Towards the Response Threshold for p-Hydroxyacetophenone in the Denitrifying Bacterium “Aromatoleum aromaticum” EbN1

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.

ABSTRACT

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.

INTRODUCTION

Aromatic compounds belong to the most prominent components of recent and fossil organic matter in the biosphere and geosphere (e.g., see references 1 and 2) and encompass industrially relevant chemicals that are also of environmental concern (e.g., see reference 3). Accordingly, the biodegradation of these compounds is relevant for diverse areas ranging from global carbon cycling to bioremediation efforts. Research has put the most emphasis on elucidating single reactions and pathways, as aromatic compounds possess only very low chemical reactivity and require special biochemical strategies for their degradation, in particular under anoxic (devoid of O₂) conditions. A wealth of overviews is available summarizing our current knowledge on pure cultures of anaerobic degradation specialists as well as the intriguing biochemistry they harbor (e.g., see references 4, 5, and 6). The significance of anaerobic degradation is evident from the fact that large parts of the biosphere are characterized by anoxic conditions. In accordance with this, a large variety of anaerobic bacteria exists, the energy yield of which is governed by their mode of energy generation viz. the redox potential of the electron acceptor used (7). Denitrification, i.e., the reduction of nitrate (NO₃⁻) to molecular nitrogen (N₂), provides an energy yield second only to oxygen respiration (8), is widespread among Bacteria (9), and contributes to the global nitrogen cycle (10).

The denitrifying betaproteobacterium “Aromatoleum aromaticum” EbN1 has emerged since its isolation (11) as a valuable model for investigating the anaerobic degradation of aromatic compounds, covering studies on specific reactions, pathway elucidation, catabolic network reconstruction, and adaptation to environmental changes on a systems biology level (12). On the basis of its complete genome (13), differential proteomics combined with targeted metabolite analysis allowed reconstructing a complex network for 22 aromatic growth substrates organized in a dozen peripheral anaerobic degradation pathways (14–19). The high substrate specificity of the pathway-specific subproteomes observed in the aforementioned studies pointed to a fine-tuned regulatory network in A. aromaticum EbN1, presupposing the capacity to discriminate between structurally similar compounds on the sensory level (12).

A. aromaticum EbN1 anaerobically degrades the growth substrates p-ethylphenol and p-hydroxyacetophenone in analogy to the route used for ethylbenzene. An initial O₂-independent hydroxylation to 1-(4-hydroxyphenyl)ethanol is followed by dehydrogenation to p-hydroxyacetophenone, which is then converted, supposedly via carboxylation, coenzyme A (CoA) activation, and thiolytic acetyl-CoA removal, to p-hydroxybenzoyl-CoA (16) (Fig. 1). All involved proteins are encoded in an operon-like catabolic gene cluster that, together with an associated efflux gene cluster, frames the gene for the σ⁵⁴-dependent sensor/regulator EtpR (formerly EbA324). As both operon-like gene clusters bear conserved σ⁵⁴-DNA binding motifs, it was proposed that EtpR coordinately regulates their transcription in response to p-ethylphenol and p-hydroxyacetophenone (16) (Fig. 1). An accordingly substrate-specific regulation was demonstrated on the basis of targeted transcript analysis as well as differential proteomic and enzymatic profiling (16, 20, 21). Furthermore, generation of an unmarked ΔetpR in-frame deletion mutation resulted in loss of anaerobic growth with p-ethylphenol and p-hydroxyacetophenone as well as respective transcript and protein formation, underpinning the essential role of EtpR (22).

FIG 1.

Scheme of the proposed transcriptional regulation of the anaerobic degradation of p-ethylphenol in the denitrifying bacterium Aromatoleum aromaticum EbN1. The highly specific regulation of the peripheral degradation route by the substrate p-ethylphenol and its conversion intermediate p-hydroxyacetophenone is proposed to be mediated by the predicted σ⁵⁴-dependent sensor/regulator EtpR. The two aromatic compounds are suggested to be recognized by the sensory domain of the EtpR protein, which thereafter activates the transcription of the genes for the catabolism as well as a presumptive associated efflux system. For practical reasons the sensitivity of the system was investigated with p-hydroxyacetophenone across a concentration range from 100 μM down to 0.1 nM by determining its depletion profiles (Fig. 2) and formation of involved transcripts (Fig. 3). Genes marked in red were selected for transcript profiling. Compound names: 1, p-ethylphenol; 2, 1-(4-hydroxyphenyl)ethanol; 3, p-hydroxyacetophenone; 4, presumptive p-hydroxybenzoylacetate; 5, p-hydroxybenzoyl-CoA; 6, benzoyl-CoA. Enzyme names: PchCF, predicted p-ethylphenol methylenehydroxylase; hped, 1-(4-hydroxyphenyl)ethanol dehydrogenase; AcsA, predicted acetyl-CoA synthetase; EbA332/5/6/7, presumptive solvent efflux system. UBS, putative upstream binding sequence of EtpR.

Transcriptional control of genes for the aerobic (with O₂) degradation of aromatic compounds has long been studied with organisms such as Pseudomonas spp., Alcaligenes spp., or Escherichia coli, unraveling a broad array of regulators (for overviews see references 23, 24, and 25). As typical for prokaryotes in general (26, 27), these transcriptional regulators are mostly of the one-component type. Major research focused on deciphering promoter interactions (28, 29), architecture of effector-specific transcriptional networks (30), and the structural basis of effector binding to the regulator (31–34). Mechano-transcriptional activators of the NtrC family bind upstream of the promoter, stimulating open complex formation of the σ⁵⁴-RNA-polymerase holoenzyme (35, 36). The best-studied NtrC family members responsive to aromatic compounds are the XylR (alkylbenzenes) and DmpR (phenol) proteins of Pseudomonas putida (37, 38). The binding affinities of these regulators are less well understood, as the latter have apparently resisted purification so far. Gene expression studies on the basis of chemostat cultivation revealed a responsiveness of the alphaproteobacterium Sphingomonas paucimobilis B90A (reclassified as S. indicum B90A^T [39]) to hexachlorocyclohexane down to 7 μM (40). A bioluminescent biosensor of Pseudomonas putida pPG7 featured a 50 nM detection limit for naphthalene from the gaseous phase (41).

The present study embarked on assessing the response threshold of A. aromaticum EbN1 cultures for p-hydroxyacetophenone by determining its depletion from nonadapted cells upon its addition at various concentrations over several orders of magnitude and by targeted transcript analyses of genes involved in its catabolism and putative associated efflux.

RESULTS

Rational of experimental design.

p-Hydroxyacetophenone was used as an effector to approximate, on the level of whole cells, the response threshold of the EtpR-based regulatory system of A. aromaticum EbN1 that substrate-specifically turns on the anaerobic degradation of p-ethylphenol. Selection of p-hydroxyacetophenone as an effector was for practical reasons, since this compound can be added to cultures in exact increments from aqueous stock solutions and dissolves well, while this is more challenging with the less water-soluble p-ethylphenol. Furthermore, both compounds initiate the expression of genes for the shared anaerobic degradation pathway with congruent specificity and scale (16). However, at present it cannot be excluded that a common conversion product serves as the true effector of EtpR rather than p-ethylphenol or p-hydroxyacetophenone.

The responsiveness of A. aromaticum EbN1 to p-hydroxyacetophenone was tested with a physiological approach using nonadapted, active cells. These were anaerobic cultures that had been adapted to limiting provision with benzoate (1 mM against a background of surplus 10 mM nitrate). The presumptive effector was added immediately upon depletion of benzoate (i.e., after 17.5 h of incubation) in a single pulse of a defined concentration (ranging from 500 μM down to 10 nM; for gene expression analysis, it ranged further down to 0.1 nM). Subsequent depletion of the effector was considered indicative of the regulatory system's responsiveness. For this purpose, a highly sensitive micro-high-performance liquid chromatography (microHPLC) method was established, providing a dynamic range for p-hydroxyacetophenone down to 5 nM (see Fig. S1 and S2 in the supplemental material).

The suitability of this experimental design was demonstrated with two types of experiments. (i) It was verified that the observed depletion profile of p-hydroxyacetophenone resulted from the de novo expression of the involved catabolic genes. For this purpose, transcription-inhibiting rifampin was added 30 min prior to a single pulse of p-hydroxyacetophenone, leading to the complete inhibition of effector depletion (Fig. S3). (ii) It was shown that the cells, upon benzoate depletion, were not energy deprived, affecting gene expression on the p-hydroxyacetophenone pulse. To this end, an additional 100 μM benzoate was provided together with the effector. While this did not affect the depletion of p-hydroxyacetophenone, it apparently had a somewhat impeding effect on the expression of target genes (e.g., ebA335) (Fig. S4). Thus, the experimental setup afforded cells with adequately energy supplies while at the same time avoided potential transcription-impeding conditions.

Depletion profiles of p-hydroxyacetophenone.

Addition of p-hydroxyacetophenone (i.e., 500 μM down to 10 nM) upon consumption of the primary substrate benzoate to cultures of A. aromaticum EbN1 resulted in its measurable depletion already after 5 min (Fig. 2A and B and Fig. S3B and S5). The maximal rates of p-hydroxyacetophenone depletion positively correlated (R² = 0.97) with the added effector concentration (Fig. 2C), e.g., 20.1 μmol/h versus 6.7 nmol/h for 100 μM versus 10 nM, indicative of a diffusion-driven effector uptake. These in vivo experiments suggested a response threshold for p-hydroxyacetophenone of below 10 nM.

FIG 2.

Growth experiments testing the responsiveness of nonadapted cells of A. aromaticum EbN1 to p-hydroxyacetophenone. Growth was supported by the primary substrate benzoate (added at limiting concentration of 1 mM), which was reproducibly depleted after 17.5 h of incubation. Immediately thereafter, p-hydroxyacetophenone was added in a single pulse at various concentrations, the responsiveness to which was assessed by determining its depletion via microHPLC. (A and B) Experiments with provision of 100 μM and 10 nM p-hydroxyacetophenone; all experiments were conducted with three biological replicates. The enlarged portions show at higher resolution the time point of addition of p-hydroxyacetophenone as well as those for sampling for subsequent targeted transcript profiling. (C) Correlation between the nine tested p-hydroxyacetophenone concentrations and the determined depletion rates; growth curves and compound depletion profiles for the seven p-hydroxyacetophenone concentrations not shown in panels A and B are provided in Fig. S5.

Targeted transcript analysis.

Complementing the aforementioned in vivo analyses, transcription profiles of genes involved in p-hydroxyacetophenone catabolism and efflux were investigated, covering the same range of effector concentrations and extending the lower limit down to 0.1 nM. To consider the operon-like structure of both gene clusters (Fig. 1), expression of genes at their beginning (acsA and ebA335) and end (pchF and ebA326) were studied. In addition, expression of hped [encoding 1-(4-hydroxyphenyl)ethanol dehydrogenase], located in the center of the larger catabolic gene cluster (16.4 kbp), was analyzed. Changes in the expression level of the respective genes were determined relative to their expression level directly prior to addition of the effector pulse. For each target gene, the transcript level at this reference time point was constant across all tested effector concentrations, affording reliable comparison between all experimental conditions (coefficient of variation of threshold cycle [C_T] values, <0.035).

The determined expression profiles (Fig. 3, Table S1) of the targeted genes mirrored the discretely pulsed p-hydroxyacetophenone (effector) concentrations and their aforementioned operon position. Accordingly, acsA and ebA335, encoded at the beginning of the respective gene clusters, were expressed most rapidly, with ∼40- and ∼11-fold increased transcript abundances already 5 min after a pulse of 100 μM effector, and also reached the highest values (∼250- and ∼46-fold) under this effector condition after 120 min. In contrast, transcripts of terminally located pchF and ebA326 showed significant fold change increases only 30 min (∼4-fold) and 15 min (∼6-fold) after the 100 μM effector pulse. In both cases maximal values were recorded after 60 min, with 14-fold and ∼26-fold, respectively. Significant increase in expression of hped was observed after 30 min and reached its highest fold change (∼46-fold) 120 min after the 100 μM effector pulse. Taken together, expression started earlier and reached a higher level the closer a targeted gene was located to the presumptive transcriptional start of its appendant operon and the higher the concentration of the pulsed effector.

FIG 3.

Time-resolved, quantitative transcript profiles of A. aromaticum EbN1 in response to different extracellular effector (p-hydroxyacetophenone) concentrations. The selected transcripts represent genes (Fig. 1) involved in the catabolism of p-hydroxyacetophenone (pchF, hped, and acsA) and its presumptive efflux (ebA335 and ebA326). Transcript abundance was determined by qRT-PCR, with the time point of 5 min prior to p-hydroxyacetophenone addition serving as a reference. Each data point is based on 3 biological replicates; 3 technical replicates were analyzed for each. Growth cultures providing samples for the nine studied p-hydroxyacetophenone concentrations as well as for the control (no addition of effector) are shown in detail in Fig. S6, and fold changes of transcript abundance are shown in Table S1.

Irrespective of gene position and effector concentration, however, transcript formation could only be observed down to 10 nM p-hydroxyacetophenone. The absence of expression at lower tested concentrations of p-hydroxyacetophenone (1 nM and 0.1 nM) complemented the microHPLC-based limit of detection for p-hydroxyacetophenone and narrowed the response threshold down to between 10 nM and 1 nM effector.

DISCUSSION

Data on naturally occurring amounts of monocyclic aromatic compounds are scarcer than one might expect. Nonetheless, several studies show that monocyclic aromatic compounds utilized by A. aromaticum EbN1 occur on a global scale at highly various concentrations in diverse environments. In surface sediments from the highly contaminated Randle Reef, Lake Ontario (Canada), concentrations of mixed m- and p-cresols ranged from 81.4 to 147.9 μmol/kg of dry weight (42). In freshwater sediments of the Potomac River (USA), phenol and p-cresol were detected at concentrations of 0.98 nM and 92.9 nM, respectively (43). Freshwater samples from the Xi River in China showed profound, season-dependent concentration variations of phenol (19.3 nM to 48.2 μM) and p-cresol (3.1 nM to 9.6 μM) (44). In a U.S.-wide census of water resources, samples were collected from 139 streams across 30 states, among which acetophenone, phenol, and p-cresol were detected at mean concentrations of 1.25, 7.44, and 0.46 nM, respectively (45). For seven boreal lake sediments in Sweden it was shown that p-hydroxyacetophenone makes up 22 to 32% of all hydroxyl phenols (46). In marine sediments, p-hydroxyacetophenone occurs in depths from 30 m to 90 m at concentrations of around 1.7 mmol/kg organic carbon (47).

Chemoreception allows bacteria to constantly monitor the chemical composition of their proximate environment and to adapt their nutritional and behavioral strategy accordingly. Proteins involved in chemoreception have been studied intensively with respect to domain architecture, specificity, and affinity (dissociation constant, K_d) of ligand-sensor interaction, as well as signal transduction (25), while comparatively little is known about the sensors' threshold of responsiveness viz. limit of detection. A well-known field of chemoreception is bacterial quorum sensing (QS) that monitors and responds to the accumulation of extracellular signals reflecting cell density and/or community composition (48, 49). The phototrophic purple nonsulfur bacterium Rhodopseudomonas palustris produced the QS signal p-coumaroyl-homoserine lactone (pC-HSL), employing the synthase RpaI. Transcription of the rpaI gene is positively controlled by the regulator RpaR in the presence of 250 nM pC-HSL (50). A whole-cell sensing system for autoinducer-2 (AI-2) based on Vibrio harveyi strain BB170 showed a limit of detection of 25 nM AI-2 (51). Binding affinities (K_d values) of chemoreceptors for organic substrates, as mostly determined by isothermal titration calorimetry, apparently reside in the lower micromolar range: 0.46 μM phenol and 4.12 μM catechol for the transcriptional regulator MopR (NtrC family) from Acinetobacter calcoaceticus (34), 39 μM citrate for McpQ from Pseudomonas putida KT2440 (52), 35.6 μM serine and 99.4 μM aspartate for Tsr and Tar, respectively, of Escherichia coli (53), and 58.6 μM pyruvate for the sensor histidine kinase BtsS of E. coli (54).

Against the background of the above-described knowledge on in situ concentrations of aromatic compounds and the binding affinities of chemoreceptors, the response threshold (1 to 10 nM) elucidated here for p-hydroxyacetophenone in A. aromaticum EbN1 sheds new light on the lower limit of responsiveness toward aromatic growth substrates. From an environmental point of view, it is interesting to approximate which in situ concentration of a given substrate suffices to transcriptionally turn on its degradation pathway. Such a threshold of responsiveness has implications for the type of habitat and/or environmental condition that provide(s) a given bacterium with nutritional opportunities for survival and eventually for proliferation. Conversely, it may approximate a threshold concentration below which a compound, which is utilizable in principle, may escape biodegradation and instead become an enduring constituent of dissolved organic matter and ultimately get preserved in the geosphere.

MATERIALS AND METHODS

Bacterial strain and cultivation conditions.

Aromatoleum aromaticum EbN1 has been subcultured and stored in our laboratory since its isolation (11). Anaerobic cultivation of A. aromaticum EbN1 was performed at 28°C in defined, bicarbonate-buffered, and ascorbate-reduced mineral medium with the electron acceptor nitrate, as previously described (11). Organic substrates (benzoate and p-hydroxyacetophenone) were provided from sterile aqueous stock solutions. For the purpose of best possible reproducibility, each growth experiment was started from the same batch of glycerol stocks of A. aromaticum EbN1 grown with benzoate (4 mM), complying with the following sequence of cultivation steps. (i) A dilution series (10⁻¹ to 10⁻⁶), likewise with benzoate (4 mM) as the sole source of carbon and energy, was inoculated from a glycerol stock and incubated for 4 days. (ii) The first preculture supplied with 2 mM benzoate (80-ml culture volume in 100-ml flat-bottomed glass bottles sealed with butyl rubber stoppers) was inoculated with 5% (vol/vol) of the 10⁻⁶ dilution and incubated for 3 days. (iii) The second preculture was carried out under the same conditions, inoculated with 5% (vol/vol) of the first preculture, and incubated for 17 h. (iv) The triplicate main cultures (see “Growth experiments,” below) with 1 mM benzoate (400-ml culture volume in 500-ml flat-bottomed glass bottles sealed with butyl rubber stoppers) were inoculated with 2% (vol/vol) of the second preculture. All chemicals were of analytical grade.

Growth experiments.

The responsiveness of A. aromaticum EbN1 to various concentrations of p-hydroxyacetophenone was studied with nonadapted cells. In each experiment, benzoate (1 mM) was initially provided as the growth-limiting sole source of organic carbon and energy. Upon its highly reproducible complete depletion after 17.5 h of incubation, a distinct pulse of p-hydroxyacetophenone was given at one of the seven tested concentrations (500 μM, 100 μM, 1 μM, 100 nM, 50 nM, 30 nM, and 10 nM). Throughout the incubation time (approximately 24 h), 3-ml samples of the culture broth were retrieved by means of sterile, N₂-flushed syringes. An aliquot of 1 ml was used for monitoring growth by measuring the optical density at 660 nm (OD₆₆₀). The remaining 2 ml was immediately centrifuged (20,000 × g, 10 min, 4°C), and the supernatant was stored at −20°C for subsequent determination of substrate depletion by micro-high-performance liquid chromatography (microHPLC). Three replicate cultures were performed per test condition.

Cultivation and cell harvesting for transcript profiling.

Cultivation was performed as described above (see “Growth experiments”) with the additional concentrations of 1 nM and 0.1 nM p-hydroxyacetophenone. At each sampling point, 5 ml culture broth was withdrawn from each of the 3 replicate cultures per tested concentration of p-hydroxyacetophenone. Samples were retrieved with sterile, N₂-flushed syringes and immediately added to 10 ml of RNAprotect bacterial reagent (Qiagen, Hilden, Germany), mixed rigorously, incubated for 5 min at room temperature, and centrifuged (4,000 × g, 10 min, 4°C). Pellets were resuspended in 0.5 ml RNAprotect bacterial reagent, transferred into 2-ml microcentrifuge tubes, and centrifuged (20,000 × g, 10 min, room temperature). Supernatants were discarded and pellets were shock frozen in liquid N₂ and stored at −80°C until further analyses. Sampling time points were 5 min prior to addition of p-hydroxyacetophenone (control), followed by 5, 15, 20, 60, and 120 min after addition, as well as after 240 min (100 μM, 10 μM, and no p-hydroxyacetophenone) and 480 min (100 μM and no p-hydroxyacetophenone) in select cases.

Quantitation of aromatic compounds by microHPLC.

Quantitative determination of the depletion profiles of benzoate and p-hydroxyacetophenone was achieved with a newly developed method employing a microHPLC (UltiMate 3000; ThermoFischer, Germering, Germany). The system was equipped with a Thermo Accucore column (C₁₈, 150 by 1 mm, 2.6-μm bead size; ThermoFisher) and an RS diode array detector (ThermoFisher); it was operated at 40°C with a flow rate of 100 μl/min. The 20-min gradient, composed of the eluent A (5% [vol/vol] acetonitrile in H₂O with 0.01% [vol/vol] H₃PO₄ [85%]) and eluent B (90% [vol/vol] acetonitrile in H₂O with 0.01% [vol/vol] H₃PO₄ [85%]) was 2.5-min constant at 3% B, 4-min linear ramping to 65% B, 1-min linear ramping to 99% B, 1.5-min constant at 99% B, 2-min linear ramping to 3% B, and finally 9-min constant at 3% B. Benzoate was detected at 229 nm with a retention time of 9.32 min and a dynamic range from 50 nM to 50 μM. p-Hydroxyacetophenone was detected at 275 nm with a retention time of 6.99 min and a dynamic range from 5 nM to 50 μM. A representative chromatogram and the respective calibration curves are provided in Fig. S1 and S2 in the supplemental material.

Preparation of total RNA.

Total RNA was prepared from all three biological replicates per sampling time point for every experiment (153 preparations in total), using saturated acidic phenol (60°C) essentially as previously described (55, 56). In brief, each cell pellet was treated twice with hot acidic phenol, and after centrifugation the aqueous phase was transferred into a 2-ml 5PRIME phase lock gel tube (Quantabio, Beverly, MA, USA). One volume of phenol-chloroform-isoamylalcohol (25:24:1) was added, and the tube was gently inverted for 5 min. After centrifugation (20,000 × g, 5 min, room temperature), nucleic acids were precipitated using ice-cold ethanol (96%) during incubation for 30 min at −80°C. After centrifugation (20,000 × g, 30 min, 4°C), the pellet was washed with ice-cold ethanol (75%) and centrifuged again (20,000 × g, 15 min, 4°C). The pellet was dried and resuspended in RNase-free water. Subsequently, every sample was subjected to DNase I (RNase-free; Qiagen) digestion. Complete removal of DNA was confirmed by PCR using genomic DNA of A. aromaticum EbN1 as a positive control. RNA quality was controlled by the Experion StdSens RNA chip (Bio-Rad, Hercules, CA, USA) operated in an Experion automated electrophoresis station (Bio-Rad). RNA concentration was determined using a TrayCell (Hellma Analytics, Müllheim, Germany) operated in a spectrophotometer (UV-1800; Shimadzu, Duisburg, Germany). Total RNA was stored in aliquots at −80°C. All chemicals used for preparation of total RNA were of molecular biology grade.

Transcript profiling by qRT-PCR.

Specific primers (Table 1) for the five target genes were designed using the Primer3 software package (version 0.4.0; www.primer3.org). cDNA generation and real-time PCR was performed with three technical replicates per RNA preparation using 50 ng of total RNA, the Brilliant III ultra-fast SYBR green quantitative reverse transcription-PCR (qRT-PCR) master mix (Agilent, Santa Clara, CA, USA), and the CFX96 real-time system (Bio-Rad). In total, 9 measurements were conducted per analyzed time point. The one-tube RT real-time PCR was carried out with one cycle of reverse transcription for 10 min at 50°C and one cycle of PCR initiation for 3 min at 95°C, followed by 40 cycles of 10 s of denaturation at 95°C, 30 s of annealing (primer specific), and 30 s of extension at 60°C, succeeded by real-time detection for 5 s. The gene-specific annealing temperatures were the following: acsA, 60°C; hped, 60°C; pchF, 54.5°C; ebA335, 60°C; and ebA326, 57.5°C. The specificity of accumulated products was verified by melting curve analysis, ranging from 60°C to 90°C in steps of 0.5°C. The RNA preparation from the samples retrieved 5 min prior to addition of p-hydroxyacetophenone was used as a reference, while all samples retrieved at a later time point during incubation represented the test states. The differences in transcript abundance were calculated according to the following equation (57): ratios = E^{ΔCT(reference − test)}. Primer-specific efficiencies (E) of the PCR for each primer pair were determined as previously reported (58).

TABLE 1.

Gene-specific primer pairs used for expression analysis of target genes by means of qRT-PCR

Primera	Target gene	Nucleotide sequence (5′ to 3′)	Product length (bp)	PCR efficiencyb
acsA_359_F	acsA	GCCAGTGCCCGGTAGATC	275	1.955
acsA_633_R		GCGGCATTCAACGAGCAG
hped_399_F	hped	CCGACAGGTTGATGCCGA	222	2.024
hped_620_R		GGGAAACACTCGCCCTGA
pchF_1336_F	pchF	GGCCGGCAACGTCATCATC	273	1.813
pchF_1099_R		CCATCCGGGAGCACCACT
ebA335_1092_F	ebA335	GCTGGGGGAGACGAA	253	1.916
ebA335_1344_R		CGCCGCCTTGTTGT
ebA326_41_F	ebA326	TGGCTGGATCTCTGCTC	275	2.162
ebA326_315_R		TTCCCGTGCGACCTG

^aF, forward primer; R, reverse primer.

^bMean value of all performed qRT-PCR experiments.

Dedicated to Fritz Widdel on the occasion of his retirement.