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Nitrification inhibitors (NIs) applied to soil reduce nitrogen fertilizer losses from agr o -ecosystems. NIs that are currently registered for use in agriculture appear to selectively inhibit ammonia-oxidizing bacteria (AOB), while their impact on other nitrifiers is limited or unknown. Ethoxyquin (EQ), a fruit 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 with that of dicyandiamide (DCD), nitrapyrin (NP), and 3,4-dimethylpyrazole-phosphate (DMPP), NIs that have been used in agricultural settings. The effect of each compound on the growth of AOB ( Nitrosomonas europaea, Nitrosospira multiformis ), ammonia-oxidizing archaea (AOA; " Candidatus Nitrosocosmicus franklandus," " Candidatus Nitrosotalea sinensis"), and a nitrite-oxidizing bacterium (NOB; Nitrobacter sp. NHB1), all being soil isolates, were determined in liquid culture over a range of concentrations by measuring nitrite production or consumption and qPCR of amoA and nxrB genes, respectively. The degradation of NIs in the liquid cultures was also determined. In all cultures, EQ was transformed to the short-lived QI (major derivative) and the persistent EQNL (minor derivative). They all showed significantly higher inhibition activity of AOA compared to AOB and NOB isolates. QI was the most potent AOA inhibitor (EC 50 = 0.3-0.7 μM) compared to EQ (EC 50 = 1-1.4 μM) and EQNL (EC 50 = 26.6-129.5 μM). The formation and concentration of QI in EQ-amended cultures correlated with the inhibition patterns for all isolates suggesting that it was primarily responsible for inhibition after application of EQ. DCD and DMPP showed greater inhibition of AOB compared to AOA or NOB, with DMPP being more potent (EC 50 = 221.9-248.7 μM vs EC 50 = 0.6-2.1 μM). NP was the only NI to which both AOA and AOB were equally sensitive with EC 50s of 0.8-2.1 and 1.0-6.7 μM, respectively. Overall, EQ, QI, and NP were the most potent NIs against AOA, NP, and DMPP were the most effective against AOB, while NP, EQ and its derivatives showed the highest activity against the NOB isolate. Our findings benchmark the activity range of known and novel NIs with practical implications for their use in agriculture and the development of NIs with broad or complementary activity against all AO., (Copyright © 2020 Papadopoulou, Bachtsevani, Lampronikou, Adamou, Katsaouni, Vasileiadis, Thion, Menkissoglu-Spiroudi, Nicol and Karpouzas.)

Auxotrophy to amino acids and vitamins is a common feature in the bacterial world shaping microbial communities through cross-feeding relations. The amino acid auxotrophy of pollutant-degrading bacteria could hamper their bioremediation potential, however, the underlying mechanisms of auxotrophy remain unexplored. We employed genome sequence-based metabolic reconstruction to identify potential mechanisms driving the amino acid auxotrophy of a Sphingomonas haloaromaticamans strain degrading the fungicide ortho -phenylphenol (OPP) and provided further verification for the identified mechanisms via in vitro bacterial assays. The analysis identified potential gaps in the biosynthesis of isoleucine, phenylalanine and tyrosine, while methionine biosynthesis was potentially effective, relying though in the presence of B12. Supplementation of the bacterium with the four amino acids in all possible combinations rescued its degrading capacity only with methionine. Genome sequence-based metabolic reconstruction and analysis suggested that the bacterium was incapable of de novo biosynthesis of B12 (missing genes for the construction of the corrin ring) but carried a complete salvage pathway for corrinoids uptake from the environment, transmembrane transportation and biosynthesis of B12. In line with this the bacterium maintained its degrading capacity and growth when supplied with environmentally relevant B12 concentrations (i.e., 0.1 ng ml -1 ). Using genome-based metabolic reconstruction and in vitro testing we unraveled the mechanism driving the auxotrophy of a pesticide-degrading S. haloaromaticamans . Further studies will investigate the corrinoids preferences of S. haloaromaticamans for optimum growth and OPP degradation., (Copyright © 2020 Perruchon, Vasileiadis, Papadopoulou and Karpouzas.)

Pesticides are intentionally applied to agricultural fields for crop protection. They can harm non-target organisms such as soil microorganisms involved in important ecosystem functions with impacts at the global scale. Within the frame of the pesticide registration process, the ecotoxicological impact of pesticides on soil microorganisms is still based on carbon and nitrogen mineralization tests, despite the availability of more extensive approaches analyzing the abundance, activity or diversity of soil microorganisms. In this study, we used a high-density DNA microarray (PhyloChip) and 16S rDNA amplicon next-generation sequencing (NGS) to analyze the impact of the organophosphate insecticide chlorpyrifos (CHL), the phenyl-urea herbicide isoproturon (IPU), or the triazole fungicide tebuconazole (TCZ) on the diversity and composition of the soil bacterial community. To our knowledge, it is the first time that the combination of these approaches are applied to assess the impact of these three pesticides in a lab-to-field experimental design. The PhyloChip analysis revealed that although no significant changes in the composition of the bacterial community were observed in soil microcosms exposed to the pesticides, significant differences in detected operational taxonomic units (OTUs) were observed in the field experiment between pesticide treatments and control for all three tested pesticides after 70 days of exposure. NGS revealed that the bacterial diversity and composition varied over time. This trend was more marked in the microcosm than in the field study. Only slight but significant transient effects of CHL or TCZ were observed in the microcosm and the field study, respectively. IPU was not found to significantly modify the soil bacterial diversity or composition. Our results are in accordance with conclusions of the Environmental Food Safety Authority (EFSA), which concluded that these three pesticides may have a low risk toward soil microorganisms.

Diphenylamine (DPA) is a common soil and water contaminant. A Pseudomonas putida strain, recently isolated from a wastewater disposal site, was efficient in degrading DPA. Thorough knowledge of the metabolic capacity, genetic stability and physiology of bacteria during biodegradation of pollutants is essential for their future industrial exploitation. We employed genomic, proteomic, transcription analyses and plasmid curing to (i) identify the genetic network of P. putida driving the microbial transformation of DPA and explore its evolution and origin and (ii) investigate the physiological response of bacterial cells during degradation of DPA. Genomic analysis identified (i) two operons encoding a biphenyl (bph) and an aniline (tdn) dioxygenase, both flanked by transposases and (ii) two operons and several scattered genes encoding the ortho -cleavage of catechol. Proteomics identified 11 putative catabolic proteins, all but BphA1 up-regulated in DPA- and aniline-growing cells, and showed that the bacterium mobilized cellular mechanisms to cope with oxidative stress, probably induced by DPA and its derivatives. Transcription analysis verified the role of the selected genes/operons in the metabolic pathway: DPA was initially transformed to aniline and catechol by a biphenyl dioxygenase (DPA-dioxygenase); aniline was then transformed to catechol which was further metabolized via the ortho -cleavage pathway. Plasmid curing of P. putida resulted in loss of the DPA and aniline dioxygenase genes and the corresponding degradation capacities. Overall our findings provide novel insights into the evolution of the DPA degradation pathway and suggests that the degradation capacity of P. putida was acquired through recruitment of the bph and tdn operons via horizontal gene transfer.