Anaerobic degradation of p-cresol (4-methylphenol) by the denitrifying betaproteobacterium Aromatoleum aromaticum EbN1 is regulated with high substrate specificity, presumed to be mediated by the predicted σ54-dependent two-component system PcrSR. An unmarked, in-frame ΔpcrSR deletion mutant showed reduced expression of the genes cmh (21-fold) and hbd (8-fold) that encode the two enzymes for initial oxidation of p-cresol to p-hydroxybenzoate compared to their expression in the wild type. The expression of cmh and hbd was restored by in trans complementation with pcrSR in the ΔpcrSR background to even higher levels than in the wild type. This is likely due to ∼200-/∼30-fold more transcripts of pcrSR in the complemented mutant. The in vivo responsiveness of A. aromaticum EbN1 to p-cresol was studied in benzoate-limited anaerobic cultures by the addition of p-cresol at various concentrations (from 100 μM down to 0.1 nM). Time-resolved transcript profiling by quantitative reverse transcription-PCR (qRT-PCR) revealed that the lowest p-cresol concentrations just affording cmh and hbd expression (response threshold) ranged between 1 and 10 nM, which is even more sensitive than the respective odor receptors of insects. A similar response threshold was determined for another alkylphenol, p-ethylphenol, which strain EbN1 anaerobically degrades via a different route and senses by the σ54-dependent one-component system EtpR. Based on these data and theoretical considerations, p-cresol or p-ethylphenol added as a single pulse (10 nM) requires less than a fraction of a second to reach equilibrium between intra- and extracellular space (∼20 molecules per cell), with an estimated Kd (dissociation constant) of <100 nM alkylphenol (p-cresol or p-ethylphenol) for its respective sensory protein (PcrS or EtpR). IMPORTANCE Alkylphenols (like p-cresol and p-ethylphenol) represent bulk chemicals for industrial syntheses. Besides massive local damage events, large-scale micropollution is likewise of environmental and health concern. Next to understanding how such pollutants can be degraded by microorganisms, it is also relevant to determine the microorganisms’ lower threshold of responsiveness. Aromatoleum aromaticum EbN1 is a specialist in anaerobic degradation of aromatic compounds, employing a complex and substrate-specifically regulated catabolic network. The present study aims at verifying the predicted role of the PcrSR system in sensing p-cresol and at determining the threshold of responsiveness for alkylphenols. The findings have implications for the enigmatic persistence of dissolved organic matter (escape from biodegradation) and for the lower limits of aromatic compounds required for bacterial growth.

The denitrifying betaproteobacterium "" EbN1 regulates the capacity to anaerobically degrade -ethylphenol (via-hydroxyacetophenone) with high substrate specificity. This process is mediated by the σ-dependent transcriptional regulator EtpR, which apparently recognizes both aromatic compounds, yielding congruent expression profiles. The responsiveness of this regulatory system was studied with -hydroxyacetophenone, which is more easily administered to cultures and traced analytically. Cultures of EbN1 were initially cultivated under nitrate-reducing conditions with a growth-limiting supply of benzoate, upon the complete depletion of which -hydroxyacetophenone was added at various concentrations (from 500 μM down to 0.1 nM). Depletion profiles of this aromatic substrate and presumptive effector were determined by highly sensitive micro-high-performance liquid chromatography (microHPLC). Irrespective of the added concentration of-hydroxyacetophenone, depletion commenced after less than 5 min and suggested a response threshold of below 10 nM. This approximation was corroborated by time-resolved transcript profiles (quantitative reverse transcription-PCR) of selected degradation and efflux relevant genes (e.g., , encoding a subunit of predicted-ethylphenol methylenehydroxylase) and narrowed down to a range of 10 to 1 nM. The most pronounced transcriptional response was observed, as expected, for genes located at the beginning of the two operon-like structures, related to catabolism (i.e., ) and potential efflux (i.e.,). Aromatic compounds are widespread microbial growth substrates with natural as well as anthropogenic sources, albeit with their concentrations and their bioavailabilities varying over several orders of magnitude. Even though degradation pathways and underlying regulatory systems have long been studied with aerobic and, to a lesser extent, with anaerobic bacteria, comparatively little is known about the effector concentration-dependent responsiveness. EbN1 is a model organism for the anaerobic degradation of aromatic compounds with the architecture of the catabolic network and its substrate-specific regulation having been intensively studied by means of differential proteogenomics. The present study aims at unraveling the minimal concentration of an aromatic growth substrate (-hydroxyacetophenone here) required to initiate gene expression for its degradation pathway and to learn in principle about the lower limit of catabolic responsiveness of an anaerobic degradation specialist.

The betaproteobacterial degradation specialist Aromatoleum aromaticum EbN1T utilizes several plant-derived 3-phenylpropanoids coupled to denitrification. In vivo responsiveness of A. aromaticum EbN1T was studied by exposing non-adapted 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 qRT-PCR of four selected 3-phenylpropanoid genes revealed a response threshold of 30–50 nM for p-coumarate and 1–10 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 (EbA5316–21) 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 wildtype cells compared to an unmarked in-frame ΔpprR mutant. In trans complementation of pprR into the ΔpprR background restored wildtype-like transcript levels. When adapted to p-coumarate, the three genotypes had similar relative transcript abundances of ebA5316–21, despite a significantly longer lag-phase of the pprR-complemented mutant (∼100-fold higher pprR transcript level than wildtype). Notably, transcript levels of ebA5316–21 were ∼10–100-fold higher in p-coumarate- versus succinate- or benzoate-adapted cells across all three genotypes. This possibly indicates the additional involvement of a yet unknown transcriptional regulator. Furthermore, physiological, transcriptional and (aromatic) acyl-CoA ester intermediate analyses of wildtype and ΔpprR mutant grown with binary substrate mixtures suggest a mode of catabolite repression of superior order to PprR. IMPORTANCE Lignin is a ubiquitous hetero-biopolymer built from of a suite of 3-phenylpropanoid subunits. It not only accounts for more than 30% of the global plant dry material, but lignin-related compounds are also 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 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 EbN1T 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.

The marine alphaproteobacterium <i>Phaeobacter inhibens</i> DSM 17395, a member of the <i>Roseobacter</i> group, was recently shown to markedly enhance growth upon deletion of its 262-kb chromid encoding biosynthesis of tropodithietic acid (TDA). To scrutinize the metabolic/regulatory adaptations that underlie enhanced growth of the Δ262 mutant, its transcriptome and proteome compared to the wild type were investigated in process-controlled bioreactors with Casamino Acids as growth substrate. Genome resequencing revealed only few additional genetic changes (a heterogenic insertion, prophage activation, and several point mutations) between wild type and Δ262 mutant, albeit with no conceivable effect on the studied growth physiology. The abundances of the vast majority of transcripts and proteins involved in the catabolic network for complete substrate oxidation to CO₂ were found to be unchanged, suggesting that the enhanced amino acid utilization of the Δ262 mutant did not require elevated synthesis of most enzymes of the catabolic network. Similarly, constituents of genetic information processing and cellular processes remained mostly unchanged. In contrast, 426 genes displayed differential expression, of which 410 were localized on the 3.2-Mb chromosome, 5 on the 65-kb chromid, and 11 on the 78-kb chromid. Notably, the branched-chain amino transferase IlvE acting on rapidly utilized Val, Ile, and Leu was upregulated. Moreover, the transportome was reconfigured, as evidenced from increased abundances of transcripts and proteins of several uptake systems for amino acids and inorganic nutrients (e.g., phosphate). Some components of the respiratory chain were also upregulated, which correlates with the higher respiration rates of the Δ262 mutant. Furthermore, chromosomally encoded transcripts and proteins that are peripherally related to TDA biosynthesis (e.g., the serine acyl transferase CysE) were strongly downregulated in the Δ262 mutant. Taken together, these observations reflect adaptations to enhanced growth as well as the functional interconnectivity of the replicons of <i>P. inhibens</i> DSM 17395.

Growth energetics and metabolic efficiency contribute to the lifestyle and habitat imprint of microorganisms. Roseobacters constitute one of the most abundant and successful marine bacterioplankton groups. Here, we reflect on the energetics and metabolic efficiency of Phaeobacter inhibens DSM 17395, a versatile heterotrophic roseobacter. Fourteen different substrates (five sugars and nine amino acids) and their degradation pathways were assessed for energetic efficiencies based on catabolic ATP yields, calculated from net formed ATP and reducing equivalents. The latter were converted into ATP by employing the most divergent coupling ratios (i.e., ions per ATP) currently known for F1Fo ATP synthases in heterotrophic bacteria. The catabolic ATP yields of the pathways studied in P. inhibens differed ∼3-fold. The actual free energy costs for ATP synthesis were estimated at 81.6 kJ per mol ATP (3.3 ions per ATP) or 104.2 kJ per mol ATP (4.3 ions per ATP), yielding an average thermodynamic efficiency of ∼37.7% or ∼29.5%, respectively. Growth performance (rates, yields) and carbon assimilation efficiency were determined for P. inhibens growing in process-controlled bioreactors with 10 different single substrates (Glc, Man, N-acetylglucosamine [Nag], Phe, Trp, His, Lys, Thr, Val, or Leu) and with 2 defined substrate mixtures. The efficiencies of carbon assimilation into biomass ranged from ∼28% to 61%, with His/Trp and Thr/Leu yielding the lowest and highest levels. These efficiencies correlated with catabolic and ATP yields only to some extent. Substrate-specific metabolic demands and/or functions, as well as the compositions of the substrate mixtures, apparently affected the energetic costs of growth. These include energetic burdens associated with, e.g., slow growth, stress, and/or the production of tropodithietic acid. IMPORTANCE Heterotrophic members of the bacterioplankton serve the marine ecosystem by transforming organic matter, an activity that is governed by the bacterial growth efficiencies (BGEs) obtained under given environmental conditions. In marine ecology, the concept of BGE refers to the carbon assimilation efficiency within natural communities. The marine bacterium studied here, Phaeobacter inhibens DSM 17395, is a copiotrophic representative of the globally abundant Roseobacter group, and the 15 catabolic pathways investigated are widespread among these marine heterotrophs. Combining pathway-specific catabolic ATP yields with in-depth quantitative physiological data could (i) provide a new baseline for the study of growth energetics and efficiency in further Roseobacter group members and other copiotrophic marine bacteria in productive coastal ecosystems and (ii) contribute to a better understanding of the factors controlling BGE (including the additional energetic burden arising from widespread secondary-metabolite formation) based on laboratory studies with pure cultures.