Degradation of cinnamic acid by the rhizosphere strain Achromobacter insolitus LCu2

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Abstract

The Achromobacter insolitus LCu2 strain, isolated from the roots of alfalfa (Medicago sativa L.), utilized cinnamic acid, as well as its methoxy derivatives ‒ vanillic and ferulic acids ‒ as the only carbon source. Weak growth was observed on m-coumaric acid, but not on o- and p-coumaric acids. Growth on cinnamic acid was slow and diauxic. The loss of substrate from the cultivation medium was 53%, the destructive efficiency was 30 μg/mg of raw biomass for 14 days. Despite the bactericidal effect of cinnamic acid, the A. insolitus LCu2 culture remained viable for a long time. Genomic analysis revealed two gene clusters, hca and mhp, responsible for dihydroxylation of the phenyl ring (hcaA1A2CDB) and its subsequent cleavage to central metabolic products (mhpACDE), as well as a transcriptional regulator (hcaR) and a putative transporter (hcaT). A putative biochemical pathway for cinnamic acid degradation by A. insolitus strain LCu2 was predicted using genomic data.

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About the authors

E. V. Kryuchkova

FRCenter “Saratov Scientific Center RAS”

Author for correspondence.
Email: kryu-lena@yandex.ru

Institute of Biochemistry and Physiology of Plants and Microorganisms

Russian Federation, 410049, Saratov

E. S. Morozova

St. Petersburg State University

Email: kryu-lena@yandex.ru
Russian Federation, 199034, St. Petersburg

V. S. Grinev

FRCenter “Saratov Scientific Center RAS”; Saratov National Research State University named after N. G. Chernyshevsky

Email: kryu-lena@yandex.ru

Institute of Biochemistry and Physiology of Plants and Microorganisms

Russian Federation, 410049, Saratov; 410012, Saratov

G. L. Burygin

FRCenter “Saratov Scientific Center RAS”; Saratov National Research State University named after N. G. Chernyshevsky

Email: kryu-lena@yandex.ru

Institute of Biochemistry and Physiology of Plants and Microorganisms

Russian Federation, 410049, Saratov; 410012, Saratov

N. E. Gogoleva

Kazan (Volga Region) Federal University; Institute of Cellular and Intracellular Symbiosis, Ural Branch of the Russian Academy of Sciences

Email: kryu-lena@yandex.ru
Russian Federation, 420008, Kazan; 460000, Orenburg

Yu. V. Gogolev

Kazan (Volga Region) Federal University; Kazan Scientific Center of the Russian Academy of Sciences

Email: kryu-lena@yandex.ru

Kazan Institute of Biochemistry and Biophysics

Russian Federation, 420008, Kazan; 420008, Kazan

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2. Fig. 1. Biphasic growth curve of A. insolitus LCu2 on 0.5 g/L cinnamic acid. Each point is the mean of three replicates; error bars show the confidence interval for p ≤ 0.05.

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3. Fig. 2. Viability of bacteria cultured for a month on cinnamic acid: 1, 2 – cultures grown on 0.5 g/l; 3, 4 – on 1.0 g/l cinnamic acid, plated on mineral-synthetic (MS1) and LB media; confidence interval for p ≤ 0.05.

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4. Fig. 3. UV spectra (a) and HPLC analysis (b) of cinnamic acid before and after cultivation of A. insolitus LCu2. Blue curve – chemical control, red – after cultivation of bacteria.

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5. Fig. 4. Phytotoxicity of supernatants with cinnamic acid in relation to wheat sprouts of the Saratovskaya 29 variety: a – average root length; b – average stem length; 1 – biological control; 2 – chemical control; 3 – LCu2 culture liquid. Error bars show the confidence interval for p ≤ 0.05; in each variant n = 25; plant age 5 days; cinnamic acid content 1 mg.

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6. Fig. 5. Structural organization of the gene cluster containing the hca and mhp genes and the putative biochemical pathway of cinnamic acid degradation by A. insolitus LCu2 strain. a: R – gntR (transcription regulator); D – mhpD; hB – hcaB; hp – QEK92583; A2 – hcaA2; A1 – hcaA1; hС – hcaC; hD – hcaD; b: HcaA1A2 – α- and β-subunits of trans/cinnamate dioxygenase; HcaC – ferredoxin; HcaD – ferredoxin reductase; HcaB – 2,3-dihydroxy-2,3-dihydrophenylpropionate dehydrogenase; QEK92583 – hypothetical protein – putatively, extradiol dioxygenase; MhpC – 2-hydroxy-6-oxonadienedioate/2-hydroxy-6-oxononatrienedioate hydroxylase; MhpD – 2-keto-4-pentonoate hydratase; MhpE – 4-hydroxy-2-oxovalerate aldolase. Ovals indicate central metabolites.

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7. Fig. 6. Bioinformatics analysis of the amino acid sequence (QEK92583) from A. insolitus LCu2: a ‒ comparison of amino acid sequence fragments of salicylate 1,2-dioxygenase from P. salicylatoxidans BN12 (AAQ91293.1) and a hypothetical protein of A. insolitus LCu2 (QEK92583). The cupin domain with three histidine residues (His119; His121 and His160) coordinating Fe2+ in the catalytic center (Matera et al., 2008) is marked with blue rectangles and stars and is designated as Motif I and Motif II; b ‒ phylogenetic analysis of proteins with cupin domains using the Maximum-Likelihood method in Mega X (Kumar et al., 2018), bootstrap 1000, sequences aligned in ClustalOmega W with default settings; root – homogentisate 1,2-dioxygenase.

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