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INTRODUCTION: Phocaeicola vulgatus (basonym Bacteroides vulgatus) belongs to the intestinal microbiome of healthy humans and animals, where it participates in the fermentative breakdown of biopolymers ingested with food. In doing so, P. vulgatus contributes to the shaping of the gut metabolome, which benefits the host health. Moreover, considering the fermentation product range (short chain fatty acids), P. vulgatus suggests itself as a potential nonstandard platform organism for a sustainable production of basic organic chemicals. Complementing a recent physiologic-proteomic report deciphering the strain's versatile fermentation network, the present study focusses on the global growth phase-dependent response of P. vulgatus. METHODS: P. vulgatus was anaerobically cultivated with glucose as sole source of carbon and energy in process-controlled bioreactors operated in parallel. Close sampling was conducted to measure growth parameters (OD, CDW, ATP-content, substrate/product profiles) as basis for determining growth stoichiometry in detail. A coarser sampling (½ODmax, ODmax, and ODstat) served the molecular analysis of the global growth phase-dependent response, studied by means of differential proteomics (soluble and membrane fractions, nanoLC-ESI-MS/MS) as well as targeted metabolite (GC-MS and LC-MS/MS) and untargeted exometabolome (FT-ICR-MS) analyses. RESULTS: The determined growth performance of P. vulgatus features 1.74 h doubling time, 0.06 gCDW/mmolGlc biomass yield, 0.36 (succinate) and 0.61 (acetate) mmolP/mmolGlc as predominant fermentation product yields, and 0.43 mmolATP/mmolC as theoretically calculated ATP yield. The fermentation pathway displayed distinct growth phase-dependent dynamics: the levels of proteins and their accompanying metabolites constituting the upper part of glycolysis peaked at ½ODmax, whereas those of the lower part of glycolysis and of the fermentation routes in particular toward the predominant products acetate and succinate were highest at ODmax and ODstat. While identified proteins of monomer biosynthesis displayed rather unspecific profiles, most of the intracellular amino acids peaked at ODmax. By contrast, proteins and metabolites related to stress response and quorum sensing showed increased abundances at ODmax and ODstat. Finally, the composition of the exometabolome expanded from 2,317 molecular formulas at ½ODmax via 4,258 at ODmax to 4,501 at ODstat, with growth phase-specific subsets increasing in parallel. CONCLUSIONS: The present study provides insights into the distinct growth phase-dependent behavior of P. vulgatus during cultivation in bioreactors on the physiological and molecular levels. This could serve as a valuable knowledge-base for further developing P. vulgatus as a nonconventional platform organism for biotechnological applications. In addition, the findings shed new light on the potential growth phase-dependent imprints of P. vulgatus on the gut microbiome environment, e.g., by indicating interactions via quorum sensing and by unraveling the complex exometabolic background against which fermentation products and secondary metabolites are formed.
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Reactores Biológicos , Fermentación , Microbioma Gastrointestinal , Glucosa , Microbioma Gastrointestinal/fisiología , Fermentación/fisiología , Glucosa/metabolismo , Reactores Biológicos/microbiología , Proteómica , Proteínas Bacterianas/metabolismo , Humanos , Bacteroides/metabolismo , Bacteroides/crecimiento & desarrollo , Anaerobiosis/fisiología , Metaboloma/fisiologíaRESUMEN
INTRODUCTION: Phocaeicola vulgatus (formerly Bacteroides vulgatus) is a prevalent member of human and animal guts, where it influences by its dietary-fiber-fueled, fermentative metabolism the microbial community as well as the host health. Moreover, the fermentative metabolism of P. vulgatus bears potential for a sustainable production of bulk chemicals. The aim of the present study was to refine the current understanding of the P. vulgatus physiology. METHODS: P. vulgatus was adapted to anaerobic growth with 14 different carbohydrates, ranging from hexoses, pentoses, hemicellulose, via an uronic acid to deoxy sugars. These substrate-adapted cells formed the basis to define the growth stoichiometries by quantifying growth/fermentation parameters and to reconstruct the catabolic network by applying differential proteomics. RESULTS: The determination of growth performance revealed, e.g., doubling times (h) from 1.39 (arabinose) to 14.26 (glucuronate), biomass yields (gCDW/mmolS) from 0.01 (fucose) to 0.27 (α-cyclodextrin), and ATP yields (m
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Fermentación , Proteómica , Fermentación/fisiología , Proteínas Bacterianas/metabolismo , Microbioma Gastrointestinal/fisiología , Anaerobiosis/fisiología , Fibras de la Dieta/metabolismo , Humanos , Bacteroidetes/metabolismo , Redes y Vías MetabólicasRESUMEN
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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Fenol , Fenoles , Fenol/metabolismo , Fenoles/metabolismo , Rhodocyclaceae/metabolismo , Biodegradación Ambiental , AnaerobiosisRESUMEN
Phaeobacter inhibens DSM 17395 is a heterotrophic member of the ubiquitous, marine Roseobacter group and specializes in the aerobic utilization of carbohydrates and amino acids via pathways widespread among roseobacters. The in vivo responsiveness of P. inhibens DSM 17395 was studied with nonadapted cells (succinate-grown), which were exposed to a single pulse (100-0.01 µM) each of N-acetylglucosamine, mannitol, xylose, leucine, phenylalanine, or tryptophan (effectors). Responsiveness was then determined by time-resolved transcript analyses (quantitative reverse transcription-PCR) of "degradation" and "uptake" genes selected based on previously reported substrate-specific proteome profiles. The transcriptional response thresholds were: 50-100 nM for nagK (N-acetylglucosamine kinase), paaA (ring 1,2-phenylacetyl-CoA epoxidase), and kynA (tryptophan 2,3-dioxygenase), 10-50 nM for xylA (xylose isomerase), and around 10 nM for mtlK (mannitol 2-dehydrogenase). A threshold for leucine could not be determined due to the elevated intrinsic presence of leucine in the exometabolome of succinate-grown cells (no effector addition). Notably, the response thresholds for presumptive carbohydrate-binding proteins of ABC-transporters were in the same range or even lower: 0.1-1 µM for c27930 (N-acetylglucosamine) and even below 10 nM for c13210 (mannitol) and xylF (xylose). These results shed new light on the sensory/regulatory sensitivity of a well-studied roseobacter for recognizing potential substrates at low ambient concentrations and on the concentration threshold below which these might escape biodegradation ("emergent recalcitrance" concept of dissolved organic matter persistence).
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Aminoácidos , Roseobacter , Acetilglucosamina/metabolismo , Aminoácidos/metabolismo , Hexosas/metabolismo , Manitol/metabolismo , Rhodobacteraceae , Roseobacter/genética , Succinatos/metabolismo , Xilosa/metabolismoRESUMEN
In all living organisms, adenosine triphosphate (ATP) and NAD(H) represent universal molecular currencies for energy and redox state, respectively, and are thus widely applicable molecular proxies for an organism's viability and activity. To this end, corresponding luciferase-based assays in combination with a microplate reader were established with the marine model bacterium Phaeobacter inhibens DSM 17395 (Escherichia coli K12 served as reference). Grey multiwell plates best balanced sensitivity and crosstalk, and optimal incubation times were 5 min and 30 min for the ATP and NAD(H) assay, respectively, together allowing limits of detection of 0.042, 0.470 and 0.710 nM for ATP, NAD+, and NADH, respectively. Quenching of bacterial cell samples involved Tris-EDTA-DTAB and bicarbonate base-DTAB for ATP and NAD(H) assays, respectively. The ATP and NAD(H) yields determined for P. inhibens DSM 17395 at » ODmax were found to reside well within the range previously reported for E. coli and other bacteria, e.g., 3.28 µmol ATP (g cellsdry)-1. Thus, the here described methods for luciferase-based determination of ATP/NAD(H) pools open a promising approach to investigate energy and redox states in marine (environmental) bacteria.
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Adenosina Trifosfato , NAD , Escherichia coli/genética , LuciferasasRESUMEN
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 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 (â¼100-fold higher pprR transcript level than the wild type). Notably, transcript levels of ebA5316-21 were â¼10- 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 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.
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Cinamatos/metabolismo , Ácidos Cumáricos/metabolismo , Lignina/metabolismo , Fenilpropionatos/metabolismo , Rhodocyclaceae/metabolismo , Biodegradación AmbientalRESUMEN
The genus Desulfonema belongs to the deltaproteobacterial family Desulfobacteraceae and comprises marine, sulfate-reducing bacteria that form filaments and move by gliding. This study reports on the complete, manually annotated genomes of Dn. limicola 5ac10T (6.91 Mbp; 6,207 CDS) and Dn. magnum 4be13T (8.03 Mbp; 9,970 CDS), integrated with substrate-specific proteome profiles (8 vs. 11). The richness in mobile genetic elements is shared with other Desulfobacteraceae members, corroborating horizontal gene transfer as major driver in shaping the genomes of this family. The catabolic networks of Dn. limicola and Dn. magnum have the following general characteristics: 98 versus 145 genes assigned (having genomic shares of 1.7 vs. 2.2%), 92.5 versus 89.7% proteomic coverage, and scattered gene clusters for substrate degradation and energy metabolism. The Dn. magnum typifying capacity for aromatic compound degradation (e.g., p-cresol, 3-phenylpropionate) requires 48 genes organized in operon-like structures (87.7% proteomic coverage; no homologs in Dn. limicola). The protein complements for aliphatic compound degradation, central pathways, and energy metabolism are highly similar between both genomes and were identified to a large extent (69-96%). The differential protein profiles revealed a high degree of substrate-specificity for peripheral reaction sequences (forming central intermediates), agreeing with the high number of sensory/regulatory proteins predicted for both strains. By contrast, central pathways and modules of the energy metabolism were constitutively formed under the tested substrate conditions. In accord with their natural habitats that are subject to fluctuating changes of physicochemical parameters, both Desulfonema strains are well equipped to cope with various stress conditions. Next to superoxide dismutase and catalase also desulfoferredoxin and rubredoxin oxidoreductase are formed to counter exposure to molecular oxygen. A variety of proteases and chaperones were detected that function in maintaining cellular homeostasis upon heat or cold shock. Furthermore, glycine betaine/proline betaine transport systems can respond to hyperosmotic stress. Gliding movement probably relies on twitching motility via type-IV pili or adventurous motility. Taken together, this proteogenomic study demonstrates the adaptability of Dn. limicola and Dn. magnum to its dynamic habitats by means of flexible catabolism and extensive stress response capacities.
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The marine alphaproteobacterium Phaeobacter inhibens DSM 17395, a member of the Roseobacter 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 P. inhibens DSM 17395.
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Antibacterianos/biosíntesis , Rhodobacteraceae/genética , Rhodobacteraceae/metabolismo , Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , Reactores Biológicos , Dióxido de Carbono , Cromosomas , Proteoma , Replicón , Transcriptoma , Tropolona/análogos & derivadosRESUMEN
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 â¼200-/â¼30-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 (â¼20 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.
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Anaerobiosis , Biodegradación Ambiental , Contaminantes Ambientales/química , Fenoles/química , Algoritmos , Regulación Bacteriana de la Expresión Génica , Modelos Teóricos , Mutación , Proteoma , Rhodocyclaceae/genética , Rhodocyclaceae/metabolismo , TranscriptomaRESUMEN
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 â¼3-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 â¼37.7% or â¼29.5%, 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 â¼28% 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.
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Aminoácidos/metabolismo , Procesos Heterotróficos/fisiología , Rhodobacteraceae/metabolismo , Azúcares/metabolismo , Adenosina Trifosfato/metabolismo , Biomasa , Reactores Biológicos , Metabolismo de los Hidratos de Carbono , Redes y Vías Metabólicas , Rhodobacteraceae/crecimiento & desarrollo , Roseobacter/metabolismo , Tropolona/análogos & derivadosRESUMEN
The denitrifying betaproteobacterium "Aromatoleum aromaticum" EbN1 regulates the capacity to anaerobically degrade p-ethylphenol (via p-hydroxyacetophenone) with high substrate specificity. This process is mediated by the σ54-dependent transcriptional regulator EtpR, which apparently recognizes both aromatic compounds, yielding congruent expression profiles. The responsiveness of this regulatory system was studied with p-hydroxyacetophenone, which is more easily administered to cultures and traced analytically. Cultures of A. aromaticum EbN1 were initially cultivated under nitrate-reducing conditions with a growth-limiting supply of benzoate, upon the complete depletion of which p-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 p-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., pchF, encoding a subunit of predicted p-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., acsA) and potential efflux (i.e., ebA335).IMPORTANCE Aromatic compounds are widespread microbial growth substrates with natural as well as anthropogenic sources, albeit with their in situ 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. A. aromaticum 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 (p-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.