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

Jannes Vagts,a Sabine Scheve,a Mirjam Kant,a Lars Wöhlbrand,a Ralf Rabusa

aGeneral and Molecular Microbiology, Institute for Chemistry and Biology of the Marine Environment (ICBM),Carl von Ossietzky University of Oldenburg, Oldenburg, Germany

ABSTRACT The denitrifying betaproteobacterium “Aromatoleum aromaticum” EbN1regulates the capacity to anaerobically degrade p-ethylphenol (via p-hydroxyacetophenone)with high substrate specificity. This process is mediated by the �54-dependent tran-scriptional regulator EtpR, which apparently recognizes both aromatic compounds,yielding congruent expression profiles. The responsiveness of this regulatory systemwas studied with p-hydroxyacetophenone, which is more easily administered to cul-tures and traced analytically. Cultures of A. aromaticum EbN1 were initially cultivatedunder nitrate-reducing conditions with a growth-limiting supply of benzoate, uponthe complete depletion of which p-hydroxyacetophenone was added at various con-centrations (from 500 �M down to 0.1 nM). Depletion profiles of this aromatic sub-strate and presumptive effector were determined by highly sensitive micro-high-performance liquid chromatography (microHPLC). Irrespective of the added concentration ofp-hydroxyacetophenone, depletion commenced after less than 5 min and suggesteda response threshold of below 10 nM. This approximation was corroborated by time-resolved transcript profiles (quantitative reverse transcription-PCR) of selected degra-dation and efflux relevant genes (e.g., pchF, encoding a subunit of predictedp-ethylphenol methylenehydroxylase) and narrowed down to a range of 10 to 1 nM.The most pronounced transcriptional response was observed, as expected, for geneslocated 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 substrateswith natural as well as anthropogenic sources, albeit with their in situ concentrationsand their bioavailabilities varying over several orders of magnitude. Even thoughdegradation pathways and underlying regulatory systems have long been studiedwith aerobic and, to a lesser extent, with anaerobic bacteria, comparatively little isknown about the effector concentration-dependent responsiveness. A. aromaticumEbN1 is a model organism for the anaerobic degradation of aromatic compoundswith the architecture of the catabolic network and its substrate-specific regulationhaving been intensively studied by means of differential proteogenomics. The pres-ent study aims at unraveling the minimal concentration of an aromatic growth sub-strate (p-hydroxyacetophenone here) required to initiate gene expression for its deg-radation pathway and to learn in principle about the lower limit of catabolicresponsiveness of an anaerobic degradation specialist.

KEYWORDS anaerobic degradation, aromatic compounds, regulation,responsiveness, sensory system

Aromatic compounds belong to the most prominent components of recent andfossil organic matter in the biosphere and geosphere (e.g., see references 1 and

2) and encompass industrially relevant chemicals that are also of environmental

Received 28 April 2018 Accepted 26 June2018

Accepted manuscript posted online 29June 2018

Citation Vagts J, Scheve S, Kant M, WöhlbrandL, Rabus R. 2018. Towards the responsethreshold for p-hydroxyacetophenone in thedenitrifying bacterium “Aromatoleumaromaticum” EbN1. Appl Environ Microbiol84:e01018-18. https://doi.org/10.1128/AEM.01018-18.

Editor Rebecca E. Parales, University ofCalifornia, Davis

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Ralf Rabus,rabus@icbm.de.

Dedicated to Fritz Widdel on the occasion ofhis retirement.

ENVIRONMENTAL MICROBIOLOGY

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concern (e.g., see reference 3). Accordingly, the biodegradation of these com-pounds is relevant for diverse areas ranging from global carbon cycling to biore-mediation efforts. Research has put the most emphasis on elucidating singlereactions and pathways, as aromatic compounds possess only very low chemicalreactivity and require special biochemical strategies for their degradation, in par-ticular under anoxic (devoid of O2) conditions. A wealth of overviews is availablesummarizing our current knowledge on pure cultures of anaerobic degradationspecialists 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 thatlarge parts of the biosphere are characterized by anoxic conditions. In accordancewith this, a large variety of anaerobic bacteria exists, the energy yield of which isgoverned by their mode of energy generation viz. the redox potential of theelectron acceptor used (7). Denitrification, i.e., the reduction of nitrate (NO3

�) tomolecular 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 hasemerged since its isolation (11) as a valuable model for investigating the anaerobicdegradation of aromatic compounds, covering studies on specific reactions, path-way elucidation, catabolic network reconstruction, and adaptation to environmen-tal changes on a systems biology level (12). On the basis of its complete genome(13), differential proteomics combined with targeted metabolite analysis allowedreconstructing a complex network for 22 aromatic growth substrates organized ina dozen peripheral anaerobic degradation pathways (14–19). The high substratespecificity of the pathway-specific subproteomes observed in the aforementionedstudies pointed to a fine-tuned regulatory network in A. aromaticum EbN1, presup-posing the capacity to discriminate between structurally similar compounds on thesensory level (12).

A. aromaticum EbN1 anaerobically degrades the growth substrates p-ethylphenol andp-hydroxyacetophenone in analogy to the route used for ethylbenzene. An initial O2-independent hydroxylation to 1-(4-hydroxyphenyl)ethanol is followed by dehydrogenationto p-hydroxyacetophenone, which is then converted, supposedly via carboxylation, coen-zyme 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 �54-dependentsensor/regulator EtpR (formerly EbA324). As both operon-like gene clusters bear conserved�54-DNA binding motifs, it was proposed that EtpR coordinately regulates their transcrip-tion in response to p-ethylphenol and p-hydroxyacetophenone (16) (Fig. 1). An accordinglysubstrate-specific regulation was demonstrated on the basis of targeted transcript analysisas well as differential proteomic and enzymatic profiling (16, 20, 21). Furthermore, gener-ation of an unmarked �etpR in-frame deletion mutation resulted in loss of anaerobicgrowth with p-ethylphenol and p-hydroxyacetophenone as well as respective transcriptand protein formation, underpinning the essential role of EtpR (22).

Transcriptional control of genes for the aerobic (with O2) degradation of aro-matic compounds has long been studied with organisms such as Pseudomonas spp.,Alcaligenes spp., or Escherichia coli, unraveling a broad array of regulators (foroverviews see references 23, 24, and 25). As typical for prokaryotes in general (26,27), these transcriptional regulators are mostly of the one-component type. Majorresearch focused on deciphering promoter interactions (28, 29), architecture ofeffector-specific transcriptional networks (30), and the structural basis of effectorbinding to the regulator (31–34). Mechano-transcriptional activators of the NtrCfamily bind upstream of the promoter, stimulating open complex formation of the�54-RNA-polymerase holoenzyme (35, 36). The best-studied NtrC family membersresponsive to aromatic compounds are the XylR (alkylbenzenes) and DmpR (phenol)proteins of Pseudomonas putida (37, 38). The binding affinities of these regulatorsare less well understood, as the latter have apparently resisted purification so far.

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Gene expression studies on the basis of chemostat cultivation revealed a respon-siveness of the alphaproteobacterium Sphingomonas paucimobilis B90A (reclassifiedas S. indicum B90AT [39]) to hexachlorocyclohexane down to 7 �M (40). A biolu-minescent biosensor of Pseudomonas putida pPG7 featured a 50 nM detection limitfor naphthalene from the gaseous phase (41).

The present study embarked on assessing the response threshold of A. aromaticumEbN1 cultures for p-hydroxyacetophenone by determining its depletion from non-adapted cells upon its addition at various concentrations over several orders ofmagnitude and by targeted transcript analyses of genes involved in its catabolism andputative associated efflux.

RESULTSRational of experimental design. p-Hydroxyacetophenone was used as an effector

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

FIG 1 Scheme of the proposed transcriptional regulation of the anaerobic degradation of p-ethylphenol in the denitrifying bacterium Aromatoleumaromaticum EbN1. The highly specific regulation of the peripheral degradation route by the substrate p-ethylphenol and its conversion intermediatep-hydroxyacetophenone is proposed to be mediated by the predicted �54-dependent sensor/regulator EtpR. The two aromatic compounds are suggestedto be recognized by the sensory domain of the EtpR protein, which thereafter activates the transcription of the genes for the catabolism as well as apresumptive associated efflux system. For practical reasons the sensitivity of the system was investigated with p-hydroxyacetophenone across aconcentration range from 100 �M down to 0.1 nM by determining its depletion profiles (Fig. 2) and formation of involved transcripts (Fig. 3). Genesmarked 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 methylenehydroxy-lase; 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.

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The responsiveness of A. aromaticum EbN1 to p-hydroxyacetophenone was testedwith a physiological approach using nonadapted, active cells. These were anaerobiccultures that had been adapted to limiting provision with benzoate (1 mM against abackground of surplus 10 mM nitrate). The presumptive effector was added immedi-ately upon depletion of benzoate (i.e., after 17.5 h of incubation) in a single pulse of adefined concentration (ranging from 500 �M down to 10 nM; for gene expressionanalysis, it ranged further down to 0.1 nM). Subsequent depletion of the effector wasconsidered indicative of the regulatory system’s responsiveness. For this purpose, ahighly sensitive micro-high-performance liquid chromatography (microHPLC) methodwas 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 exper-iments. (i) It was verified that the observed depletion profile of p-hydroxyacetophenoneresulted 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-hydroxy-acetophenone, leading to the complete inhibition of effector depletion (Fig. S3). (ii) It wasshown that the cells, upon benzoate depletion, were not energy deprived, affecting geneexpression on the p-hydroxyacetophenone pulse. To this end, an additional 100 �Mbenzoate was provided together with the effector. While this did not affect the depletionof p-hydroxyacetophenone, it apparently had a somewhat impeding effect on the expres-sion of target genes (e.g., ebA335) (Fig. S4). Thus, the experimental setup afforded cells withadequately energy supplies while at the same time avoided potential transcription-impeding conditions.

Depletion profiles of p-hydroxyacetophenone. Addition of p-hydroxyacetophenone(i.e., 500 �M down to 10 nM) upon consumption of the primary substrate benzoate tocultures 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-hydroxyacetophenonedepletion positively correlated (R2 � 0.97) with the added effector concentration (Fig.2C), e.g., 20.1 �mol/h versus 6.7 nmol/h for 100 �M versus 10 nM, indicative of adiffusion-driven effector uptake. These in vivo experiments suggested a responsethreshold for p-hydroxyacetophenone of below 10 nM.

Targeted transcript analysis. Complementing the aforementioned in vivo anal-yses, transcription profiles of genes involved in p-hydroxyacetophenone catabolismand efflux were investigated, covering the same range of effector concentrationsand extending the lower limit down to 0.1 nM. To consider the operon-like structureof both gene clusters (Fig. 1), expression of genes at their beginning (acsA andebA335) and end (pchF and ebA326) were studied. In addition, expression of hped[encoding 1-(4-hydroxyphenyl)ethanol dehydrogenase], located in the center of thelarger catabolic gene cluster (16.4 kbp), was analyzed. Changes in the expressionlevel of the respective genes were determined relative to their expression leveldirectly prior to addition of the effector pulse. For each target gene, the transcriptlevel at this reference time point was constant across all tested effector concen-trations, affording reliable comparison between all experimental conditions (coef-ficient of variation of threshold cycle [CT] values, �0.035).

The determined expression profiles (Fig. 3, Table S1) of the targeted genesmirrored the discretely pulsed p-hydroxyacetophenone (effector) concentrationsand their aforementioned operon position. Accordingly, acsA and ebA335, encodedat 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 of100 �M effector, and also reached the highest values (�250- and �46-fold) underthis effector condition after 120 min. In contrast, transcripts of terminally locatedpchF and ebA326 showed significant fold change increases only 30 min (�4-fold)and 15 min (�6-fold) after the 100 �M effector pulse. In both cases maximal valueswere recorded after 60 min, with 14-fold and �26-fold, respectively. Significantincrease in expression of hped was observed after 30 min and reached its highest

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fold change (�46-fold) 120 min after the 100 �M effector pulse. Taken together,expression started earlier and reached a higher level the closer a targeted gene waslocated to the presumptive transcriptional start of its appendant operon and thehigher the concentration of the pulsed effector.

Irrespective of gene position and effector concentration, however, transcript formationcould only be observed down to 10 nM p-hydroxyacetophenone. The absence of expres-sion at lower tested concentrations of p-hydroxyacetophenone (1 nM and 0.1 nM) com-plemented the microHPLC-based limit of detection for p-hydroxyacetophenone and nar-rowed the response threshold down to between 10 nM and 1 nM effector.

FIG 2 Growth experiments testing the responsiveness of nonadapted cells of A. aromaticum EbN1 top-hydroxyacetophenone. Growth was supported by the primary substrate benzoate (added at limitingconcentration of 1 mM), which was reproducibly depleted after 17.5 h of incubation. Immediatelythereafter, p-hydroxyacetophenone was added in a single pulse at various concentrations, the respon-siveness to which was assessed by determining its depletion via microHPLC. (A and B) Experiments withprovision of 100 �M and 10 nM p-hydroxyacetophenone; all experiments were conducted with threebiological replicates. The enlarged portions show at higher resolution the time point of addition ofp-hydroxyacetophenone as well as those for sampling for subsequent targeted transcript profiling. (C)Correlation between the nine tested p-hydroxyacetophenone concentrations and the determined de-pletion rates; growth curves and compound depletion profiles for the seven p-hydroxyacetophenoneconcentrations not shown in panels A and B are provided in Fig. S5.

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FIG 3 Time-resolved, quantitative transcript profiles of A. aromaticum EbN1 in response to differentextracellular effector (p-hydroxyacetophenone) concentrations. The selected transcripts represent genes(Fig. 1) involved in the catabolism of p-hydroxyacetophenone (pchF, hped, and acsA) and its presumptiveefflux (ebA335 and ebA326). Transcript abundance was determined by qRT-PCR, with the time point of 5min prior to p-hydroxyacetophenone addition serving as a reference. Each data point is based on 3

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DISCUSSION

Data on naturally occurring amounts of monocyclic aromatic compounds are scarcerthan one might expect. Nonetheless, several studies show that monocyclic aromaticcompounds utilized by A. aromaticum EbN1 occur on a global scale at highly variousconcentrations in diverse environments. In surface sediments from the highly contam-inated Randle Reef, Lake Ontario (Canada), concentrations of mixed m- and p-cresolsranged from 81.4 to 147.9 �mol/kg of dry weight (42). In freshwater sediments of thePotomac River (USA), phenol and p-cresol were detected at concentrations of 0.98 nMand 92.9 nM, respectively (43). Freshwater samples from the Xi River in China showedprofound, season-dependent concentration variations of phenol (19.3 nM to 48.2 �M)and p-cresol (3.1 nM to 9.6 �M) (44). In a U.S.-wide census of water resources, sampleswere 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 thatp-hydroxyacetophenone makes up 22 to 32% of all hydroxyl phenols (46). In marinesediments, p-hydroxyacetophenone occurs in depths from 30 m to 90 m at concen-trations of around 1.7 mmol/kg organic carbon (47).

Chemoreception allows bacteria to constantly monitor the chemical composition oftheir proximate environment and to adapt their nutritional and behavioral strategyaccordingly. Proteins involved in chemoreception have been studied intensively withrespect to domain architecture, specificity, and affinity (dissociation constant, Kd) ofligand-sensor interaction, as well as signal transduction (25), while comparatively littleis known about the sensors’ threshold of responsiveness viz. limit of detection. Awell-known field of chemoreception is bacterial quorum sensing (QS) that monitors andresponds to the accumulation of extracellular signals reflecting cell density and/orcommunity composition (48, 49). The phototrophic purple nonsulfur bacterium Rho-dopseudomonas palustris produced the QS signal p-coumaroyl-homoserine lactone(pC-HSL), employing the synthase RpaI. Transcription of the rpaI gene is positivelycontrolled by the regulator RpaR in the presence of 250 nM pC-HSL (50). A whole-cellsensing system for autoinducer-2 (AI-2) based on Vibrio harveyi strain BB170 showed alimit of detection of 25 nM AI-2 (51). Binding affinities (Kd values) of chemoreceptors fororganic substrates, as mostly determined by isothermal titration calorimetry, apparentlyreside in the lower micromolar range: 0.46 �M phenol and 4.12 �M catechol for thetranscriptional regulator MopR (NtrC family) from Acinetobacter calcoaceticus (34), 39�M citrate for McpQ from Pseudomonas putida KT2440 (52), 35.6 �M serine and 99.4�M aspartate for Tsr and Tar, respectively, of Escherichia coli (53), and 58.6 �M pyruvatefor the sensor histidine kinase BtsS of E. coli (54).

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

FIG 3 Legend (Continued)biological replicates; 3 technical replicates were analyzed for each. Growth cultures providing samples forthe nine studied p-hydroxyacetophenone concentrations as well as for the control (no addition ofeffector) are shown in detail in Fig. S6, and fold changes of transcript abundance are shown in Table S1.

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MATERIALS AND METHODSBacterial strain and cultivation conditions. Aromatoleum aromaticum EbN1 has been subcul-

tured and stored in our laboratory since its isolation (11). Anaerobic cultivation of A. aromaticumEbN1 was performed at 28°C in defined, bicarbonate-buffered, and ascorbate-reduced mineralmedium with the electron acceptor nitrate, as previously described (11). Organic substrates (ben-zoate and p-hydroxyacetophenone) were provided from sterile aqueous stock solutions. For thepurpose of best possible reproducibility, each growth experiment was started from the same batchof glycerol stocks of A. aromaticum EbN1 grown with benzoate (4 mM), complying with the followingsequence of cultivation steps. (i) A dilution series (10�1 to 10�6), likewise with benzoate (4 mM) asthe 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-bottomedglass bottles sealed with butyl rubber stoppers) was inoculated with 5% (vol/vol) of the 10�6 dilutionand 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 maincultures (see “Growth experiments,” below) with 1 mM benzoate (400-ml culture volume in 500-mlflat-bottomed glass bottles sealed with butyl rubber stoppers) were inoculated with 2% (vol/vol) ofthe second preculture. All chemicals were of analytical grade.

Growth experiments. The responsiveness of A. aromaticum EbN1 to various concentrations ofp-hydroxyacetophenone was studied with nonadapted cells. In each experiment, benzoate (1 mM) wasinitially provided as the growth-limiting sole source of organic carbon and energy. Upon its highlyreproducible complete depletion after 17.5 h of incubation, a distinct pulse of p-hydroxyacetophenonewas given at one of the seven tested concentrations (500 �M, 100 �M, 1 �M, 100 nM, 50 nM, 30 nM, and10 nM). Throughout the incubation time (approximately 24 h), 3-ml samples of the culture broth wereretrieved by means of sterile, N2-flushed syringes. An aliquot of 1 ml was used for monitoring growth bymeasuring 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 ofsubstrate depletion by micro-high-performance liquid chromatography (microHPLC). Three replicatecultures were performed per test condition.

Cultivation and cell harvesting for transcript profiling. Cultivation was performed as describedabove (see “Growth experiments”) with the additional concentrations of 1 nM and 0.1 nM p-hydroxy-acetophenone. At each sampling point, 5 ml culture broth was withdrawn from each of the 3 replicatecultures 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, 10min, 4°C). Pellets were resuspended in 0.5 ml RNAprotect bacterial reagent, transferred into 2-mlmicrocentrifuge tubes, and centrifuged (20,000 � g, 10 min, room temperature). Supernatants werediscarded and pellets were shock frozen in liquid N2 and stored at �80°C until further analyses. Samplingtime 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 �M, 10 �M, and no p-hydroxyacetophenone)and 480 min (100 �M and no p-hydroxyacetophenone) in select cases.

Quantitation of aromatic compounds by microHPLC. Quantitative determination of the depletionprofiles of benzoate and p-hydroxyacetophenone was achieved with a newly developed methodemploying a microHPLC (UltiMate 3000; ThermoFischer, Germering, Germany). The system was equippedwith a Thermo Accucore column (C18, 150 by 1 mm, 2.6-�m bead size; ThermoFisher) and an RS diodearray detector (ThermoFisher); it was operated at 40°C with a flow rate of 100 �l/min. The 20-mingradient, 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 constantat 3% B, 4-min linear ramping to 65% B, 1-min linear ramping to 99% B, 1.5-min constant at 99% B, 2-minlinear ramping to 3% B, and finally 9-min constant at 3% B. Benzoate was detected at 229 nm with aretention time of 9.32 min and a dynamic range from 50 nM to 50 �M. p-Hydroxyacetophenone wasdetected at 275 nm with a retention time of 6.99 min and a dynamic range from 5 nM to 50 �M. Arepresentative chromatogram and the respective calibration curves are provided in Fig. S1 and S2 in thesupplemental material.

Preparation of total RNA. Total RNA was prepared from all three biological replicates per samplingtime 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 acidicphenol, and after centrifugation the aqueous phase was transferred into a 2-ml 5PRIME phase lock geltube (Quantabio, Beverly, MA, USA). One volume of phenol-chloroform-isoamylalcohol (25:24:1) wasadded, and the tube was gently inverted for 5 min. After centrifugation (20,000 � g, 5 min, roomtemperature), nucleic acids were precipitated using ice-cold ethanol (96%) during incubation for 30 minat �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 inRNase-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 apositive 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 wasdetermined 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 usedfor preparation of total RNA were of molecular biology grade.

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Transcript profiling by qRT-PCR. Specific primers (Table 1) for the five target genes were designedusing the Primer3 software package (version 0.4.0; www.primer3.org). cDNA generation and real-timePCR was performed with three technical replicates per RNA preparation using 50 ng of total RNA, theBrilliant 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 wereconducted per analyzed time point. The one-tube RT real-time PCR was carried out with one cycle ofreverse transcription for 10 min at 50°C and one cycle of PCR initiation for 3 min at 95°C, followed by 40cycles 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 accumulatedproducts was verified by melting curve analysis, ranging from 60°C to 90°C in steps of 0.5°C. The RNApreparation from the samples retrieved 5 min prior to addition of p-hydroxyacetophenone was used asa 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 � EΔCT(reference � test). Primer-specific efficiencies (E) of the PCR for each primer pair were determinedas previously reported (58).

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.01018-18.

SUPPLEMENTAL FILE 1, PDF file, 0.9 MB.

ACKNOWLEDGMENTSWe are grateful to Christina Hinrichs for technical assistance.J.V., L.W., and R.R. conceived the study; J.V., S.S., and M.K. performed the cultivation

experiments; S.S. conducted the microHPLC analyses; J.V. and S.S. did the RNA work;J.V., L.W., and R.R. wrote the manuscript.

This study was supported by the Deutsche Forschungsgemeinschaft (DFG)within the framework of the research training group Molecular Basis of SensoryBiology (GRK 1885).

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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 1.955acsA_633_R GCGGCATTCAACGAGCAGhped_399_F hped CCGACAGGTTGATGCCGA 222 2.024hped_620_R GGGAAACACTCGCCCTGApchF_1336_F pchF GGCCGGCAACGTCATCATC 273 1.813pchF_1099_R CCATCCGGGAGCACCACTebA335_1092_F ebA335 GCTGGGGGAGACGAA 253 1.916ebA335_1344_R CGCCGCCTTGTTGTebA326_41_F ebA326 TGGCTGGATCTCTGCTC 275 2.162ebA326_315_R TTCCCGTGCGACCTGaF, forward primer; R, reverse primer.bMean value of all performed qRT-PCR experiments.

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