Experimental Evolution Reveals a Novel Ene Reductase That Detoxifies α,β-Unsaturated Aldehydes in Listeria monocytogenes
ABSTRACT The plant essential oil component trans-cinnamaldehyde (t-CIN) exhibits antibacterial activity against a broad range of foodborne pathogenic bacteria, including L. monocytogenes, but its mode of action is not fully understood. In this study, several independent mutants of L. monocytogenes w...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-06-01
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| Series: | Microbiology Spectrum |
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| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.04877-22 |
| _version_ | 1797803435692654592 |
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| author | Lei Sun Ann Van Loey Carolien Buvé Chris W. Michiels |
| author_facet | Lei Sun Ann Van Loey Carolien Buvé Chris W. Michiels |
| author_sort | Lei Sun |
| collection | DOAJ |
| description | ABSTRACT The plant essential oil component trans-cinnamaldehyde (t-CIN) exhibits antibacterial activity against a broad range of foodborne pathogenic bacteria, including L. monocytogenes, but its mode of action is not fully understood. In this study, several independent mutants of L. monocytogenes with increased t-CIN tolerance were obtained via experimental evolution. Whole-genome sequencing (WGS) analysis revealed single-nucleotide-variation mutations in the yhfK gene, encoding an oxidoreductase of the short-chain dehydrogenases/reductases superfamily, in each mutant. The deletion of yhfK conferred increased sensitivity to t-CIN and several other α,β-unsaturated aldehydes, including trans-2-hexenal, citral, and 4-hydroxy-2-nonenal. The t-CIN tolerance of the deletion mutant was restored via genetic complementation with yhfK. Based on a gas chromatography-mass spectrometry (GC-MS) analysis of the culture supernatants, it is proposed that YhfK is an ene reductase that converts t-CIN to 3-phenylpropanal by reducing the C=C double bond of the α,β-unsaturated aldehyde moiety. YhfK homologs are widely distributed in Bacteria, and the deletion of the corresponding homolog in Bacillus subtilis also caused increased sensitivity to t-CIN and trans-2-hexenal, suggesting that this protein may have a conserved function to protect bacteria against toxic α,β-unsaturated aldehydes in their environments. IMPORTANCE While bacterial resistance against clinically used antibiotics has been well studied, less is known about resistance against other antimicrobials, such as natural compounds that could replace traditional food preservatives. In this work, we report that the food pathogen Listeria monocytogenes can rapidly develop an elevated tolerance against t-cinnamaldehyde, a natural antimicrobial from cinnamon, by single base pair changes in the yhfK gene. The enzyme encoded by this gene is an oxidoreductase, but its substrates and precise role were hitherto unknown. We demonstrate that the enzyme reduces the double bond in t-cinnamaldehyde and thereby abolishes its antibacterial activity. Furthermore, the mutations linked to t-CIN tolerance increased bacterial sensitivity to a related compound, suggesting that they modify the substrate specificity of the enzyme. Since the family of oxidoreductases to which YhfK belongs is of great interest in the mediation of stereospecific reactions in biocatalysis, our work may also have unanticipated application potential in this field. |
| first_indexed | 2024-03-13T05:20:48Z |
| format | Article |
| id | doaj.art-ff39c9e711f84823a5b3239b62e2f9f3 |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-03-13T05:20:48Z |
| publishDate | 2023-06-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-ff39c9e711f84823a5b3239b62e2f9f32023-06-15T13:18:33ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972023-06-0111310.1128/spectrum.04877-22Experimental Evolution Reveals a Novel Ene Reductase That Detoxifies α,β-Unsaturated Aldehydes in Listeria monocytogenesLei Sun0Ann Van Loey1Carolien Buvé2Chris W. Michiels3Department of Microbial and Molecular Systems and Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Leuven, BelgiumDepartment of Microbial and Molecular Systems and Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Leuven, BelgiumDepartment of Microbial and Molecular Systems and Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Leuven, BelgiumDepartment of Microbial and Molecular Systems and Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Leuven, BelgiumABSTRACT The plant essential oil component trans-cinnamaldehyde (t-CIN) exhibits antibacterial activity against a broad range of foodborne pathogenic bacteria, including L. monocytogenes, but its mode of action is not fully understood. In this study, several independent mutants of L. monocytogenes with increased t-CIN tolerance were obtained via experimental evolution. Whole-genome sequencing (WGS) analysis revealed single-nucleotide-variation mutations in the yhfK gene, encoding an oxidoreductase of the short-chain dehydrogenases/reductases superfamily, in each mutant. The deletion of yhfK conferred increased sensitivity to t-CIN and several other α,β-unsaturated aldehydes, including trans-2-hexenal, citral, and 4-hydroxy-2-nonenal. The t-CIN tolerance of the deletion mutant was restored via genetic complementation with yhfK. Based on a gas chromatography-mass spectrometry (GC-MS) analysis of the culture supernatants, it is proposed that YhfK is an ene reductase that converts t-CIN to 3-phenylpropanal by reducing the C=C double bond of the α,β-unsaturated aldehyde moiety. YhfK homologs are widely distributed in Bacteria, and the deletion of the corresponding homolog in Bacillus subtilis also caused increased sensitivity to t-CIN and trans-2-hexenal, suggesting that this protein may have a conserved function to protect bacteria against toxic α,β-unsaturated aldehydes in their environments. IMPORTANCE While bacterial resistance against clinically used antibiotics has been well studied, less is known about resistance against other antimicrobials, such as natural compounds that could replace traditional food preservatives. In this work, we report that the food pathogen Listeria monocytogenes can rapidly develop an elevated tolerance against t-cinnamaldehyde, a natural antimicrobial from cinnamon, by single base pair changes in the yhfK gene. The enzyme encoded by this gene is an oxidoreductase, but its substrates and precise role were hitherto unknown. We demonstrate that the enzyme reduces the double bond in t-cinnamaldehyde and thereby abolishes its antibacterial activity. Furthermore, the mutations linked to t-CIN tolerance increased bacterial sensitivity to a related compound, suggesting that they modify the substrate specificity of the enzyme. Since the family of oxidoreductases to which YhfK belongs is of great interest in the mediation of stereospecific reactions in biocatalysis, our work may also have unanticipated application potential in this field.https://journals.asm.org/doi/10.1128/spectrum.04877-22Listeria monocytogenesnatural antimicrobialsfood preservativesantimicrobial resistanceene reductaseshort-chain dehydrogenase/reductase |
| spellingShingle | Lei Sun Ann Van Loey Carolien Buvé Chris W. Michiels Experimental Evolution Reveals a Novel Ene Reductase That Detoxifies α,β-Unsaturated Aldehydes in Listeria monocytogenes Microbiology Spectrum Listeria monocytogenes natural antimicrobials food preservatives antimicrobial resistance ene reductase short-chain dehydrogenase/reductase |
| title | Experimental Evolution Reveals a Novel Ene Reductase That Detoxifies α,β-Unsaturated Aldehydes in Listeria monocytogenes |
| title_full | Experimental Evolution Reveals a Novel Ene Reductase That Detoxifies α,β-Unsaturated Aldehydes in Listeria monocytogenes |
| title_fullStr | Experimental Evolution Reveals a Novel Ene Reductase That Detoxifies α,β-Unsaturated Aldehydes in Listeria monocytogenes |
| title_full_unstemmed | Experimental Evolution Reveals a Novel Ene Reductase That Detoxifies α,β-Unsaturated Aldehydes in Listeria monocytogenes |
| title_short | Experimental Evolution Reveals a Novel Ene Reductase That Detoxifies α,β-Unsaturated Aldehydes in Listeria monocytogenes |
| title_sort | experimental evolution reveals a novel ene reductase that detoxifies α β unsaturated aldehydes in listeria monocytogenes |
| topic | Listeria monocytogenes natural antimicrobials food preservatives antimicrobial resistance ene reductase short-chain dehydrogenase/reductase |
| url | https://journals.asm.org/doi/10.1128/spectrum.04877-22 |
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