Biphenyl 2,3-Dioxygenase in Pseudomonas alcaliphila JAB1 Is Both Induced by Phenolics and Monoterpenes and Involved in Their Transformation

Abstract

The involvement of bacterial aromatic ring-hydroxylating dioxygenases (ARHDs) in the degradation of aromatic pollutants, such as polychlorinated biphenyls (PCBs), has been well studied. However, there is considerable speculation as to the origin of this ability. One hypothesis is centered on a connection between the ability to degrade aromatic pollutants and the necessity of soil bacteria to cope with and/or utilize secondary plant metabolites (SPMs). To investigate this connection, we researched the involvement of biphenyl 2,3-dioxygenase (BPDO), an ARHD essential for the degradation of PCBs, in the metabolism of SPMs in the soil bacterium Pseudomonas alcaliphila JAB1, a versatile degrader of PCBs. We demonstrated the ability of the strain JAB1 to transform a variety of SPMs, namely the flavonoids apigenin, flavone, flavanone, naringenin, fisetin, quercetin, morin, and catechin, caffeic acid, trans-cinnamic acid, and the monoterpenes (S)-limonene and (R)-carvone. Of those, the transformation of flavone, flavanone, and (S)-limonene was conditioned by the activity of JAB1-borne BPDO and thus was researched in more detail, and we found evidence for the limonene monooxygenase activity of the BPDO. Furthermore, the bphA gene in the strain JAB1 was demonstrated to be induced by a wide range of SPMs, with monoterpenes being the strongest inducers of the SPMs tested. Thus, our findings contribute to the growing body of evidence that ARHDs not only play a role in the catabolism of aromatic pollutants, but also of natural plant-derived aromatics, and this study supports the hypothesis that ARHDs participate in ecological processes mediated by SPMs.

Keywords: aromatic ring-hydroxylating dioxygenases; biphenyl dioxygenase; monoterpenes; phenolics; secondary plant metabolites.

Figure 1

Architecture of the biphenyl/PCB degradation pathway (bph operon) in the wild-type JAB1 (A) and in the bphA-null mutant JAB1ΔbphA (B). The position of the bph operon in the JAB1 annotated whole genome sequence (Genbank accession no. CP016162.1) is indicated. The deduced protein sequences encoded by all genes shown were 100% identical with their homologues from Pseudomonas furukawaii KF707, except BphB with the identity score of 99%. The gene gntR encodes for a putative GntR-family transcription regulator; bphA and bphE – large and small subunit of the two-component biphenyl dioxygenase, respectively; bphX – protein with unknown function; bphF and bphG – ferredoxin and ferredoxin reductase, respectively; bphB – cis-2,3-dihydrobiphenyl-2,3-diol dehydrogenase; bphC – 2,3-dihydroxybiphenyl-1,2-dioxygenase; bphK – glutathione S-transferase; bphH – 2-hydroxypenta-2,4-dienoate hydratase; bphJ – acetaldehyde dehydrogenase (acetylating); bphI – 4-hydroxy-2-oxovalerate aldolase; and bphD – 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase.

Figure 2

Secondary plant metabolites used in this study.

Figure 3

Degradation of secondary plant metabolites (SPMs) by strains JAB1 and JAB1ΔbphA. The ability of strains to degrade SPMs was tested by resting cell assay (RCA), depletion rates R in % of the corresponding abiotic controls are shown. Cells were incubated for 24 h in the presence of individual SPMs (0.25 mM). Error bars represent SD from three replicates. Asterisks (^∗) show a depletion rate significantly different from the abiotic control, daggers (^†) indicate a depletion by the wild-type JAB1 that is significantly different from the strain JAB1ΔbphA. One-way ANOVA (p < 0.05) was used to analyze the data.

Figure 4

Products of flavone (A), flavanone (B), and (S)-limonene (C) degradation mediated by the JAB1-borne biphenyl 2,3-dioxygenase (BPDO). Compounds: I, 2-(m,n-dihydroxyphenyl)chromane-4-one; II, methyl 4-oxo-4H-chromene-2-carboxylate; III, 4-oxo-4H-chromene-2-carboxylic acid; IV, 2-(m,n-dihydroxyphenyl)chromane-3,4-diol; V, perillyl alcohol; VI, perillyl aldehyde; VII, perillic acid; VIII, carveol; IX, carvone; and X, limonene 1,2-epoxide.

Figure 5

Relative levels of bphA transcripts in JAB1 strain exposed to SPMs. JAB1 cells were exposed to individual 0.25 mM SPMs for 1 and 3 h; the level of bphA transcripts was quantified by quantitative reverse transcription-PCR (RT-qPCR). The 16S rRNA transcript was used as a reference. The levels of bphA transcripts are shown as the average fold change in the non-induced control (cells without SPMs added, induction rate equals 1) with 95%-confidence intervals. The obtained c_p values were processed by the common base method according to Ganger et al. (2017). Asterisks (^∗) denote a significant difference from the control as tested by t test (α = 0.05).