Skip to content
1887

Abstract

A novel strain, 681, was isolated from a moss sample taken from the Chrutzelried woods in Canton Zürich, Switzerland. The strain showed potent activity against several fungi and oomycetes. It was affiliated to the genus by 16S rRNA gene sequence phylogeny. Genome sequencing showed a G+C content of 59.9 mol%. The highest average nucleotide identity was 86.63%, and the highest digital DNA–DNA hybridization value was 32.2% (with ln5), considerably below the thresholds for species delineation. Multi-locus phylogeny using 81 concatenated sequences indicated that the strain represented a new species within the subgroup. This study details its characterization as a new species. The 681 genome was screened using antiSMASH to reveal candidate secondary metabolite clusters for the strong antifungal activity exhibited by 681. Of the 15 clusters identified, we disrupted the seven best and tested for activity against . The pyrrolnitrin, rhizoxin, novel PKS-NRPS and acaterin gene clusters contributed significantly to the observed antifungal activity. Phenotypic analyses found that strain 681 cells were aerobic, Gram-negative, motile rods (mean length 2.60 µm and mean width of 0.67 µm) with one to three polar flagella. Optimal growth was at 30 °C, but growth also occurred at 8 °C. The pH range was 6–7, with some growth at pH 8. Robust growth occurred at 0–3% (w/v) NaCl and weak growth at 4% (w/v) NaCl, with the optimum at 1% (w/v) NaCl. Strain 681 was oxidase positive, hydrolysed arginine under anaerobic conditions, showed intense fluorescence on King’s medium B and produced a hypersensitive response in tobacco leaves. Following our investigations, we propose the designation sp. nov. for this new species. The type strain, 681, is available from the DSMZ and LMG/BCCM culture collections under the designations DSM 115721 and LMG 33039, respectively.

Funding
This study was supported by the:
  • Gebert Rüf Stiftung (Award GRS-076/19)
    • Principle Award Recipient: KirstyAgnoli
Loading

Article metrics loading...

/content/journal/ijsem/10.1099/ijsem.0.006624
2025-01-08
2025-01-15
Loading full text...

Full text loading...

References

  1. Lalucat J, Gomila M, Mulet M, Zaruma A, García-Valdés E. Past, present and future of the boundaries of the Pseudomonas genus: Proposal of Stutzerimonas gen. Nov. Syst Appl Microbiol 2022; 45:126289 [View Article] [PubMed]
    [Google Scholar]
  2. Gomila M, Peña A, Mulet M, Lalucat J, García-Valdés E. Phylogenomics and systematics in Pseudomonas. Front Microbiol 2015; 6:214 [View Article] [PubMed]
    [Google Scholar]
  3. Moore ERB, Mau M, Arnscheidt A, Böttger EC, Hutson RA et al. The determination and comparison of the 16S rRNA gene sequences of species of the genus Pseudomonas (sensu stricto) and estimation of the natural intrageneric relationships. Syst Appl Microbiol 1996; 19:478–492 [View Article]
    [Google Scholar]
  4. Mulet M, Lalucat J, García-Valdés E. DNA sequence-based analysis of the Pseudomonas species. Environ Microbiol 2010; 12:1513–1530 [View Article] [PubMed]
    [Google Scholar]
  5. Flury P, Aellen N, Ruffner B, Péchy-Tarr M, Fataar S et al. Insect pathogenicity in plant-beneficial Pseudomonads: phylogenetic distribution and comparative genomics. ISME J 2016; 10:2527–2542 [View Article] [PubMed]
    [Google Scholar]
  6. Xin XF, Kvitko B, He SY. Pseudomonas syringae: what it takes to be a pathogen. Nat Rev Microbiol 2018; 16:316–328 [View Article] [PubMed]
    [Google Scholar]
  7. Campos VL, Valenzuela C, Yarza P, Kämpfer P, Vidal R et al. Pseudomonas arsenicoxydans sp nov., an arsenite-oxidizing strain isolated from the Atacama desert. Syst Appl Microbiol 2010; 33:193–197 [View Article] [PubMed]
    [Google Scholar]
  8. Rojas-Solis D, Larsen J, Lindig-Cisneros R. Arsenic and mercury tolerant rhizobacteria that can improve phytoremediation of heavy metal contaminated soils. PeerJ 2023; 11:e14697 [View Article] [PubMed]
    [Google Scholar]
  9. Andersen SM, Johnsen K, Sørensen J, Nielsen P, Jacobsen CS. Pseudomonas frederiksbergensis sp. nov., isolated from soil at a coal gasification site. Int J Syst Evol Microbiol 2000; 50 Pt 6:1957–1964 [View Article] [PubMed]
    [Google Scholar]
  10. Cámara B, Strömpl C, Verbarg S, Spröer C, Pieper DH et al. Pseudomonas reinekei sp. nov., Pseudomonas moorei sp. nov. and Pseudomonas mohnii sp. nov., novel species capable of degrading chlorosalicylates or isopimaric acid. Int J Syst Evol Microbiol 2007; 57:923–931 [View Article] [PubMed]
    [Google Scholar]
  11. Gross H, Loper JE. Genomics of secondary metabolite production by Pseudomonas spp. Nat Prod Rep 2009; 26:1408–1446 [View Article] [PubMed]
    [Google Scholar]
  12. Nguyen DD, Melnik AV, Koyama N, Lu X, Schorn M et al. Indexing the Pseudomonas specialized metabolome enabled the discovery of poaeamide B and the bananamides. Nat Microbiol 2016; 2:16197 [View Article] [PubMed]
    [Google Scholar]
  13. Cesa-Luna C, Geudens N, Girard L, Roo V, Maklad HR et al. Charting the lipopeptidome of nonpathogenic Pseudomonas. mSystems 2023; 8:e0098822 [View Article]
    [Google Scholar]
  14. Zhou L, Höfte M, Hennessy RC. Does regulation hold the key to optimizing lipopeptide production in Pseudomonas for biotechnology?. Front Bioeng Biotechnol 2024; 12:1363183 [View Article]
    [Google Scholar]
  15. Ma Z, Geudens N, Kieu NP, Sinnaeve D, Ongena M et al. Biosynthesis, chemical structure, and structure-activity relationship of orfamide lipopeptides produced by Pseudomonas protegens and related species. Front Microbiol 2016; 7:382 [View Article]
    [Google Scholar]
  16. Bonnichsen L, Bygvraa Svenningsen N, Rybtke M, de Bruijn I, Raaijmakers JM et al. Lipopeptide biosurfactant viscosin enhances dispersal of Pseudomonas fluorescens SBW25 biofilms. Microbiology 2015; 161:2289–2297 [View Article]
    [Google Scholar]
  17. Nielsen TH, Thrane C, Christophersen C, Anthoni U, Sørensen J. Structure, production characteristics and fungal antagonism of tensin - a new antifungal cyclic lipopeptide from Pseudomonas fluorescens strain 96.578. J Appl Microbiol 2000; 89:992–1001 [View Article] [PubMed]
    [Google Scholar]
  18. Zachow C, Jahanshah G, de Bruijn I, Song C, Ianni F et al. The novel lipopeptide poaeamide of the endophyte Pseudomonas poae RE*1-1-14 is involved in pathogen suppression and root colonization. Mol Plant Microbe Interact 2015; 28:800–810 [View Article] [PubMed]
    [Google Scholar]
  19. Omoboye OO, Geudens N, Duban M, Chevalier M, Flahaut C et al. Pseudomonas sp. COW3 produces new bananamide-type cyclic lipopeptides with antimicrobial activity against Pythium myriotylum and Pyricularia oryzae. Molecules 2019; 24:4170 [View Article]
    [Google Scholar]
  20. Nielsen TH, Sørensen D, Tobiasen C, Andersen JB, Christophersen C et al. Antibiotic and biosurfactant properties of cyclic lipopeptides produced by fluorescent Pseudomonas spp. from the sugar beet rhizosphere. Appl Environ Microbiol 2002; 68:3416–3423 [View Article] [PubMed]
    [Google Scholar]
  21. Naganuma S, Sakai K, Hasumi K, Endo A. Acaterin, a novel inhibitor of acyl-CoA: cholesterol acyltransferase produced by Pseudomonas sp. A92. J Antibiot 1992; 45:1216–1221 [View Article]
    [Google Scholar]
  22. Vieweg L, Reichau S, Schobert R, Leadlay PF, Süssmuth RD. Recent advances in the field of bioactive tetronates. Nat Prod Rep 2014; 31:1554–1584 [View Article] [PubMed]
    [Google Scholar]
  23. Deora A, Hatano E, Tahara S, Hashidoko Y. Inhibitory effects of furanone metabolites of a rhizobacterium, Pseudomonas jessenii, on phytopathogenic Aphanomyces cochlioides and Pythium aphanidermatum. Plant Pathol 2010; 59:84–99 [View Article]
    [Google Scholar]
  24. Miller JH. Experiments in Molecular Genetics Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 1972
    [Google Scholar]
  25. Freire B, Ladra S, Parama JR. Memory-efficient assembly using Flye. IEEE/ACM trans comput. Biol Bioinform 2022; 19:3564–3577 [View Article] [PubMed]
    [Google Scholar]
  26. Li W, O’Neill KR, Haft DH, DiCuccio M, Chetvernin V et al. RefSeq: Expanding the prokaryotic genome annotation pipeline reach with protein family model curation. Nucleic Acids Res 2021; 49:D1020–D1028 [View Article] [PubMed]
    [Google Scholar]
  27. Haft DH, DiCuccio M, Badretdin A, Brover V, Chetvernin V et al. RefSeq: an update on prokaryotic genome annotation and curation. Nucleic Acids Res 2018; 46:D851–D860 [View Article] [PubMed]
    [Google Scholar]
  28. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP et al. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res 2016; 44:6614–6624 [View Article] [PubMed]
    [Google Scholar]
  29. Yoon S-H, Ha S-M, Kwon S, Lim J, Kim Y et al. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol 2017; 67:1613–1617 [View Article] [PubMed]
    [Google Scholar]
  30. Alanjary M, Steinke K, Ziemert N. AutoMLST: an automated web server for generating multi-locus species trees highlighting natural product potential. Nucleic Acids Res 2019; 47:W276–W282 [View Article] [PubMed]
    [Google Scholar]
  31. Meier-Kolthoff JP, Carbasse JS, Peinado-Olarte RL, Göker M. TYGS and LPSN: a database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes. Nucleic Acids Res 2022; 50:D801–D807 [View Article] [PubMed]
    [Google Scholar]
  32. Meier-Kolthoff JP, Auch AF, Klenk H-P, Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 2013; 14:60 [View Article] [PubMed]
    [Google Scholar]
  33. Meier-Kolthoff JP, Göker M. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat Commun 2019; 10:2182 [View Article] [PubMed]
    [Google Scholar]
  34. Yoon SH, Ha SM, Lim J, Kwon S, Chun J. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek 2017; 110:1281–1286 [View Article] [PubMed]
    [Google Scholar]
  35. Lee I, Ouk Kim Y, Park SC, Chun J. OrthoANI: An improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol 2016; 66:1100–1103 [View Article] [PubMed]
    [Google Scholar]
  36. Blin K, Shaw S, Kloosterman AM, Charlop-Powers Z, van Wezel GP et al. antiSMASH 6.0: improving cluster detection and comparison capabilities. Nucleic Acids Res 2021; 49:W29–W35 [View Article] [PubMed]
    [Google Scholar]
  37. Blin K, Medema MH, Kazempour D, Fischbach MA, Breitling R et al. antiSMASH 2.0--a versatile platform for genome mining of secondary metabolite producers. Nucleic Acids Res 2013; 41:W204–12 [View Article] [PubMed]
    [Google Scholar]
  38. Laslett D, Canback B. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res 2004; 32:11–16 [View Article] [PubMed]
    [Google Scholar]
  39. Liu B, Zheng D, Zhou S, Chen L, Yang J. VFDB 2022: a general classification scheme for bacterial virulence factors. Nucleic Acids Res 2022; 50:D912–D917 [View Article] [PubMed]
    [Google Scholar]
  40. Chen LH, Yang J, Yu J, Ya ZJ, Sun LL et al. VFDB: a reference database for bacterial virulence factors. Nucleic Acids Research 2004; 33:D325–D328 [View Article]
    [Google Scholar]
  41. Zierep PF, Ceci AT, Dobrusin I, Rockwell-Kollmann SC, Günther S. SeMPI 2.0 - A web server for PKS and NRPS predictions combined with metabolite screening in natural product databases. Metabolites 2020; 11:13 [View Article] [PubMed]
    [Google Scholar]
  42. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 2012; 9:676–682 [View Article] [PubMed]
    [Google Scholar]
  43. Schwyn B, Neilands JB. Universal chemical assay for the detection and determination of siderophores. Anal Biochem 1987; 160:47–56 [View Article] [PubMed]
    [Google Scholar]
  44. Agnoli K, Schwager S, Uehlinger S, Vergunst A, Viteri DF et al. Exposing the third chromosome of Burkholderia cepacia complex strains as a virulence plasmid. Mol Microbiol 2012; 83:362–378 [View Article] [PubMed]
    [Google Scholar]
  45. Lelliott RA, Billing E, Hayward AC. A determinative scheme for the fluorescent plant pathogenic Pseudomonads. J Appl Bacteriol 1966; 29:470–489 [View Article] [PubMed]
    [Google Scholar]
  46. KOVACS N. Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature 1956; 178:703 [View Article] [PubMed]
    [Google Scholar]
  47. Woodward EJ, Robinson K. An improved formulation and method of preparation of crystal violet pectate medium for detection of pectolytic Erwinia. Lett Appl Microbiol 1990; 10:171–173 [View Article]
    [Google Scholar]
  48. Thornley MJ. The differentiation of Pseudomonas from other gram-negative bacteria on the basis of arginine metabolism. J Appl Bacteriol 1960; 23:37–52 [View Article]
    [Google Scholar]
  49. KLEMENT Z. Rapid detection of the pathogenicity of phytopathogenic Pseudomonads. Nature 1963; 199:299–300 [View Article] [PubMed]
    [Google Scholar]
  50. Shastri S, Spiewak HL, Sofoluwe A, Eidsvaag VA, Asghar AH et al. An efficient system for the generation of marked genetic mutants in members of the genus Burkholderia. Plasmid 2017; 89:49–56 [View Article] [PubMed]
    [Google Scholar]
  51. Kim M, Oh HS, Park SC, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 2014; 64:346–351 [View Article] [PubMed]
    [Google Scholar]
  52. Riesco R, Trujillo ME. Update on the proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol 2024; 74:006300 [View Article] [PubMed]
    [Google Scholar]
  53. Craig L, Forest KT, Maier B. Type IV pili: dynamics, biophysics and functional consequences. Nat Rev Microbiol 2019; 17:429–440 [View Article] [PubMed]
    [Google Scholar]
  54. Tripathi RK, Gottlieb D. Mechanism of action of the antifungal antibiotic pyrrolnitrin. J Bacteriol 1969; 100:310–318 [View Article] [PubMed]
    [Google Scholar]
  55. Pacheco-Moreno A, Stefanato FL, Ford JJ, Trippel C, Uszkoreit S et al. Pan-genome analysis identifies intersecting roles for Pseudomonas specialized metabolites in potato pathogen inhibition. Elife 2021; 10:e71900 [View Article] [PubMed]
    [Google Scholar]
  56. Johnston I, Osborn LJ, Markley RL, McManus EA, Kadam A et al. Identification of essential genes for Escherichia coli aryl polyene biosynthesis and function in biofilm formation. NPJ Biofilms Microbiomes 2021; 7:56 [View Article] [PubMed]
    [Google Scholar]
  57. Schalk IJ, Guillon L. Pyoverdine biosynthesis and secretion in Pseudomonas aeruginosa: implications for metal homeostasis. Environ Microbiol 2013; 15:1661–1673 [View Article] [PubMed]
    [Google Scholar]
  58. Segade Y, Montaos MA, Rodríguez J, Jiménez C. A short stereoselective synthesis of prepiscibactin using A SmI2-mediated reformatsky reaction and Zn2+-induced asymmetric thiazolidine formation. Org Lett 2014; 16:5820–5823 [View Article] [PubMed]
    [Google Scholar]
  59. Yoshida M, Takeuchi H, Ishida Y, Yashiroda Y, Yoshida M et al. Synthesis, structure determination, and biological evaluation of destruxin E. Org Lett 2010; 12:3792–3795 [View Article]
    [Google Scholar]
  60. Zhang Q, Ji Y, Xiao Q, Chng S, Tong Y et al. Role of Vfr in the regulation of antifungal compound production by Pseudomonas fluorescens FD6. Microbiol Res 2016; 188–189:106–112 [View Article] [PubMed]
    [Google Scholar]
  61. Kirner S, Hammer PE, Hill DS, Altmann A, Fischer I et al. Functions encoded by pyrrolnitrin biosynthetic genes from Pseudomonas fluorescens. J Bacteriol 1998; 180:1939–1943 [View Article] [PubMed]
    [Google Scholar]
  62. Arima K, Fukuta A, Imanaka H, Kousaka M, Tamura G. Pyrrolnitrin new antibiotic substance produced by Pseudomonas. Agric Biol Chem 1964; 28: [View Article]
    [Google Scholar]
  63. Chernin L, Brandis A, Ismailov Z, Chet I. Pyrrolnitrin production by an Enterobacter agglomerans strain with a broad spectrum of antagonistic activity towards fungal and bacterial phytopathogen. Curr Microbiol 1996; 32:208–212 [View Article]
    [Google Scholar]
  64. Costa R, van Aarle IM, Mendes R, van Elsas JD. Genomics of pyrrolnitrin biosynthetic loci: evidence for conservation and whole-operon mobility within gram-negative bacteria. Environ Microbiol 2009; 11:159–175 [View Article] [PubMed]
    [Google Scholar]
  65. Pawar S, Chaudhari A, Prabha R, Shukla R, Singh DP. Microbial pyrrolnitrin: natural metabolite with immense practical utility. Biomolecules 2019; 9:443 [View Article] [PubMed]
    [Google Scholar]
  66. Partida-Martinez LP, Hertweck C. Pathogenic fungus harbours endosymbiotic bacteria for toxin production. Nature 2005; 437:884–888 [View Article] [PubMed]
    [Google Scholar]
  67. Richter I, Radosa S, Cseresnyés Z, Ferling I, Büttner H et al. Toxin-producing endosymbionts shield pathogenic fungus against micropredators. mBio 2022; 13:e0144022 [View Article] [PubMed]
    [Google Scholar]
  68. Klapper M, Schlabach K, Paschold A, Zhang S, Chowdhury S et al. Biosynthesis of Pseudomonas-derived butenolides. Angew Chem Int Ed Engl 2020; 59:5607–5610 [View Article] [PubMed]
    [Google Scholar]
  69. Hatano E, Hashidoko Y, Deora A, Fukushi Y, Tahara S. Isolation and structure elucidation of Peronosporomycetes hyphal branching-inducing factors produced by Pseudomonas jessenii EC-S101. Biosci Biotechnol Biochem 2007; 71:1601–1605 [View Article] [PubMed]
    [Google Scholar]
  70. Andreolli M, Zapparoli G, Angelini E, Lucchetta G, Lampis S et al. Pseudomonas protegens MP12: A plant growth-promoting endophytic bacterium with broad-spectrum antifungal activity against grapevine phytopathogens. Microbiol Res 2019; 219:123–131 [View Article] [PubMed]
    [Google Scholar]
  71. Liu F, Yang S, Xu F, Zhang Z, Lu Y et al. Characteristics of biological control and mechanisms of Pseudomonas chlororaphis zm-1 against peanut stem rot. BMC Microbiol 2022; 22:9 [View Article]
    [Google Scholar]
  72. Ranjbariyan A, Shams-Ghahfarokhi M, Razzaghi-Abyaneh M. Antifungal activity of a soil isolate of Pseudomonas chlororaphis against medically important dermatophytes and identification of a phenazine-like compound as its bioactive metabolite. J Mycol Med 2014; 24:e57–64 [View Article] [PubMed]
    [Google Scholar]
  73. Ntana F, Hennessy RC, Zervas A, Stougaard P. Pseudomonas nunensis sp. nov. isolated from a suppressive potato field in Greenland. Int J Syst Evol Microbiol 2023; 73: [View Article] [PubMed]
    [Google Scholar]
  74. Michelsen CF, Stougaard P. A novel antifungal Pseudomonas fluorescens isolated from potato soils in Greenland. Curr Microbiol 2011; 62:1185–1192 [View Article] [PubMed]
    [Google Scholar]
/content/journal/ijsem/10.1099/ijsem.0.006624
Loading
/content/journal/ijsem/10.1099/ijsem.0.006624
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error