Biology subjects

Larsen, T. O.

Publications and source records attributed to Larsen, T. O..

7 recordsLinked to original sources

bioRxiv2026

Beyond Antimicrobial Activity: Soil Bacteria Reveal a Biotransformation Fate for the Lanthipeptide Nisin

Natural products are central mediators of microbial interactions. However, once released into the environment, they also become available for neighboring microorganisms capable of degrading and modifying them through biotransformation. These biotransformations may fundamentally reshape metabolomes and influence community behavior, yet our understanding of these processes remains limited. Ribosomally synthesized peptides are particularly compelling in this context because their structural complexity and potent antimicrobial activity coexist with the potential to yield essential nutrients and reduced bioactivity through biotransformation. Identifying the pathways underlying these biotransformations is essential for understanding mechanisms that support microbial coexistence and nutrient recycling in soil microbiomes. Here, we used nisin as a model peptide to investigate biotransformation by soil bacteria. Selective isolation under nisin-rich, carbon-limited conditions yielded two Gram-negative isolates, Burkholderia stabilis and Pseudomonas fragi. Using growth assays and liquid chromatography-mass spectrometry, we found that both isolates grow in the presence of nisin while biotransforming and depleting the peptide. Burkholderia stabilis completely converted nisin through sequential cleavage of the C-terminus, hinge region and lanthionine ring C, whereas Pseudomonas fragi showed more limited processing restricted to the C-terminal region. Although these biotransformations dismantled structural features required for nisins antimicrobial activity, the intrinsic resistance of both isolates suggests a role beyond detoxification. We further detected nisin biosynthetic genes in the source environment, supporting nisins ecological relevance and suggesting that these bacteria may participate in its turnover in soil. Together, these findings reveal extensive microbial processing of nisin and support a role for antimicrobial peptide recycling in soil microbiomes. ImportanceNatural products are often studied through the lens of their biological activities, but much less attention has been paid to what happens to these molecules after they enter complex microbial communities. Using the lantibiotic nisin as a model system, we show that soil bacteria can extensively biotransform and deplete an antimicrobial peptide through extracellular enzymatic activity. The presence of both nisin-producing and nisin-biotransforming microorganisms in the same soil environment suggests that antimicrobial peptides may be continuously produced and recycled in nature. Our findings highlight biotransformation as an important but underexplored process governing the persistence, turnover, and ecological roles of microbial natural products.

microbiology↗

bioRxiv2024

RoCi - A Single Step Multi-Copy Integration System Based on Rolling-Circle Replication

Fungi are often used as cell factories for homologous and heterologous production of enzymes and metabolites. One strategy to obtain high yielding strains is to enhance the expression level of the gene(s) responsible for production of the product by inserting multiple copies of the gene-expression cassette. Typically, this is achieved by transforming non-homologous end-joining proficient strains with large amounts of a DNA vector, which randomly integrates in multiple copies at different loci, or more often, into a single locus with copies arranged as mixed orientation repeats. The majority of strains produced in this manner are unstable and substantial screening is necessary to identify strains with high and stable production. Moreover, the randomness of the insertion processes makes it difficult to determine how and where the copies are positioned in the genome. To this end, we envisioned that the instability of gene clusters made by the classical method is mostly due to the presence of a mixture of directly and inverted repeats. In such clusters, hairpins formed by inverted repeats may cause frequent recombinogenic lesions during replication to induce gene-expression cassette copy-loss by direct-repeat recombination. It is therefore possible that strains with gene-expression cassette clusters made solely by direct repeats would be more stable. Using Aspergillus nidulans as a model, we tested this idea and developed RoCi, a simple and efficient method to facilitate integration of multiple directly repeated gene-expression cassettes into a defined genomic locus through rolling-circle replication without pre-engineering requirements for strain preparation. In addition, we demonstrate that RoCi can be performed without E. coli based cloning, making it compatible with medium-high throughput experiments. Analyzing strains produced by RoCi, we have constructed strains bearing up to 68 mRFP GECs and we show that an mRFP multi-copy gene-array supports high and stable mRFP production for at least [~]150 generations on solid medium. In liquid culture we observed a minor average copy loss at 1 L scale. This loss could be eliminated by extending the gene-expression cassette with a crippled selection marker. To demonstrate the strength of the method, we used it to produce stable and high yielding cell factories for production of the specialized metabolite cordycepin on solid medium and of the enzyme {beta}-glucuronidase in submerged culture. Finally, we show that RoCi can also be applied in the industrial workhorses A. niger and A. oryzae indicating that RoCi is generally applicable in fungi.

synthetic biology↗

bioRxiv2024

Insights into the nature of the microalgal toxins from the Chrysochromulina leadbeateri blooms in Northern Norwegian fjords

In May-June 2019, the microalga Chrysochromulina leadbeateri caused a massive fish-killing event in several fjords in Northern Norway, resulting in the largest direct impact ever on aquaculture in northern Europe due to toxic algae. Motivated by the fact that no algal toxins have previously been described from C. leadbeateri, we set out to investigate the chemical nature and toxicity of secondary metabolites in extracts of two strains (UIO 393, UIO 394) isolated from the 2019 bloom, as well as one older strain (UIO 035) isolated during a bloom in Northern Norway in 1991. Initial LC-DAD-MS/MS-based molecular networking analysis of the crude MeOH extracts of the cultivated strains showed that their profiles of small organic molecules, including a large number of known lipids, were very similar, suggesting that the same class of toxin(s) were likely the causative agents of the two harmful algal bloom (HAB) events. Next, bioassay-guided fractionation using the RTgill-W1 cell line and metabolomics analysis pointed to a major compound affording [M+H]+ ions at m/z 1399.8333 as a possible toxin, corresponding to a compound with the formula C67H127ClO27. Moreover, our study unveiled a series of minor analogues exhibiting distinct patterns of chlorination and sulfation, together defining a new family of compounds, which we propose to name leadbeaterins. Remarkably, these suspected toxins were detected in situ in samples collected during the 2019 bloom close to Tromso, thereby substantiating their likely role in fish kills. The elemental compositions of the putative C. leadbeateri ichthyotoxins strongly indicate them to be long linear polyhydroxylated polyketides, structurally similar to sterolysins, reported from a number of dinoflagellates.

ecology↗

bioRxiv2023

Unveiling the Microbial Diversity and Associated Secondary Metabolism on Black Apples

Black apples are the late-stage microbial decomposition of apples after having fallen to the ground. This phenomenon is highly comparable from year to year, with the filamentous fungus Monilinia fructigena most commonly being the first invader, followed by Penicillium expansum. Motivated by the fact that only little chemistry has been reported from apple microbiomes, we set out to investigate the chemical diversity and potential ecological roles of secondary metabolites (SMs) in a total of 38 black apples. Metabolomics analyses were conducted on either whole apples or small excisions of fungal biomass derived from black apples. Annotation of fungal SMs in black apple extracts was aided by cultivation of 15 recently isolated fungal strains on 9 different substrates in an OSMAC approach, leading to identification of 3319 unique chemical features. Only 6.8% were attributable to known compounds based on analysis of HPLC-HRMS/MS data using spectral library matching tools. Of the 1606 features detected in the black apple extracts, 32% could be assigned as fungal-derived, due to their presence in the OSMAC-based training dataset. Notably, the detection of several antifungal compounds clearly indicates the importance of such compounds for invasion of and control of other microbial competitors on apples. In conclusion, the diversity and abundance of microbial SMs on black apples was found to be much higher than that typically observed for other environmental microbiomes. Detection of SMs known to be produced by the six fungal species tested also highlights a succession of fungal growth following the initial invader M. fructigena. ImportanceMicrobial secondary metabolites constitute a significant reservoir of biologically potent and clinically valuable chemical scaffolds. However, their usefulness is hampered by rapidly developing resistance, resulting in reduced profitability of such research endeavours. Hence, it is vital that the ecological role of such microbial secondary metabolites be considered to understand how best to utilise such compounds as chemotherapeutics. Here, we explore an under-investigated environmental microbiome in the case of black apples; a veritable "low-hanging fruit", with relatively high abundances and diversity of microbially produced secondary metabolites. Using both a targeted and untargeted metabolomics approach, the interplay between metabolites, other microbes and the apple host itself was investigated. This study highlights the surprisingly low incidence of known secondary metabolites in such a system, highlighting the need to study the functionality of secondary metabolites in microbial interactions and complex microbiomes.

ecology↗

bioRxiv2023

Enhanced niche colonisation and competition during bacterial adaptation to a fungus

Bacterial-fungal interactions (BFIs) influence microbial community performance of most ecosystems and elicit specific microbial behaviours, including stimulating specialised metabolite production. Using a simple BFI system encompassing the Gram-positive bacterium Bacillus subtilis and the black mould fungus Aspergillus niger, we established a co-culture experimental evolution method to investigate bacterial adaptation to the presence of a fungus. In the evolving populations, B. subtilis was rapidly selected for enhanced production of the lipopeptide surfactin and accelerated surface spreading ability, leading to inhibition of fungal expansion and acidification of the environment. These phenotypes were explained by specific mutations in the DegS-DegU two-component system. In the presence of surfactin, fungal hyphae exhibited bulging cells with delocalised secretory vesicles and RlmA-dependent cell wall stress induction. Increased surfactin production typically enhances the competitive success of bacteria against fungi, which likely explains the primary adaption path in the presence of A. niger. Significance statementExperimental evolution and co-cultivation of different microbes are important and useful techniques for discovering new traits and unravelling cryptic regulatory connections. We combined these methods by evolving the Gram-positive bacterium Bacillus subtilis in the presence of the black mould fungus Aspergillus niger that were previously shown to engage in an intricate and physical interaction. Both are ubiquitous, environmentally and industrially relevant model microbes in the colonisation of rhizo- and endosphere and in the production of enzymes. Our results demonstrate how laboratory adaptation can be exploited to improve biocontrol properties of bacteria.

microbiology↗

bioRxiv2020

Genomic and chemical diversity of Bacillus subtilis secondary metabolites against plant pathogenic fungi

Bacillus subtilis produces a wide range of secondary metabolites providing diverse plant-growth-promoting and biocontrol abilities. These secondary metabolites include non-ribosomal peptides (NRPs) with strong antimicrobial properties, causing either cell lysis, pore formation in fungal membranes, inhibition of certain enzymes, or bacterial protein synthesis. However, the natural products of B. subtilis are mostly studied either in laboratory strains or in individual isolates and therefore, a comparative overview of B. subtilis secondary metabolites is missing. In this study, we have isolated 23 B. subtilis strains from eleven sampling sites, compared the fungal inhibition profiles of wild types and their NRPs mutants, followed the production of targeted lipopeptides, and determined the complete genomes of 13 soil isolates. We discovered that non-ribosomal peptide production varied among B. subtilis strains co-isolated from the same soil samples. In vitro antagonism assays revealed that biocontrol properties depend on the targeted plant pathogenic fungus and the tested B. subtilis isolate. While plipastatin alone is sufficient to inhibit Fusarium sp., a combination of plipastatin and surfactin is required to hinder the growth of Botrytis cinerea. Detailed genomic analysis revealed that altered NRP production profiles in certain isolates is due to missing core genes, nonsense mutation, or potentially altered gene regulation. Our study combines microbiological antagonism assays with chemical NRPs detection and biosynthetic gene cluster predictions in diverse B. subtilis soil isolates to provide a broader overview of the secondary metabolite chemodiversity of B. subtilis. IMPORTANCESecondary or specialized metabolites with antimicrobial activities define the biocontrol properties of microorganisms. Members of the Bacillus genus produce a plethora of secondary metabolites, of which non-ribosomally produced lipopeptides in particular display strong antifungal activity. To facilitate prediction of the biocontrol potential of new Bacillus subtilis isolates, we have explored the in vitro antifungal inhibitory profiles of recent B. subtilis isolates, combined with analytical natural product chemistry, mutational analysis, and detailed genome analysis of biosynthetic gene clusters. Such a comparative analysis helped to explain why selected B. subtilis isolates lack production of certain secondary metabolites.

microbiology↗

bioRxiv2019

Depiction of secondary metabolites and antifungal activity of Bacillus velezensis DTU001

For a safe and sustainable environment, effective microbes as biocontrol agents are in high demand. We have isolated a new Bacillus velezensis strain DTU001, investigated its antifungal spectrum, sequenced its genome, and uncovered the production of lipopeptides in HPLC-HRMS analysis. To test the antifungal efficacy, extracts of B. velezensis DTU001 was tested against a range of twenty human or plant pathogenic fungi. We demonstrate that inhibitory potential of B. velezensis DTU001 against selected fungi is superior in comparison to single lipopeptide, either iturin or fengycin. The isolate showed analogous biofilm formation to other closely related Bacilli. To further support the biocontrol properties of the isolate, coculture with Candida albicans demonstrated that B. velezensis DTU001 exhibited excellent antiproliferation effect against C. albicans. In summary, the described isolate is a potential antifungal agent with a broad antifungal spectrum that might assist our aims to avoid hazardous pathogenic fungi and provide alternative to toxicity caused by chemicals.

microbiology↗