, Kateřina Čapková, Antti Mattila, Esa Haapaniemi, Perttu Permi, Ivar Mysterud, Olav M. Skulberg, Jan Karlsen, David P. Fewer, Kaarina Sivonen, Hanne Hjorth Tønnesen, Pavel Hrouzek
Applied and Environmental Microbiology Feb 2019, 85 (4) e02675-18; DOI: 10.1128/AEM.02675-18
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    • ABSTRACT
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KEYWORDS

biosynthesis
cyanobacteria
fatty acyl-AMP ligase
lipopeptides
nonribosomal peptide synthetase

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