eThe betaproteobacteria "Aromatoleum aromaticum" pCyN1 and "Thauera" sp. strain pCyN2 anaerobically degrade the plant-derived aromatic hydrocarbon p-cymene (4-isopropyltoluene) under nitrate-reducing conditions. Metabolite analysis of p-cymene-adapted "A. aromaticum" pCyN1 cells demonstrated the specific formation of 4-isopropylbenzyl alcohol and 4-isopropylbenzaldehyde, whereas with "Thauera" sp. pCyN2, exclusively 4-isopropylbenzylsuccinate and tentatively identified (4-isopropylphenyl)itaconate were observed. 4-Isopropylbenzoate in contrast was detected with both strains. Proteogenomic investigation of p-cymene-versus succinate-adapted cells of the two strains revealed distinct protein profiles agreeing with the different metabolites formed from p-cymene. "A. aromaticum" pCyN1 specifically produced (i) a putative p-cymene dehydrogenase (CmdABC) expected to hydroxylate the benzylic methyl group of p-cymene, (ii) two dehydrogenases putatively oxidizing 4-isopropylbenzyl alcohol (Iod) and 4-isopropylbenzaldehyde (Iad), and (iii) the putative 4-isopropylbenzoate-coenzyme A (CoA) ligase (Ibl). The p-cymene-specific protein profile of "Thauera" sp. pCyN2, on the other hand, encompassed proteins homologous to subunits of toluene-activating benzylsuccinate synthase (termed [4-isopropylbenzyl]succinate synthase IbsABCDEF; identified subunits, IbsAE) and protein homologs of the benzylsuccinate -oxidation (Bbs) pathway (termed BisABCDEFGH; all identified except for BisEF). This study reveals that two related denitrifying bacteria employ fundamentally different peripheral degradation routes for one and the same substrate, p-cymene, with the two pathways apparently converging at the level of 4-isopropylbenzoyl-CoA.
BackgroundSulfate-reducing bacteria (SRB) are key players of the carbon- and sulfur-cycles in the sediments of the world’s oceans. Habitat relevant SRBs are often members of the Desulfosarcina-Desulfococcus clade belonging to the deltaproteobacterial family of Desulfobacteraceae. Despite this environmental recognition, their molecular (genome-based) physiology and their potential to contribute to organic carbon mineralization as well as to adapt to changing environmental conditions have been scarcely investigated. A metabolically versatile representative of this family is Desulfococcus multivorans that is able to completely oxidize (to CO2) a variety of organic acids, including fatty acids up to C14, as well as aromatic compounds.ResultsIn this study the complete 4.46 Mbp and manually annotated genome of metabolically versatile Desulfococcus multivorans DSM 2059 is presented with particular emphasis on a proteomics-driven metabolic reconstruction. Proteomic profiling covered 17 substrate adaptation conditions (6 aromatic and 11 aliphatic compounds) and comprised 2D DIGE, shotgun proteomics and analysis of the membrane protein-enriched fractions. This comprehensive proteogenomic dataset allowed for reconstructing a metabolic network of degradation pathways and energy metabolism that consists of 170 proteins (154 detected; ~91 % coverage). Peripheral degradation routes feed via central benzoyl-CoA, (modified) β-oxidation or methylmalonyl-CoA pathways into the Wood-Ljungdahl pathway for complete oxidation of acetyl-CoA to CO2. Dissimilatory sulfate reduction is fueled by a complex electron transfer network composed of cytoplasmic components (e.g., electron transfer flavoproteins) and diverse membrane redox complexes (Dsr, Qmo, Hmc, Tmc, Qrc, Nuo and Rnf). Overall, a high degree of substrate-specific formation of catabolic enzymes was observed, while most complexes involved in electron transfer appeared to be constitutively formed.ConclusionsA highly dynamic genome structure in combination with substrate-specifically formed catabolic subproteomes and a constitutive subproteome for energy metabolism and electron transfer appears to be a common trait of Desulfobacteraceae members.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3236-7) contains supplementary material, which is available to authorized users.
BackgroundAt high concentrations of organic substrates, microbial utilization of preferred substrates (i.e., supporting fast growth) often results in diauxic growth with hierarchical substrate depletion. Unlike the carbon catabolite repression-mediated discriminative utilization of carbohydrates, the substrate preferences of non-carbohydrate-utilizing bacteria for environmentally relevant compound classes (e.g., aliphatic or aromatic acids) are rarely investigated. The denitrifying alphaproteobacterium Magnetospirillum sp. strain pMbN1 anaerobically degrades a wide variety of aliphatic and aromatic compounds and is unique for anaerobic degradation of 4-methylbenzoate. The latter proceeds via a distinct reaction sequence analogous to the central anaerobic benzoyl-CoA pathway to intermediates of central metabolism. Considering the presence of these two different anaerobic “aromatic ring degrading” pathways, substrate preferences of Magnetospirillum sp. strain pMbN1 were investigated. Anaerobic growth and substrate consumption were monitored in binary and ternary mixtures of 4-methylbenzoate, benzoate and succinate, in conjuction with time-resolved abundance profiling of selected transcripts and/or proteins related to substrate uptake and catabolism.ResultsDiauxic growth with benzoate preference was observed for binary and ternary substrate mixtures containing 4-methylbenzoate and succinate (despite adaptation of Magnetospirillum sp. strain pMbN1 to one of the latter two substrates). On the contrary, 4-methylbenzoate and succinate were utilized simultaneously from a binary mixture, as well as after benzoate depletion from the ternary mixture. Apparently, simultaneous repression of 4-methylbenzoate and succinate utilization from the ternary substrate mixture resulted from (i) inhibition of 4-methylbenzoate uptake, and (ii) combined inhibition of succinate uptake (via the two transporters DctPQM and DctA) and succinate conversion to acetyl-CoA (via pyruvate dehydrogenase). The benzoate-mediated repression of C4-dicarboxylate utilization in Magnetospirillum sp. strain pMbN1 differs from that recently described for “Aromatoleum aromaticum” EbN1 (involving only DctPQM).ConclusionsThe preferential or simultaneous utilization of benzoate and other aromatic acids from mixtures with aliphatic acids may represent a more common nutritional behavior among (anaerobic) degradation specialist than previously thought. Preference of Magnetospirillum sp. strain pMbN1 for benzoate from mixtures with 4-methylbenzoate, and thus temporal separation of the benzoyl-CoA (first) and 4-methylbenzoyl-CoA (second) pathway, may reflect a catabolic tuning towards metabolic efficiency and the markedly broader range of aromatic substrates feeding into the central anaerobic benzoyl-CoA pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0269-4) contains supplementary material, which is available to authorized users.
BackgroundThe denitrifying betaproteobacterium "Aromatoleum aromaticum" EbN1 anaerobically utilizes a multitude of aromatic compounds via specific peripheral degradation routes. Compound-specific formation of these catabolic modules is assumed to be mediated by specific transcriptional activators. In case of the recently elucidated p-ethylphenol/p-hydroxyacetophenone pathway, the highly substrate-specific regulation was implicated to involve the predicted σ54-dependent, NtrC-type regulator EbA324. The latter was suggested to control the expression of the two neighboring gene clusters encoding the catabolic enzymes as well as a corresponding putative solvent efflux system. In the present study, a molecular genetic approach was used to study the predicted function of EbA324.ResultsAn unmarked in frame ΔebA324 (here renamed as ΔetpR; p-ethylphenol regulator) deletion mutation was generated. The ΔetpR mutant was unable to grow anaerobically with either p-ethylphenol or p-hydroxyacetophenone. Growth similar to the wild type was restored in the ΔetpR mutant background by in trans expression of plasmid-born etpR. Furthermore, expression of the "p-ethylphenol" gene clusters as well as corresponding protein formation was shown to depend on the presence of both, EtpR and either p-ethylphenol or p-hydroxyacetophenone. In the wild type, the etpR gene appears to be constitutively expressed and its expression level not to be modulated upon effector presence. Comparison with the regulatory domains of known phenol- and alkylbenzene-responsive NtrC-type regulators of Pseudomonas spp. and Thauera aromatica allowed identifying >60 amino acid residues in the regulatory domain (in particular positions 149 to 192 of EtpR) that may contribute to the effector specificity viz. presumptively restricted effector spectrum of EtpR.ConclusionsThis study provides experimental evidence for the genome predicted σ54-dependent regulator EtpR (formerly EbA324) of "A. aromaticum" EbN1 to be responsive to p-ethylphenol, as well as its degradation intermediate p-hydroxyacetophenone, and to control the expression of genes involved in the anaerobic degradation of these two aromatic growth substrates. Overall, the presented results advance our understanding on the regulation of anaerobic aromatic compound catabolism, foremost based on the sensory discrimination of structurally similar substrates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0571-9) contains supplementary material, which is available to authorized users.
The constitutions and absolute configurations of two previously unknown intermediates, (1S,2S,4S)‐2‐hydroxy‐4‐isopropylcyclohexane‐1‐carboxylate and (S)‐3‐isopropylpimelate, of anaerobic degradation of p‐cymene in the bacterium Aromatoleum aromaticum pCyN1 are reported. These intermediates (as CoA esters) are involved in the further degradation of 4‐isopropylbenzoyl‐CoA formed by methyl group hydroxylation and subsequent oxidation of p‐cymene. Proteogenomics indicated 4‐isopropylbenzoyl‐CoA degradation involves (i) a novel member of class I benzoyl‐CoA reductase (BCR) as known from Thauera aromatica K172 and (ii) a modified β‐oxidation pathway yielding 3‐isopropylpimeloyl‐CoA analogously to benzoyl‐CoA degradation in Rhodopseudomonas palustris. Reference standards of all four diastereoisomers of 2‐hydroxy‐4‐isopropylcyclohexane‐1‐carboxylate as well as both enantiomers of 3‐isopropylpimelate were obtained by stereoselective syntheses via methyl 4‐isopropyl‐2‐oxocyclohexane‐1‐carboxylate. The stereogenic center carrying the isopropyl group was established using a rhodium‐catalyzed asymmetric conjugate addition. X‐ray crystallography revealed that the thermodynamically most stable stereoisomer of 2‐hydroxy‐4‐isopropylcyclohexane‐1‐carboxylate is formed during p‐cymene degradation. Our findings imply that the reductive dearomatization of 4‐isopropylbenzoyl‐CoA by the BCR of A. aromaticum pCyN1 stereospecifically forms (S)‐4‐isopropyl‐1,5‐cyclohexadiene‐1‐carbonyl‐CoA.
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