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Publications2020-12-04T10:56:47+00:00

Publications

Crouzet J, Arguelles-Arias A, Dhondt-Cordelier S, Cordelier S, Pršic J, Hoff G, Mazeyrat-Gourbeyre F, Baillieul F, Clément C, Ongena M and Dorey S (2020) “Biosurfactants in Plant Protection Against Diseases: Rhamnolipids and Lipopeptides Case Study.” Frontiers in Bioengineering and Biotechnology. 8:1014.

Biosurfactants are amphiphilic surface-active molecules that are produced by a variety of microorganisms including fungi and bacteria. PseudomonasBurkholderia, and Bacillus species are known to secrete rhamnolipids and lipopeptides that are used in a wide range of industrial applications. Recently, these compounds have been studied in a context of plant-microbe interactions. This mini-review describes the direct antimicrobial activities of these compounds against plant pathogens. We also provide the current knowledge on how rhamnolipids and lipopeptides stimulate the plant immune system leading to plant resistance to phytopathogens. Given their low toxicity, high biodegradability and ecological acceptance, we discuss the possible role of these biosurfactants as alternative strategies to reduce or even replace pesticide use in agriculture.

https://www.frontiersin.org/articles/10.3389/fbioe.2020.01014/full

Andric S, Meyer T and Ongena M (2020) Bacillus Responses to Plant-Associated Fungal and Bacterial Communities. Frontiers in Microbiology. 11:1350.

Some members of root-associated Bacillus species have been developed as biocontrol agents due to their contribution to plant protection by directly interfering with the growth of pathogens or by stimulating systemic resistance in their host. As rhizosphere-dwelling bacteria, these bacilli are surrounded and constantly interacting with other microbes via different types of communications. With this review, we provide an updated vision of the molecular and phenotypic responses of Bacillus upon sensing other rhizosphere microorganisms and/or their metabolites. We illustrate how Bacillus spp. may react by modulating the production of secondary metabolites, such as cyclic lipopeptides or polyketides. On the other hand, some developmental processes, such as biofilm formation, motility, and sporulation may also be modified upon interaction, reflecting the adaptation of Bacillus multicellular communities to microbial competitors for preserving their ecological persistence. This review also points out the limited data available and a global lack of knowledge indicating that more research is needed in order to, not only better understand the ecology of bacilli in their natural soil niche, but also to better assess and improve their promising biocontrol potential.

https://www.frontiersin.org/articles/10.3389/fmicb.2020.01350/full

Oni, F.E., Geudens, N., Onyeka, J.T., Olorunleke, O.F., Salami, A.E., Omoboye, O.O., Arias, A.A., Adiobo, A., De Neve, S., Ongena, M., Martins, J.C. and Höfte, M. (2020b) Cyclic lipopeptide-producing Pseudomonas koreensis group strains dominate the cocoyam rhizosphere of a Pythium root rot suppressive soil contrasting with P. putida prominence in conducive soils. Environmental Microbiology 00.

Pseudomonas isolates from tropical environments have been underexplored and may form an untapped reservoir of interesting secondary metabolites. In this study, we compared Pseudomonas and cyclic lipopeptide (CLP) diversity in the rhizosphere of a cocoyam root rot disease (CRRD) suppressive soil in Boteva, Cameroon with those from four conducive soils in Cameroon and Nigeria. Compared with other soils, Boteva andosols were characterized by high silt, organic matter, nitrogen and calcium. Besides, the cocoyam rhizosphere at Boteva was characterized by strains belonging mainly to the Pkoreensis and Pputida (sub)groups, with representations in the PfluorescensPchlororaphisPjessenii and Pasplenii (sub)groups. In contrast, Pputida isolates were prominent in conducive soils. Regarding CLP diversity, Boteva was characterized by strains producing 11 different CLP types with cocoyamide A producers, belonging to the Pkoreensis group, being the most abundant. However, putisolvin III‐V producers were the most dominant in the rhizosphere of conducive soils in both Cameroon and Nigeria. Furthermore, we elucidated the chemical structure of putisolvin derivatives—putisolvin III‐V, and described its biosynthetic gene cluster. We show that high Pseudomonas and metabolic diversity may be driven by microbial competition, which likely contributes to soil suppressiveness to CRRD.

https://sfamjournals.onlinelibrary.wiley.com/doi/abs/10.1111/1462-2920.15127

Oni, F.E., Geudens, N., Adiobo, A., Omoboye, O.O., Enow, E.A., Onyeka, J.T., Salami, A.E., De Mot, R., Martins, J.C. and Höfte, M. (2020a) “Biosynthesis and antimicrobial activity of pseudodesmin and viscosinamide cyclic lipopeptides produced by pseudomonads associated with the cocoyam rhizosphere.” Microorganisms 8, 1–26.

Pseudomonas cyclic lipopeptides (CLPs) are encoded non-ribosomally by biosynthetic gene clusters (BGCs) and possess diverse biological activities. In this study, we conducted chemical structure and BGC analyses with antimicrobial activity assays for two CLPs produced by Pseudomonas strains isolated from the cocoyam rhizosphere in Cameroon and Nigeria. LC-MS and NMR analyses showed that the Pseudomonas sp. COR52 and A2W4.9 produce pseudodesmin and viscosinamide, respectively. These CLPs belong to the Viscosin group characterized by a nonapeptidic moiety with a 7-membered macrocycle. Similar to other Viscosin-group CLPs, the initiatory non-ribosomal peptide synthetase (NRPS) gene of the viscosinamide BGC is situated remotely from the other two NRPS genes. In contrast, the pseudodesmin genes are all clustered in a single genomic locus. Nano- to micromolar levels of pseudodesmin and viscosinamide led to the hyphal distortion and/or disintegration of Rhizoctonia solani AG2-2 and Pythium myriotylum CMR1, whereas similar levels of White Line-Inducing Principle (WLIP), another member of the Viscosin group, resulted in complete lysis of both soil-borne phytopathogens. In addition to the identification of the biosynthetic genes of these two CLPs and the demonstration of their interaction with soil-borne pathogens, this study provides further insights regarding evolutionary divergence within the Viscosin group.

https://www.mdpi.com/2076-2607/8/7/1079

Girard, L., Lood, C., Rokni-zadeh, H., Noort, V. Van, Lavigne, R. and De Mot, R. (2020b) “Reliable identification of environmental Pseudomonas isolates using the rpoD gene.” Microorganisms 8, 1–12.

The taxonomic affiliation of Pseudomonas isolates is currently assessed by using the 16S rRNA gene, MultiLocus Sequence Analysis (MLSA), or whole genome sequencing. Therefore, microbiologists are facing an arduous choice, either using the universal marker, knowing that these affiliations could be inaccurate, or engaging in more laborious and costly approaches. The rpoD gene, like the 16S rRNA gene, is included in most MLSA procedures and has already been suggested for the rapid identification of certain groups of Pseudomonas. However, a comprehensive overview of the rpoD-based phylogenetic relationships within the Pseudomonas genus is lacking. In this study, we present the rpoD-based phylogeny of 217 type strains of Pseudomonas and defined a cutoff value of 98% nucleotide identity to differentiate strains at the species level. To validate this approach, we sequenced the rpoD of 145 environmental isolates and complemented this analysis with whole genome sequencing. The rpoD sequence allowed us to accurately assign Pseudomonas isolates to 20 known species and represents an excellent first diagnostic tool to identify new Pseudomonas species. Finally, rpoD amplicon sequencing appears as a reliable and low-cost alternative, particularly in the case of large environmental studies with hundreds or thousands of isolates.

https://www.mdpi.com/2076-2607/8/8/1166

Girard, L., Höfte, M. and De Mot, R., (2020a) “Lipopeptide families at the interface between pathogenic and beneficial Pseudomonas-plant interactions”. Critical Reviews in Microbiology 46, 397–419.

Lipopeptides (LPs) are a prominent class of molecules among the steadily growing spectrum of specialized metabolites retrieved from Pseudomonas, in particular soil-dwelling and plant-associated isolates. Among the multiple LP families, pioneering research focussed on phytotoxic and antimicrobial cyclic lipopeptides (CLPs) of the ubiquitous plant pathogen Pseudomonas syringae (syringomycin and syringopeptin). Their non-ribosomal peptide synthetases (NRPSs) are embedded in biosynthetic gene clusters (BGCs) that are tightly co-clustered on a pathogenicity island. Other members of the P. syringae group (Pseudomonas cichorii) and some species of the Pseudomonas asplenii group and Pseudomonas fluorescens complex have adopted these biosynthetic strategies to co-produce their own mycin and peptin variants, in some strains supplemented with an analogue of the P. syringae linear LP (LLP), syringafactin. This capacity is not confined to phytopathogens but also occurs in some biocontrol strains, which indicates that these LP families not solely function as general virulence factors. We address this issue by scrutinizing the structural diversity and bioactivities of LPs from the mycin, peptin, and factin families in a phylogenetic and evolutionary perspective. BGC functional organization (including associated regulatory and transport genes) and NRPS modular architectures in known and candidate LP producers were assessed by genome mining.

https://www.tandfonline.com/doi/full/10.1080/1040841X.2020.1794790

De Vleeschouwer, M., Van Kersavond, T., Verleysen, Y., Sinnaeve, D., Coenye, T., Martins, J. C., & Madder, A. (2020). “Identification of the Molecular Determinants Involved in Antimicrobial Activity of Pseudodesmin A, a Cyclic Lipopeptide From the Viscosin Group.” Frontiers in Microbiology, 11(646).

Cyclic lipo(depsi)peptides (CLiPs) from Pseudomonas constitute a class of natural products involved in a broad range of biological functions for their producers. They also display interesting antimicrobial potential including activity against Gram-positive bacteria. Literature has indicated that these compounds can induce membrane permeabilization, possibly through pore-formation, leading to the general view that the cellular membrane constitutes the primary target in their mode of action. In support of this view, we previously demonstrated that the enantiomer of pseudodesmin A, a member of the viscosin group of CLiPs, shows identical activity against a test panel of six Gram-positive bacterial strains. Here, a previously developed total organic synthesis route is used and partly adapted to generate 20 novel pseudodesmin A analogs in an effort to derive links between molecular constitution, structure and activity. From these, the importance of a macrocycle closed by an ester bond as well as a critical length of β-OH fatty acid chain capping the N-terminus is conclusively demonstrated, providing further evidence for the importance of peptide-membrane interactions in the mode of action. Moreover, an alanine scan is used to unearth the contribution of specific amino acid residues to biological activity. Subsequent interpretation in terms of a structural model describing the location and orientation of pseudodesmin A in a membrane environment, allows first insight in the peptide-membrane interactions involved. The biological screening also identified residue positions that appear less sensitive to conservative modifications, allowing the introduction of a non-perturbing tryptophan residue which will pave the way toward biophysical studies using fluorescence spectroscopy.

https://www.frontiersin.org/articles/10.3389/fmicb.2020.00646/full

Kune, C., McCann, A., La Rocca, R., Arias, A.A., Tiquet, M., Van Kruining, D., Martinez, P.M., Ongena, M., Eppe, G., Quinton, L., Far, J. and De Pauw, E., (2019) “Rapid Visualization of Chemically Related Compounds Using Kendrick Mass Defect As a Filter in Mass Spectrometry Imaging”. Analytical Chemistry 91, 13112–13118.

Kendrick mass defect (KMD) analysis is widely used for helping the detection and identification of chemically related compounds based on exact mass measurements. We report here the use of KMD as a criterion for filtering complex mass spectrometry data set. The method allow automated, easy and efficient data processing, enabling the reconstruction of 2D distributions of families of homologous compounds from MSI images. We show that KMD filtering, based on in-house software, is suitable and robust for high resolution (full width at half-maximum, fwhm, at m/z 410 of 20 000) and very high-resolution (fwhm, at m/z 410 of 160 000) MSI data. This method has been successfully applied to two different types of samples, bacteria cocultures, and brain tissue sections.

https://pubs.acs.org/doi/10.1021/acs.analchem.9b03333

Geudens N., Kovács B., Sinnaeve D., Oni, F.E., Höfte M. and Martins J.C. (2019). “Conformation and dynamics of the cyclic lipopeptide viscosinamide at the water-lipid interface” Molecules 24(12).

Cyclic lipodepsipeptides or CLiPs from Pseudomonas are secondary metabolites that mediate a wide range of biological functions for their producers, and display antimicrobial and anticancer activities. Direct interaction of CLiPs with the cellular membranes is presumed to be essential in causing these. To understand the processes involved at the molecular level, knowledge of the conformation and dynamics of CLiPs at the water-lipid interface is required to guide the interpretation of biophysical investigations in model membrane systems. We used NMR and molecular dynamics to study the conformation, location and orientation of the Pseudomonas CLiP viscosinamide in a water/dodecylphosphocholine solution. In the process, we demonstrate the strong added value of combining uniform, isotope-enriched viscosinamide and protein NMR methods. In particular, the use of techniques to determine backbone dihedral angles and detect and identify long-lived hydrogen bonds, establishes that the solution conformation previously determined in acetonitrile is maintained in water/dodecylphosphocholine solution. Paramagnetic relaxation enhancements pinpoint viscosinamide near the water-lipid interface, with its orientation dictated by the amphipathic distribution of hydrophobic and hydrophilic residues. Finally, the experimental observations are supported by molecular dynamics simulations. Thus a firm structural basis is now available for interpreting biophysical and bioactivity data relating to this class of compounds.

https://www.mdpi.com/1420-3049/24/12/2257

Omoboye, O. O., Geudens, N., Duban, M., Chevalier, M., Flahaut, C., Martins, J. C., Leclere, V. Oni, F.E. & Höfte, M. (2019). “Pseudomonas sp. COW3 Produces New Bananamide-Type Cyclic Lipopeptides with Antimicrobial Activity against Pythium myriotylum and Pyricularia oryzae.“ Molecules, 24(22).

Pseudomonas species are metabolically robust, with capacity to produce secondary metabolites including cyclic lipopeptides (CLPs). Herein we conducted a chemical analysis of a crude CLP extract from the cocoyam rhizosphere-derived biocontrol strain Pseudomonas sp. COW3. We performed in silico analyses on its whole genome, and conducted in vitro antagonistic assay using the strain and purified CLPs. Via LC-MS and NMR, we elucidated the structures of four novel members of the bananamide group, named bananamides D-G. Besides variability in fatty acid length, bananamides D-G differ from previously described bananamides A-C and MD-0066 by the presence of a serine and aspartic acid at position 6 and 2, respectively. In addition, bananamide G has valine instead of isoleucine at position 8. Kendrick mass defect (KMD) allowed the assignment of molecular formulae to bananamides D and E. We unraveled a non-ribosomal peptide synthetase cluster banA, banB and banC which encodes the novel bananamide derivatives. Furthermore, COW3 displayed antagonistic activity and mycophagy against Pythium myriotylum, while it mainly showed mycophagy on Pyricularia oryzae. Purified bananamides D-G inhibited the growth of P. myriotylum and P. oryzae and caused hyphal distortion. Our study shows the complementarity of chemical analyses and genome mining in the discovery and elucidation of novel CLPs. In addition, structurally diverse bananamides differ in their antimicrobial activity.

https://www.mdpi.com/1420-3049/24/22/4170

Oni F. E., Geudens N., Omoboye O.O., Bertier L., Hua H.G.K., Abiodo A., Sinnaeve D., Martins J.C. and Höfte M. (2019). “Fluorescent Pseudomonas and cyclic lipopeptide diversity in the rhizosphere of cocoyam (Xanthosoma sagittifolium).” Environmental Microbiology.

Cocoyam (Xanthosoma sagittifolium (L.)), an important tuber crop in the tropics, is severely affected by the cocoyam root rot disease (CRRD) caused by Pythium myriotylum. The white cocoyam genotype is very susceptible while the red cocoyam has some field tolerance to CRRD. Fluorescent Pseudomonas isolates obtained from the rhizosphere of healthy red and white cocoyams from three different fields in Cameroon were taxonomically characterized. The cocoyam rhizosphere was enriched with P. fluorescens complex and P. putida isolates independent of the plant genotype. LC-MS and NMR analyses revealed that 50% of the Pseudomonas isolates produced cyclic lipopeptides (CLPs) including entolysin, lokisin, WLIP, putisolvin and xantholysin together with eight novel CLPs. In general, CLP types were linked to specific taxonomic groups within the fluorescent pseudomonads. Representative CLP-producing bacteria showed effective control against CRRD while purified CLPs caused hyphal branching or hyphal leakage in P. myriotylum. The structure of cocoyamide A, a CLP which is predominantly produced by P. koreensis group isolates within the P. fluorescens complex is described. Compared to the white cocoyam, the red cocoyam rhizosphere appeared to support a more diverse CLP spectrum. It remains to be investigated whether this contributes to the field tolerance displayed by the red cocoyam.

https://onlinelibrary.wiley.com/doi/abs/10.1111/1462-2920.14520

Geudens, N. and J. C. Martins (2018). “Cyclic lipodepsipeptides from Pseudomonas spp. – Biological Swiss-Army Knives.” Frontiers in Microbiology 9(1867).

Cyclic lipodepsipeptides produced by Pseudomonas spp. (Ps-CLPs) are biosurfactants that constitute a diverse class of versatile bioactive natural compounds with promising application potential. While chemically diverse, they obey a common structural blue-print, allowing the definition of 14 distinct groups with multiple structurally homologous members. In addition to antibacterial and antifungal properties the reported activity profile of Ps-CLPs includes their effect on bacterial motility, biofilm formation, induced defense responses in plants and their insecticidal activity and anti-proliferation effects on human cancer cell-lines. To further validate their status of potential bioactive substances, we assessed the results of 774 biological tests on 51 Ps-CLPs available from literature. From this, a fragmented overview emerges. While Ps-CLPs taken as a group demonstrate a broad activity profile, reports on individual Ps-CLPs mostly test on activities that reflect the scientific focus of the individual authors. The current data is therefore found to be to sparse to correlate structure and biological function. Consequently, generalizations with respect to activity across the Gram-positive and Gram-negative divide should be nuanced. As the discovery of novel Ps-CLPs accelerates, current challenges to complete and maintain a useful overview of biological activity are discussed.

https://www.frontiersin.org/articles/10.3389/fmicb.2018.01867/full