Responsiveness of Aromatoleum aromaticum EbN1T to Lignin-Derived Phenylpropanoids

Abstract

The betaproteobacterial degradation specialist Aromatoleum aromaticum EbN1^T utilizes several plant-derived 3-phenylpropanoids coupled to denitrification. In vivo responsiveness of A. aromaticum EbN1^T was studied by exposing nonadapted cells to distinct pulses (spanning 100 µM to 0.1 nM) of 3-phenylpropanoate, cinnamate, 3-(4-hydroxyphenyl)propanoate, or p-coumarate. Time-resolved, targeted transcript analyses via quantitative reverse transcription-PCR of four selected 3-phenylpropanoid genes revealed a response threshold of 30 to 50 nM for p-coumarate and 1 to 10 nM for the other three tested 3-phenylpropanoids. At these concentrations, transmembrane effector equilibration is attained by passive diffusion rather than active uptake via the ABC transporter, presumably serving the studied 3-phenylpropanoids as well as benzoate. Highly substrate-specific enzyme formation (EbA5316 to EbA5321 [EbA5316-21]) for the shared peripheral degradation pathway putatively involves the predicted TetR-type transcriptional repressor PprR. Accordingly, relative transcript abundances of ebA5316-21 are lower in succinate- and benzoate-grown wild-type cells than in an unmarked in-frame ΔpprR mutant. In trans-complementation of pprR into the ΔpprR background restored wild-type-like transcript levels. When adapted to p-coumarate, the three genotypes had relative transcript abundances similar to those of ebA5316-21 despite a significantly longer lag phase of the pprR-complemented mutant (∼100-fold higher pprR transcript level than the wild type). Notably, transcript levels of ebA5316-21 were ∼10- to 100-fold higher in p-coumarate- than succinate- or benzoate-adapted cells across all three genotypes. This indicates the additional involvement of an unknown transcriptional regulator. Furthermore, physiological, transcriptional, and (aromatic) acyl-coenzyme A ester intermediate analyses of the wild type and ΔpprR mutant grown with binary substrate mixtures suggest a mode of catabolite repression of superior order to PprR.IMPORTANCE Lignin is a ubiquitous heterobiopolymer built from a suite of 3-phenylpropanoid subunits. It accounts for more than 30% of the global plant dry material, and lignin-related compounds are increasingly released into the environment from anthropogenic sources, i.e., by wastewater effluents from the paper and pulp industry. Hence, following biological or industrial decomplexation of lignin, vast amounts of structurally diverse 3-phenylpropanoids enter terrestrial and aquatic habitats, where they serve as substrates for microbial degradation. This raises the question of what signaling systems environmental bacteria employ to detect these nutritionally attractive compounds and to adjust their catabolism accordingly. Moreover, determining in vivo response thresholds of an anaerobic degradation specialist such as A. aromaticum EbN1^T for these aromatic compounds provides insights into the environmental fate of the latter, i.e., when they could escape biodegradation due to too low ambient concentrations.

Keywords: (aromatic) acyl-CoA ester; Aromatoleum aromaticum EbN1T; anaerobic degradation; deletion mutation; diauxie; phenylpropanoids; physiology; regulation; responsiveness; sensory system; transcript profiling.

FIG 1

Scheme of the proposed transcriptional regulation of anaerobic 3-phenylpropanoid degradation in the denitrifying bacterium Aromatoleum aromaticum EbN1^T. Gene expression is proposed to be mediated by the predicted one-component transcriptional repressor PprR. PprR is assumed to bind shared degradation intermediates of different 3-phenylpropanoids to its N-terminal TetR domain. Upon effector binding, PprR disengages from the promoter region of the 3-phenylpropanoid catabolic gene cluster, allowing initiation of transcription (ebA5316-21; pprA-F). Compound names are the following: 1, benzoate; 2, 3-phenylpropanoate or 3-(4-hydroxyphenyl)propanoate; 3, cinnamate or p-coumarate; 4, hydrocinnamoyl-CoA or 3-(4-hydroxyphenyl)propanoyl-CoA; 5, cinnamoyl-CoA or p-coumaroyl-CoA; 6, 3-hydroxy-3-phenylpropanoyl-CoA or 3-hydroxy-3-(4-hydroxyphenyl)propanoyl-CoA; 7, benzoylacetyl-CoA or 4-hydroxybenzoylacetyl-CoA; 8, 4-hydroxybenzoyl-CoA; 9, benzoyl-CoA; 10, cyclohexa-1,5-diene-1-carbonyl-CoA. R, H or OH. green−, presence or absence of double bond. OM, outer membrane; PP, periplasm; IM, inner membrane.

FIG 2

Time-resolved, quantitative transcript profiles of A. aromaticum EbN1^T in response to different extracellular effector concentrations. Tested 3-phenylpropanoid effectors were 3-phenylpropanoate, 3-(4-hydroxyphenyl)propanoate, cinnamate, and p-coumarate. The selected transcripts represent genes (Fig. 1) coding for enzymes involved in the anaerobic degradation of 3-phenylpropanoids (ebA5316, ebA5317, ebA5319, and ebA5321). Relative transcript abundances were determined by means of qRT-PCR, with the time point of 5 min prior to effector addition serving as a reference. Each data point is based on 3 biological replicates with 3 technical replicates analyzed for each. Growth data of the cultures providing the RNA samples for all tested conditions are shown in detail in Fig. S1 to S4.

FIG 3

Relationship between 3-phenylpropanoid and benzoate utilization by A. aromaticum EbN1^T in terms of uptake, catabolism, and transcriptional regulation. (A) Structural and functional representation of the chromosomal locus (27) comprising the genes related to anaerobic catabolism of 3-phenylpropanoids and benzoate (20). Note the proposed shared role of the predicted ABC-type transporter for the uptake of these aromatic carboxylates. (B) Differential profiles of the encoded proteins are in accord with the proposed polyspecificity of this ABC uptake system. Proteomic profiling was based on membrane protein-enriched as well as soluble protein fractions. In case of protein identification in both fractions, the one yielding the highest MASCOT score is shown here; further details are provided in Table S7.

FIG 4

Characterization of the wild type (blue), ΔpprR mutant (pink), and pprR-complemented mutant (gray-blue) of A. aromaticum EbN1^T. All three genotypes were grown with 8 mM succinate (A), 4 mM benzoate (B), or 2 mM p-coumarate (C). (D) Increased transcript level of pprR in the pprR-complemented mutant (gray-blue) compared to the wild type (blue) of A. aromaticum EbN1^T; transcript levels are normalized against gyrB.

FIG 5

Relative transcript abundances of 3-phenylpropanoid genes in A. aromaticum EbN1^T grown with succinate (A), benzoate (B), or p-coumarate (C). Cells were harvested at half-maximum optical density, and transcripts of target genes were normalized against gyrB. Each data point is based on 3 biological replicates with 3 technical replicates analyzed for each. Colors: blue, wild type; pink, ΔpprR mutant; gray-blue, pprR-complemented mutant.

FIG 6

Anaerobic cultivation of A. aromaticum EbN1^T (wild type) for targeted transcript analysis in response to a binary mixture of 1 mM p-coumarate and either 1 mM benzoate (A) or 3 mM succinate (B). Growth was monitored by measuring the optical density at 660 nm (OD₆₆₀). Sampling time points for transcript analysis are indicated by gray dashed lines. Fold changes of each gene at each time point were normalized against gyrB. Each data point is based on 3 biological replicates with 3 technical replicates analyzed for each.

FIG 7

Quantification of (aromatic) acyl-CoA metabolites in cells of A. aromaticum EbN1^T (wild type and ΔpprR mutant) adapted to anaerobic growth with p-coumarate, benzoate, or succinate. Shades of red represent the number of molecules per cell. For each substrate condition and genotype at least three biological replicates were analyzed. Compound names: 5 to 10, see the legend to Fig. 1; 11, 6-hydroxycyclohex-1-ene-1-carbonyl-CoA; 12, 6-oxocyclohex-1-ene-1-carbonyl-CoA; 13, 3-hydroxypimelyl-CoA.

FIG 8

Correlation of response threshold with K_d values of known SBPs. (A) Response threshold (red) for 3-phenylpropanoids determined in this study. K_d values (gray) theoretically inferred for the predicted SBP EbA5316 from the in vivo response thresholds. Abbreviations: 3PP, 3-phenylpropanoate; 3HP, 3-(4-hydroxyphenyl)propanoate; Cin, cinnamate; p-Cou, p-coumarate. (B) K_d values of known SBPs as retrieved from published experimental studies (K_d values, further details, and references are compiled in Table S10). Note that the category “others” comprises SBPs for vitamins, cofactors, trace elements, and signaling molecules. Black lines, medians.