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Benzoate mediates repression of C(4)-dicarboxylate utilization in "Aromatoleum aromaticum" EbN1.

ABSTRACT: Diauxic growth was observed in anaerobic C(4)-dicarboxylate-adapted cells of "Aromatoleum aromaticum" EbN1 due to preferred benzoate utilization from a substrate mixture of a C(4)-dicarboxylate (succinate, fumarate, or malate) and benzoate. Differential protein profiles (two-dimensional difference gel electrophoresis [2D DIGE]) revealed dynamic changes in abundance for proteins involved in anaerobic benzoate catabolism and C(4)-dicarboxylate uptake. In the first active growth phase, benzoate utilization was paralleled by maximal abundance of proteins involved in anaerobic benzoate degradation (e.g., benzoyl-coenzyme A [CoA] reductase) and minimal abundance of DctP (EbA4158), the periplasmic binding protein of a predicted C(4)-dicarboxylate tripartite ATP-independent periplasmic (TRAP) transporter (DctPQM). The opposite was observed during subsequent succinate utilization in the second active growth phase. The increased dctP (respectively, dctPQM) transcript and DctP protein abundance following benzoate depletion suggests that repression of C(4)-dicarboxylate uptake seems to be a main determinant for the observed diauxie.

SUBMITTER: Trautwein K

PROVIDER: S-EPMC3256680 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Benzoate mediates repression of C(4)-dicarboxylate utilization in "Aromatoleum aromaticum" EbN1.

Trautwein Kathleen K Grundmann Olav O Wöhlbrand Lars L Eberlein Christian C Boll Matthias M Rabus Ralf R

Journal of bacteriology 20111111 2

Diauxic growth was observed in anaerobic C(4)-dicarboxylate-adapted cells of "Aromatoleum aromaticum" EbN1 due to preferred benzoate utilization from a substrate mixture of a C(4)-dicarboxylate (succinate, fumarate, or malate) and benzoate. Differential protein profiles (two-dimensional difference gel electrophoresis [2D DIGE]) revealed dynamic changes in abundance for proteins involved in anaerobic benzoate catabolism and C(4)-dicarboxylate uptake. In the first active growth phase, benzoate uti ...[more]

PMID: 22081395

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Project description:Aromatoleum aromaticum EbN1T anaerobically degrades phenol, p-cresol, and p-ethylphenol, each via a distinct peripheral pathway. The compound-specific regulation of each pathway is proposed to occur on the transcriptional level in the case of phenol supposedly mediated by the one-component system PheR. To confirm its predicted function, we generated an unmarked, in-frame deletion mutant (ΔpheR). This mutant did not express the ppsA1 gene, which encodes the A1 subunit of phenol-activating phenylphosphate synthase. The expression of ppsA1 was restored by in trans complementation of pheR into the ΔpheR background. The responsiveness to phenol was studied in vivo in benzoate-limited anaerobic cultures by adding, upon benzoate depletion, single defined pulses of phenol (from 100 µM down to 0.1 nM). Time-resolved, targeted transcript profiling by qRT-PCR revealed a response threshold for ppsA1 expression of 30‒50 nM phenol. Notably, ppsA1 expression could not be induced by p-cresol or p-ethylphenol. Conversely, lack of expression was also observed for the additional target genes cmh (p-cresol degradation) and acsA1 (p-ethylphenol degradation) applying phenol or p-ethylphenol as well as phenol or p-cresol as stimuli. Thus, the sensory proteins PheR, PcrS, and EtpR should be highly selective for phenol, p-cresol, and p-ethylphenol, respectively. The implicated incapability of cross-stimulus binding was corroborated by comparing the predicted 3D structural models of the proteins' sensory domains. While the ligand-binding pockets share the conserved hydroxy group-anchoring histidine and tryptophane, their distal faces in PcrS and EtpR are, compared to PheR, enlarged to accommodate the bulkier methyl (p-cresol) and ethyl group (p-ethylphenol), respectively. IMPORTANCE Aromatic compounds are globally abundant organic molecules with a multitude of natural and anthropogenic sources, underpinning the relevance of their biodegradation. A. aromaticum EbN1T is a well-studied environmental betaproteobacterium specialized on the anaerobic degradation of aromatic compounds. The here studied responsiveness toward phenol in conjunction with the apparent high ligand selectivity (non-promiscuity) of its PheR sensor and those of the related p-cresol (PcrS) and p-ethylphenol (EtpR) sensors are in accord with the substrate-specificity and biochemical distinctiveness of the associated degradation pathways. Furthermore, the present findings advance our general understanding of the substrate-specific regulation of the strain's remarkable degradation network and of the concentration thresholds below which phenolic compounds become essentially undetectable and as a consequence should escape substantial biodegradation. Furthermore, the findings may inspire biomimetic sensor designs for detecting and quantifying phenolic contaminants in wastewater or environments.

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Project description:Members of the genus Aromatoleum thrive in diverse habitats and use a broad range of recalcitrant organic molecules coupled to denitrification or O2 respiration. To gain a holistic understanding of the model organism A. aromaticum EbN1T, we studied its catabolic network dynamics in response to 3-(4-hydroxyphenyl)propanoate, phenylalanine, 3-hydroxybenzoate, benzoate, and acetate utilized under nitrate-reducing versus oxic conditions. Integrated multi-omics (transcriptome, proteome, and metabolome) covered most of the catabolic network (199 genes) and allowed for the refining of knowledge of the degradation modules studied. Their substrate-dependent regulation showed differing degrees of specificity, ranging from high with 3-(4-hydroxyphenyl)propanoate to mostly relaxed with benzoate. For benzoate, the transcript and protein formation were essentially constitutive, contrasted by that of anoxia-specific versus oxia-specific metabolite profiles. The matrix factorization of transcriptomic data revealed that the anaerobic modules accounted for most of the variance across the degradation network. The respiration network appeared to be constitutive, both on the transcript and protein levels, except for nitrate reductase (with narGHI expression occurring only under nitrate-reducing conditions). The anoxia/nitrate-dependent transcription of denitrification genes is apparently controlled by three FNR-type regulators as well as by NarXL (all constitutively formed). The resequencing and functional reannotation of the genome fostered a genome-scale metabolic model, which is comprised of 655 enzyme-catalyzed reactions and 731 distinct metabolites. The model predictions for growth rates and biomass yields agreed well with experimental stoichiometric data, except for 3-(4-hydroxyphenyl)propanoate, with which 4-hydroxybenzoate was exported. Taken together, the combination of multi-omics, growth physiology, and a metabolic model advanced our knowledge of an environmentally relevant microorganism that differs significantly from other bacterial model strains. IMPORTANCE Aromatic compounds are abundant constituents not only of natural organic matter but also of bulk industrial chemicals and fuel components of environmental concern. Considering the widespread occurrence of redox gradients in the biosphere, facultative anaerobic degradation specialists can be assumed to play a prominent role in the natural mineralization of organic matter and in bioremediation at contaminated sites. Surprisingly, differential multi-omics profiling of the A. aromaticum EbN1T studied here revealed relaxed regulatory stringency across its four main physiological modi operandi (i.e., O2-independent and O2-dependent degradation reactions versus denitrification and O2 respiration). Combining multi-omics analyses with a genome-scale metabolic model aligned with measured growth performances establishes A. aromaticum EbN1T as a systems-biology model organism and provides unprecedented insights into how this bacterium functions on a holistic level. Moreover, this experimental platform invites future studies on eco-systems and synthetic biology of the environmentally relevant betaproteobacterial Aromatoleum/Azoarcus/Thauera cluster.

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Benzoate mediates repression of C(4)-dicarboxylate utilization in "Aromatoleum aromaticum" EbN1.

Publications

Benzoate mediates repression of C(4)-dicarboxylate utilization in "Aromatoleum aromaticum" EbN1.

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