Biology subjects

Karpouzas, D. G.

Publications and source records attributed to Karpouzas, D. G..

7 recordsLinked to original sources

bioRxiv2026

A new automated pipeline for whole genome shotgun sequencing analysis and hazard characterization of microbial pesticides

Microbial pesticides are increasingly important for sustainable crop protection, yet hazard analysis and risk assessment remain challenging and require specific characterization of properties relevant to both safety and biological activity. Whole-genome sequencing (WGS) can support the identification of microorganisms at high resolution and characterize potential hazards associated with infectivity, pathogenicity, antimicrobial resistance, and toxic metabolite production. However, the routine use of WGS in this context requires accessible, reproducible, and interpretable workflows. Here, we present a publicly available web-based workflow for WGS-supported hazard analysis of microbial biocontrol agents. The workflow accepts assembled genomes as well as short-read, long-read and hybrid sequencing data, and performs genome quality assessment, taxonomic assignment, genome annotation, AMR detection, mobile-element screening, pathogenicity prediction and secondary-metabolite analysis, and compiles the results into an HTML report. For bacterial agents, we implement a transparent rule-based risk-classification module integrating taxonomic identity, PathogenFinder2 predictions, CARD/RGI resistance evidence, WHO priority taxa, and medically important antimicrobial categories. Secondary-metabolite assessment combines antiSMASH with local BLAST searches and EFSA-aligned identity/coverage thresholds to support product-level interpretation of biosynthetic gene clusters. Comparison with the currently used MOpS workflow developed by EFSA demonstrates the added benefits of the proposed workflow for a guided hazard analysis of microbial pesticides. The workflow is intended as a community-accessible pre-assessment tool that complements regulatory platforms by improving transparency, reproducibility, and early identification of potential hazards in microbial biocontrol candidates, envisioned to make WGS an integral part of the risk assessment of microbial pesticides. Key pointsO_LIThe RATION-GUI provides a public, reproducible workflow for whole-genome sequencing- supported hazard characterization of bacterial and fungal microbial pesticides. C_LIO_LIThe workflow integrates genome quality, taxonomy, annotation, antimicrobial resistance, pathogenicity-related evidence, mobile elements, and secondary-metabolite potential. C_LIO_LIDomain-specific outputs are translated into structured hazard indicators, evidence summaries, and recommended follow-up actions without replacing expert regulatory judgment. C_LIO_LICase studies demonstrate how the workflow improves transparency, traceability, and interpretation of genomic evidence for microbial pesticide risk assessment. C_LI

bioinformatics↗

bioRxiv2026

Common nitrification inhibitors exhibit varied physiological mechanisms on an ammonia-oxidizing microorganism

Microbial ammonia oxidation, the first and rate-limiting step of nitrification, plays a central role in soil nitrogen cycling. It is most relevant in agricultural soils as nitrifiers compete with crops for ammonia-based fertilizers. Therefore, synthetic nitrification inhibitors are widely used alongside fertilizers to reduce the activities of dominant drivers of this process, i.e. ammonia-oxidizing archaea (AOA) and bacteria (AOB). However, the physiological responses of ammonia oxidizers remain poorly resolved. Here the response of the AOA Nitrososphaera viennensis to the nitrification inhibitors 3,4-dimethylpyrazole phosphate (DMPP) and allylthiourea (ATU) were investigated using a combination of functional genomics, physiological assays, and relief experiments. The results overturn earlier assumptions that DMPP and ATU act by chelating free copper. Both compounds affected ammonia oxidation and triggered broader shifts in energy metabolism and stress-response pathways, which diverged markedly between the two inhibitors. We propose a competitive inhibition of the ammonia monooxygenase complex with DMPP as it can be alleviated by additional ammonia and elicits activation of urea acquisition, while ATU acted as a non-competitive inhibitor generally inducing quiescence. Both modes of inhibition were associated with clear transcriptomic and proteomic signals that will be advantageous for the identification of mechanisms of other nitrification inhibitors in the future. Key word: Ammonia-oxidizing archaea, nitrification, nitrification inhibitors, archaea, nitrogen cycle

microbiology↗

bioRxiv2024

Isolation of a novel Sphingomonas strain able to degrade the pleuromutilin tiamulin: omic analysis reveals its transformation pathway

Tiamulin (TIA) is a commonly used veterinary antibiotic, persistent in the animal digestive system and downstream receiving environments like soil after manuring. We aimed to isolate and characterize TIA-degrading bacteria for bioaugmentation strategies towards mitigating TIA environmental pressure. A strain able to degrade TIA and use it as sole C source was isolated from a soil exhibiting enhanced biodegradation of the antibiotic. The isolate degraded TIA at concentrations up to 100 {micro}g ml-1, with pH and temperature optima of 6.5-7.5 and 16-25{degrees}C respectively. Phylogenomic analysis deemed the isolate to be a new Sphingomonas species (83.87 % ANI with S. laterariae, [≤] 95 %), which was named Candidatus Sphingomonas perruchonii. Genomics and transcriptomics revealed the TIA-driven induction of features conducive with its antiotrophic character; genes encoding for drug efflux pumps and the catabolism of xenobiotics. These included ribosome protective ABC-F transporters and efflux pumps able to protect the ribosome and microbial cells from TIA, oxygenases (e.g. P450 cytochrome) and hydrolases (e.g. alpha/beta hydrolases and amidohydrolases) possibly contributing to its degradation. LC-MS/MS analysis detected putative transformation products (TPs) of TIA, leading us to propose a transformation pathway. This involved a primary oxidation of the tricyclic moiety of TIA to a mono-hydroxylated derivative (TIA-O), potentially mediated by the highly upregulated monoxygenases, which was either further oxidized to TIA-O2 or hydrolysed, by the upregulated hydrolase or amidohydrolases, to 2-diethylamino-ethyl-thio acetic acid, both not degrading further. Further tests and in vitro functional analysis will verify the role of these genes in the transformation of TIA. IMPORTANCEAnthropogenic antibiotic pressure in environmental settings has been acknowledged as a major threat for the public health and the environment, through the: (i) associated ecotoxicological impact; and (ii) the increase of antibiotic resistance dispersal among non-pathogenic and pathogenic bacteria, restricting available choices for treating infections in health care. In situ microbial biotransformation of antibiotics and eventual removal is an environmentally friendly method currently under intense investigation, which was recently proposed for the reduction of antibiotic selective pressure. The present study provides mechanistic insights on the genomic constituents of antibiotrophy, namely, the tolerance against and the biodegradation/transformation of the antibiotic in question. We characterize the capacity of a bacterial isolate to transform tiamulin, a heavily used antibiotic in pig farming, and propose associated biotransformation mechanisms, through an integrated approach of standard microbiological methods combined with and multi-omics (genomics, transcriptomics, metabolomics).

genomics↗

bioRxiv2024

A fast-track, high-throughput screening platform for biological nitrification inhibitors discovery based on soil relevant ammonia oxidizing strains

Nitrogen cycling is critical for ecosystem functioning, with nitrification playing a central role. Excessive nitrification, triggered by heavy nitrogen fertilisation, contributes to reduced nitrogen use efficiency, nitrate leaching, and nitrous oxide emissions. While synthetic nitrification inhibitors (SNIs) help reduce these impacts, their erratic performance and environmental risks have shifted focus to biological nitrification inhibitors (BNIs) as sustainable alternatives. In vitro bioassays with ammonia-oxidising microorganisms (AOM) are valuable for BNI discovery and research, but often have low throughput and rely on a limited number of mostly non-soil-relevant or genetically modified ammonia-oxidizing bacteria (AOB) strains, lacking validation with established BNIs. We present a refined fast-track, high-throughput assay for BNI screening, utilizing soil-relevant, ecophysiologically and phylogenetically diverse AOB (Nitrosospira multiformis, Nitrosomonas ureae, Nitrosomonas communis) and ammonia-oxidizing archaea (AOA) strains (Nitrososphaera viennensis, "Ca. Nitrosocosmicus franklandianus"), achieving consistent cellular activity and density. The assay was validated with established SNIs and BNIs, showing differences in inhibition efficacy and strain sensitivity, consistent with literature. As a proof of concept, root exudates from diverse wheat genotypes were screened, demonstrating distinct inhibition profiles. The proposed system advances previously available screening systems and, when integrated with realistic soil tests, will facilitate the discovery of novel BNIs and BNI-producing plant genotypes.

microbiology↗

bioRxiv2023

Assessing the activity of different plant-derived molecules and potential biological nitrification inhibitors on a range of soil ammonia- and nitrite- oxidizing strains

Nitrification is associated with significant losses of fertilizer-derived ammonium from agroecosystems. The use of biological nitrification inhibitors (BNIs), in place of synthetic nitrification inhibitors (SNIs), holds a great potential to effectively reduce N losses and conforms with the current move towards ecological-intensified agriculture. Knowledge of the activity of BNIs to soil nitrifiers is limited and is mostly based on bioassays with a single Nitrosomonas europaea strain. We determined the in vitro activity of multiple plant-derived compounds as BNIs like (i) root-derived compounds (sakuranetin, MHPP, and zeanone); (ii) other phytochemicals (caffeic acid, quinic acid, chlorogenic acid and shikimic acid), and (iii) analogues of statins (simvastatin), triazoles (1-butyl-4-propyl-triazole, 1,4-dibutyltriazole) and zeanone (2-methoxy-1,4-naphthoquinone), on ecophysiologically and phylogenetically distinct soil-derived ammonia-oxidizing bacteria (AOB) (Nitrosospira multiformis and N. europaea), ammonia-oxidizing archaea (AOA) (Candidatus Nitrosotalea sinensis and Candidatus Nitrosocosmicus franklandianus), and a nitrite-oxidizing bacterium (NOB) (Nitrobacter sp. NHB1). AOA were more sensitive than AOB to BNIs. Sensitivity within AOA group was BNI-dependent, unlike AOB for which N. multiformis was consistently more sensitive than N. europaea. Interestingly, several compounds were inhibitory to Nitrobacter sp. with MHPP and caffeic acid being more potent to the NOB compared to the AOB and AOA strains, an observation with potentially serious implications for soil quality and agricultural production. Overall, zeanone, MHPP and caffeic acid were the most potent BNIs towards AOB, zeanone and 2-methoxy-1,4-naphthoquinone were the most effective compounds against AOA, while caffeic acid was the most potent BNI on Nitrobacter sp. We provide pioneering evidence for the activity range of multiple BNIs on soil nitrifiers, stress the need for revisiting the biological screening systems currently used for BNI determination and we sought for a more thorough monitoring of the impact of BNI candidates on a range of both target and non-target microorganisms.

microbiology↗

bioRxiv2020

Comparison of the in vitro activity of novel and established nitrification inhibitorsapplied in agriculture: challenging the effectiveness of the currently availablecompounds

Nitrification inhibitors (NIs) applied to soil reduce nitrogen fertilizer losses from agricultural ecosystems. Currently available NIs appear to selectively inhibit ammonia-oxidizing bacteria (AOB), while their impact on other groups of nitrifiers is limited. Ethoxyquin (EQ), a preservative shown to inhibit ammonia-oxidizers (AO) in soil, is rapidly transformed to 2,6-dihydro-2,2,4-trimethyl-6-quinone imine (QI) and 2,4-dimethyl-6-ethoxy-quinoline (EQNL). We compared the inhibitory potential of EQ and its derivatives in vitro with other established NIs that have been applied in an agricultural setting (dicyandiamide (DCD), nitrapyrin (NP), 3,4-dimethylpyrazole phosphate (DMPP)) by evaluating their impact on the activity and growth of five soil-derived strains (two AOB (Nitrosomonas europaea, Nitrosospira multiformis), two ammonia-oxidizing archaea (AOA) ("Candidatus Nitrosocosmicus franklandus", "Candidatus Nitrosotalea sinensis"), and one nitrite-oxidizing bacterium (NOB) (Nitrobacter sp.)). NIs degradation was also determined. AOA were more sensitive than AOB or NOB to EQ and its derivatives. Despite its transient character, QI was primarily responsible for AO inhibition by EQ, and the most potent NI against AOA. For AOB, QI was more potent than DCD but less than nitrapyrin and DMPP. AOA and NOB showed higher tolerance to the persistent compounds DCD and DMPP. Our findings benchmark the activity range of known and novel NIs with practical implications for their use, and the development of novel NIs with broad or complementary activity against all AO.

microbiology↗

bioRxiv2020

Nutritional inter-dependencies and a carbazole-dioxygenase are key elements of a bacterial consortium relying on a Sphingomonas for the degradation of the fungicide thiabendazol

BackgroundThiabendazole (TBZ), is a benzimidazole fungicide and anthelminthic whose high persistence and toxicity pose a serious environmental threat. In our quest for environmental mitigation we previously isolated the first TBZ-degrading bacterial consortium and provided preliminary evidence for its composition and the degrading role of a Sphingomonas. Here, we employed a multi-omic approach combined with DNA-stable isotope probing (SIP) to determine the genetic make-up of the key consortium members, to disentangle nutritional and metabolic interdependencies, to identify the transformation pathway of TBZ and to understand the genetic network driving its transformation. ResultsTime-series SIP in combination with amplicon sequencing analysis verified the key role of Sphingomonas in TBZ degradation by assimilating over 80% of the 13C-labelled phenyl moiety of TBZ. Non-target mass spectroscopy (MS) analysis showed the accumulation of thiazole-4-carboxamidine as a single dead-end transformation product and no phenyl-containing derivative, in line with the phenyl moiety assimilation in the SIP analysis. Time series metagenomic analysis of the consortium supplemented with TBZ or succinate led to the assembly of 18 metagenome-assembled genomes (MAGs) with >80% completeness, six (Sphingomonas 3X21F,{gamma} -Proteobacterium 34A, Bradyrhizobiaceae 9B and Hydrogenophaga 19A, 13A, and 23F) being dominant. Meta-transcriptomic and -proteomic analysis suggested that Sphingomonas mobilize a carbazole dioxygenase (car) operon during the initial cleavage of TBZ to thiazole-4-carboxamidine and catechol, the latter is further transformed by enzymes encoded in a catechol ortho-cleavage (cat) operon; both operons being up-regulated during TBZ degradation. Computational docking analysis of the terminal oxygenase component of car, CarAa, showed high affinity to TBZ, comparable to carbazole, reinforcing its high potency for TBZ transformation. These results suggest no interactions between consortium members in TBZ transformation, performed solely by Sphingomonas. In contrast, gene expression network analysis revealed strong interactions between Sphingomonas MAG 3X12F and Hydrogenophaga MAG 23F, with Hydrogenophaga activating its cobalamin biosynthetic pathway and Sphingomonas its cobalamin salvage pathway along TBZ degradation. ConclusionsOur findings suggest interactions between consortium members which align with the "black queen hypothesis": Sphingomonas detoxifies TBZ, releasing consortium members by a toxicant; in return for this, Hydrogenophaga 23F provides cobalamin to the auxotrophic Sphingomonas.

systems biology↗