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8 recordsLinked to original sources
PubMed2026

A UG5 reverse transcriptase-nitrilase antiviral module confers phage immunity in the plant symbiont Sinorhizobium meliloti.

Bacteriophages exert strong selective pressure on soil- and rhizosphere-associated bacteria, including plant-associated symbionts. Reverse transcriptase-associated defense systems of the UG family are widespread across bacterial lineages, yet their ecological roles remain largely undefined. Within this family, UG5 systems are distinguished by reverse transcriptases fused to or associated with a nitrilase domain. Here, we combine phylogenetic, metagenomic, and functional analyses to investigate the evolutionary context and antiviral activity of UG5-associated systems. Phylogenetic analysis of 728 nitrilase domains places UG5-associated nitrilases within a well-supported UG-related radiation encompassing the UG1, UG5, and UG6 families, with UG1 nested within a broader UG5 lineage. Metagenomic analysis further revealed UG5-associated reverse transcriptases in soil- and rhizosphere-derived metagenomes. Based on this observation, we characterized a UG5-large reverse transcriptase (RT)-associated system, here designated DRT11, encoded on the pSymA megaplasmid of Sinorhizobium meliloti RMO17, a nitrogen-fixing symbiont of Medicago sativa. Despite lacking the transmembrane protein typical of canonical UG5-large architectures, DRT11 confers protection against naturally occurring M. sativa rhizosphere phages with podovirus-like morphology. Phage infection assays reveal protection at low multiplicities of infection, consistent with an abortive-infection-like mechanism. Moreover, mutational analyses demonstrate that antiviral activity requires only the RT and its fused C-terminal nitrilase domain, establishing DRT11 as a minimal UG5-associated antiviral system.IMPORTANCEIn this study, we report the functional characterization of a UG5-large reverse transcriptase-associated defense system (DRT11) encoded on the pSymA megaplasmid of the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Using a combination of phylogenetic, metagenomic, genomic, and experimental approaches, we demonstrate that DRT11 functions as a bona fide antiviral defense module, providing protection against naturally occurring rhizosphere phages through a minimal reverse transcriptase-nitrilase architecture. This work establishes direct functional evidence for antiviral activity within the UG5 family and clarifies the evolutionary placement of UG5-associated systems within the broader UG radiation.

Phylogeny↗
PubMed2026

Nitrilase-mediated degradation of insecticides flonicamid and thiacloprid by immobilized engineered Escherichia coli with a novel pathway.

The nitrile‑containing insecticides flonicamid (FLO) and thiacloprid (THI) are widely used in agriculture, posing risks to the environment and animal health. Nitrilase is a key catalyst for the degradation of nitrile compounds, and immobilized engineered bacteria are preferred in wastewater treatment. However, immobilized engineered bacteria expressing nitrilase have never been investigated for pollutant degradation. Here, engineered Escherichia coli pET28a‑VbNitA harboring the nitrilase gene VbNitA was immobilized by calcium alginate encapsulation. FLO was degraded into N-(4-trifluoromethylnicotinoyl)glycinamide and 4-(trifluoromethyl)nicotinol glycine by the immobilized cells via VbNitA. THI was converted to THI‑amide and THI‑imine using the same system. Notably, this is the first report of a nitrilase converting THI to THI‑amide and of THI‑imine as a biodegradation intermediate. Compared with free cells, the immobilized E. coli pET28a‑VbNitA showed higher tolerance to high temperature, alkaline, and acidic environments, and better long-term storage stability. The substrate inhibition model showed that the optimal initial concentrations of FLO and THI for degradation by immobilized E. coli pET28a‑VbNitA were 45.13 and 127.50 μmol/L, respectively. FLO was degraded more rapidly than THI by the immobilized cells. Molecular docking revealed that both FLO and THI formed stable interactions with VbNitA, with FLO positioned closer to Cys165 of the catalytic triad. This study presents a novel THI degradation pathway and provides a new, efficient immobilized biocatalyst for the remediation of wastewater with nitrile‑containing insecticides.

Escherichia coli↗
PubMed1978

[Use of gas-liquid chromatography for the study of nitrilases and amidases (author's transl)].

A gas-liquid chromatographic procedure is described for the determination of nitrilasic and amidasic activities. This method allows to monitor the kinetics of the hydrolysis of volatile nitriles and amides and because of its sensitivity, to determine the Michaelis constants Km of the acetonitrilase and the acetamidase of Brevibacterium R 312. For these enzymes, a correlation is shown between the kinetics monitored by proton nuclear magnetic resonance spectroscopy and gas-liquid chromatography.

Amidohydrolases↗
PubMed1976

[Nitrilase activity in several bacteria].

Eighteen strains of Bacteria from the genus Bacillus, Bacteridium, Micrococcus and Brevibacterium were isolated. They have a very general nitrilase activity that acts on all the substrates with nitrile function.

Actinomycetales↗
PubMed1977

Microbial metabolism of aromatic nitriles. Enzymology of C-N cleavage by Nocardia sp. (Rhodochrous group) N.C.I.B. 11216.

1. An organism utilizing benzonitrile as sole carbon and nitrogen source was isolated by the enrichment-culture technique and identified as a Nocardia sp. of the rhodochrous group. 2. Respiration studies indicate that nitrile degradation proceeds through benzoic acid and catechol. 3. Cell-free extracts of benzonitrile-grown cells contain an enzyme that catalyses the conversion of benzonitrile directly into benzoic acid without intermediate formation of benzamide. 4. This nitrilase enzyme was purified by DEAE-cellulose chromatography and gel filtration on Sephadex G-100 in the presence and absence of substrate. The purity of the enzyme was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing on polyacrylamide gel. 5. The enzyme shows a time-dependent substrate-activation process in which the substrate catalyses the association of inactive subunits of mol.wt. 45000 to form the polymeric 12-unit active enzyme of mol.wt. 560000. The time required for complete association is highly dependent on the concentration of the enzyme, temperature and pH. 6. The associated enzyme has a pH optimum of 8.0 and K(m) with benzonitrile as substrate of 4mm. The activation energy of the reaction as deduced from the Arrhenius plot is 51.8kJ/mol. 7. Enzyme activity is inhibited by thiol-specific reagents and several metal ions. 8. Studies with different substrates indicate that the nitrilase is specific for nitrile groups directly attached to the benzene ring. Various substituents in the ring are compatible with activity, though ortho-substitution, except by fluorine, renders the nitrile invulnerable to attack. 9. The environmental implications of these findings and the possible significance of the enzyme in the regulation of metabolism are discussed.

Aminohydrolases↗
PubMed1977

Fungal degradation of aromatic nitriles. Enzymology of C-N cleavage by Fusarium solani.

1. A strain of the fungus Fusarium solani able to use benzonitrile as sole source of carbon and nitrogen was isolated by elective culture. 2. Respiration studies indicate that the nitrile, after degradation to benzoate, is catabolized via catechol or alternatively via p-hydroxybenzoate and 3,4-dihydroxybenzoate. 3. Cell-free extracts of benzonitrile-grown cells contain an enzyme mediating the conversion of benzonitrile into benzoate and ammonia. 4. The nitrilase enzyme was purified by DEAE-cellulose chromatography, (NH(4))(2)SO(4) precipitation and gel filtration on Sephadex G-200. The homogeneity of the purified enzyme preparation was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing on polyacrylamide gel. 5. The enzyme showed a broad pH optimum between pH7.8 and 9.1 and a K(m) with benzonitrile as substrate of 0.039mm. The activation energy of the reaction deduced from an Arrhenius plot was 48.4kJ/mol. 6. The enzyme was susceptible to inhibition by thiol-specific reagents and certain heavy metal ions. 7. Gel filtration gave a value of 620000 for the molecular weight of the intact enzyme. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis demonstrated that the enzyme was composed of eight subunits of mol.wt. 76000. 8. Rates of enzymic attack on various substrates indicated that the nitrilase has a fairly broad specificity and that the fungus probably plays an important role in the biodegradation of certain nitrilic herbicides in the environment.

Aminohydrolases↗