1

Towards the Response Threshold for p-Hydroxyacetophenone 1

in the Denitrifying Bacterium "Aromatoleum aromaticum" EbN1 2

3

Jannes Vagts, Sabine Scheve, Mirjam Kant, Lars Wöhlbrand, Ralf Rabus 4

5

General and Molecular Microbiology, Institute for Chemistry and Biology of the Marine Environment 6

(ICBM), Carl von Ossietzky University of Oldenburg, Oldenburg, Germany 7

8

Address correspondence to Ralf Rabus, rabus@icbm.de 9

10

Running title: Responsiveness towards p-hydroxyacetophenone 11

12

ABSTRACT The denitrifying betaproteobacterium "Aromatoleum aromaticum" EbN1 13

regulates the capacity to anaerobically degrade p-ethylphenol (via p-hydroxyacetophenone) 14

with high substrate-specificity. This process is mediated by the 54-dependent transcriptional 15

regulator EtpR which apparently recognizes both aromatic compounds, yielding congruent 16

expression profiles. The responsiveness of this regulatory system was studied with p-17

hydroxyacetophenone, which is more easily administered to cultures and traced analytically. 18

Cultures of "A. aromaticum" EbN1 were initially cultivated under nitrate-reducing conditions 19

with a growth-limiting supply of benzoate, upon the complete depletion of which p-20

hydroxyacetophenone was added at varying concentrations (from 500 µM down to 0.1 nM). 21

Depletion profiles of this aromatic substrate and presumptive effector, respectively, were 22

determined by highly sensitive microHPLC. Irrespective of the added concentration of p-23

hydroxyacetophenone, depletion commenced after less than 5 min and suggested a 24

response threshold of below 10 nM. This approximation was corroborated by time-resolved 25

transcript profiles (qRT-PCR) of selected degradation and efflux relevant genes (e.g., pchF 26

encoding a subunit of predicted p-ethylphenol methylenehydroxylase) and narrowed down to 27

a range of 10 – 1 nM. Most pronounced transcriptional response was observed as expected 28

AEM Accepted Manuscript Posted Online 29 June 2018Appl. Environ. Microbiol. doi:10.1128/AEM.01018-18Copyright © 2018 American Society for Microbiology. All Rights Reserved.

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for genes located at the beginning of the two operon-like structures, related to catabolism 29

(i.e., acsA) and potential efflux (i.e., ebA335). 30

31

32

IMPORTANCE Aromatic compounds are widespread microbial growth substrates with 33

natural as well as anthropogenic sources, albeit with their in situ concentrations and thereby 34

bioavailabilities varying over several orders of magnitude. Even though degradation 35

pathways and underlying regulatory systems have long been studied with aerobic and to a 36

lower extent also with anaerobic bacteria, comparatively little is known about the effector 37

concentration-dependent responsiveness. "A. aromaticum" EbN1 is a model organism for the 38

anaerobic degradation of aromatic compounds with the architecture of the catabolic network 39

and its substrate-specific regulation intensively studied by means of differential 40

proteogenomics. The present study aims at unraveling the minimal concentration of an 41

aromatic growth substrate (here p-hydroxyacetophenone) required to initiate gene 42

expression for its degradation pathway and thereby at learning in principle about the lower 43

limit of catabolic responsiveness of an anaerobic degradation specialist. 44

45

KEYWORDS "Aromatoleum aromaticum" EbN1, anaerobic degradation, aromatic 46

compounds, regulation, sensory system, responsiveness 47

48

49

INTRODUCTION 50

Aromatic compounds belong to the most prominent components of recent and fossil organic 51

matter in the biosphere and geosphere (e.g., 1, 2), and encompass industrially relevant 52

chemicals that are also of environmental concern (e.g., 3). Accordingly, the biodegradation of 53

these compounds is relevant for diverse areas ranging from global carbon cycling to 54

bioremediation efforts. Research has put most emphasis on elucidating single reactions and 55

pathways, as aromatic compounds possess only very low chemical reactivity and require 56

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special biochemical strategies for their degradation, in particular under anoxic (devoid of O2) 57

conditions. A wealth of overviews is available summarizing our current knowledge on pure 58

cultures of anaerobic degradation specialists as well as the intriguing biochemistry they 59

harbor (e.g., 4, 5, 6). The significance of anaerobic degradation is evident from the fact that 60

large parts of the biosphere are characterized by anoxic conditions. In accord, a large variety 61

of anaerobic bacteria exists, the energy yield of which is governed by their mode of energy 62

generation viz. the redox potential of the electron acceptor used (7). Denitrification, i.e. the 63

reduction of nitrate (NO3) to molecular nitrogen (N2), provides an energy yield second only to 64

oxygen respiration (8), is widespread among Bacteria (9), and contributes to the global 65

nitrogen cycle (10). 66

The denitrifying betaproteobacterium "Aromatoleum aromaticum" EbN1 has emerged 67

since its isolation (11) as a valuable model for investigating the anaerobic degradation of 68

aromatic compounds, covering studies on specific reactions, pathway elucidation, catabolic 69

network reconstruction and adaptation to environmental changes on a systems biology level 70

(12). On the basis of its complete genome (13), differential proteomics combined with 71

targeted metabolite analysis allowed reconstructing a complex network for 22 aromatic 72

growth substrates organized in a dozen peripheral anaerobic degradation pathways (14-19). 73

The high substrate-specificity of the pathway-specific subproteomes observed in the 74

aforementioned studies pointed to a fine-tuned regulatory network in "A. aromaticum" EbN1 75

presupposing the capacity to discriminate between structurally similar compounds on the 76

sensory level (12). 77

"A. aromaticum" EbN1 anaerobically degrades the growth substrates p-ethylphenol and 78

p-hydroxyacetophenone in analogy to the route used for ethylbenzene. I.e., an initial O2-79

independent hydroxylation to 1-(4-hydroxyphenyl)ethanol is followed by dehydrogenation to 80

p-hydroxyacetophenone, which is then converted supposedly via carboxylation, CoA-81

activation and thiolytic acetyl-CoA removal to p-hydroxybenzoyl-CoA (16) (Fig. 1). All 82

involved proteins are encoded in an operon-like "catabolic" gene cluster that together with an 83

associated "efflux" gene cluster frames the gene for the 54-dependent sensor/regulator EtpR 84

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(formerly EbA324). As both operon-like gene clusters bear conserved 54-DNA binding 85

motifs, it was proposed that EtpR coordinately regulates their transcription in response to p-86

ethylphenol and p-hydroxyacetophenone (16) (Fig. 1). An accordingly substrate-specific 87

regulation was demonstrated on the basis of targeted transcript analysis as well as 88

differential proteomic and enzymatic profiling (16, 20, 21). Furthermore, generation of an 89

unmarked etpR in frame deletion mutation resulted in loss of anaerobic growth with p-90

ethylphenol and p-hydroxyacetophenone as well as respective transcript and protein 91

formation, underpinning the essential role of EtpR (22). 92

Transcriptional control of genes for the aerobic (with O2) degradation of aromatic 93

compounds has long been studied with organisms such as Pseudomonas spp., Alcaligenes 94

spp. or Escherichia coli, unraveling a broad array of regulators (for overview see e.g., 23, 24, 95

25). As typical for prokaryotes in general (26, 27) these transcriptional regulators are to the 96

most part of the one-component type. Major research foci were on deciphering promotor 97

interactions (e.g., 28, 29), architecture of effector-specific transcriptional networks (e.g., 30), 98

and the structural basis of effector-binding to the regulator (e.g., 31, 32-34). Mechano-99

transcriptional activators of the NtrC-family bind upstream of the promotor, stimulating open 100

complex formation of the 54-RNA-polymerase holoenzyme (35, 36). Best-studied NtrC-101

family members responsive to aromatic compounds are the XylR (alkylbenzenes) and DmpR 102

(phenol) proteins of Pseudomonas putida (37, 38). The binding affinities of these regulators 103

are less well understood, as the latter apparently resist purification so far. Gene expression 104

studies on the basis of chemostat cultivation revealed a responsiveness of the 105

alphaproteobacterium Sphingomonas paucimobilis B90A (reclassified as S. indicum B90AT; 106

(39)) to hexachlorocyclohexane down to 7 µM (40). A bioluminescent biosensor of 107

Pseudomonas putida pPG7 featured a 50 nM detection limit for naphthalene from the 108

gaseous phase (41). 109

The present study embarked on assessing the response threshold of "A. aromaticum" 110

EbN1 cultures for p-hydroxyacetophenone, by determining its depletion from non-adapted 111

cells upon its addition at varying concentrations over several orders of magnitude and by 112

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targeted transcript analyses of genes involved in its catabolism and putative "associated 113

efflux". 114

115

116

RESULTS 117

Rational of experimental design. p-Hydroxyacetophenone was used as effector to 118

approximate on the level of whole cells the response threshold of the EtpR-based regulatory 119

system of "A. aromaticum" EbN1 that substrate-specifically "turns on" the anaerobic 120

degradation of p-ethylphenol. Selection of p-hydroxyacetophenone as effector was for 121

practical reasons, since this compound can be added to cultures in exact increments from 122

aqueous stock solutions and dissolves well, while this is more challenging with the less 123

water-soluble p-ethylphenol. Furthermore, both compounds initiate the expression of genes 124

for the shared anaerobic degradation pathway with congruent specificity and scale (16). 125

However, at present it cannot be excluded that a common conversion product serves as the 126

true effector of EtpR, rather than p-ethylphenol or p-hydroxyacetophenone. 127

The responsiveness of "A. aromaticum" EbN1 to p-hydroxyacetophenone was tested 128

with a physiological approach using non-adapted, active cells. These were anaerobic 129

cultures that had been adapted to limiting provision with benzoate (1 mM against a 130

background of surplus 10 mM nitrate). The presumptive effector was added immediately 131

upon depletion of benzoate (i.e., after 17.5 h of incubation) in a single pulse of a defined 132

concentration (ranging from 500 µM down to 10 nM; for gene expression analysis further 133

down to 0.1 nM). Subsequent depletion of the effector was considered indicative of the 134

regulatory system's responsiveness. For this purpose a highly sensitive microHPLC method 135

was established providing a dynamic range for p-hydroxyacetophenone down to 5 nM (Figs. 136

S1 and S2). 137

The suitability of this experimental design was demonstrated with two types of 138

experiments. (i) It was verified that the observed depletion profile of p-hydroxyacetophenone 139

resulted from the de novo expression of the involved "catabolic" genes. For this purpose, 140

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transcription-inhibiting rifampicin was added 30 min prior to a single pulse of p-141

hydroxyacetophenone leading to the complete inhibition of effector depletion (Fig. S3). (ii) It 142

was shown that the cells upon benzoate depletion were not energy deprived affecting gene 143

expression upon the p-hydroxyacetophenone pulse. To this end, additional 100 µM benzoate 144

was provided together with the effector. While this did not affect the depletion of p-145

hydroxyacetophenone, it had apparently a somewhat impeding effect on the expression of 146

target genes (e.g., ebA335) (Fig. S4). Thus, the experimental set up afforded adequately 147

energy supplied cells while at the same time avoided potentially transcription impeding 148

conditions. 149

Depletion profiles of p-hydroxyacetophenone. Addition of p-hydroxyacetophenone 150

(i.e., 500 µM down to 10 nM) upon consumption of the primary substrate benzoate to 151

cultures of "A. aromaticum" EbN1 resulted in its measurable depletion already after 5 min 152

(Figs 2AB, S3B, S5). The maximal rates of p-hydroxyacetophenone depletion positively 153

correlated (R2 = 0.97) with the added effector concentration (Fig. 2C), e.g. 20.1 µmol/h vs. 154

6.7 nmol/h for 100 µM vs. 10 nM, indicative of a diffusion-driven effector uptake. These in 155

vivo experiments suggested a response threshold for p-hydroxyacetophenone of below 10 156

nM. 157

Targeted transcript analysis. Complementing the aforementioned in vivo analyses, 158

transcription profiles of genes involved in p-hydroxyacetophenone catabolism and efflux were 159

investigated, covering the same range of effector concentrations and extending the lower 160

limit down to 0.1 nM. To consider the operon-like structure of both gene clusters (Fig. 1), 161

expression of genes at their beginning (acsA and ebA335) and end (pchF and ebA326) were 162

studied. In addition, expression of hped, located in the center of the larger "catabolic" gene 163

cluster (16.4 kbp), was analyzed. Changes in the expression level of the respective genes 164

were determined relative to their expression level directly prior to addition of the effector 165

pulse. For each target gene, the transcript-level at this reference time point was constant 166

across all tested effector concentrations, affording reliable comparison between all 167

experimental conditions (cv of CT-values <0.035). 168

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The determined expression profiles (Fig. 3, Table S1) of the targeted genes mirrored 169

the discretely pulsed p-hydroxyacetophenone (effector) concentrations and their 170

aforementioned operon position. Accordingly, acsA and ebA335, encoded at the beginning of 171

the respective gene clusters, were expressed most rapidly with 40- and 11-fold increased 172

transcript abundances already 5 min after a pulse of 100 µM effector and also reached 173

highest values (250- and 46-fold) under this effector condition after 120 min. In contrast, 174

transcripts of terminally located pchF and ebA326 showed significant fold change increases 175

only 30 min (4-fold) and 15 min (6-fold) after the 100 µM effector pulse. In both cases 176

maximal values were recorded after 60 min with 14-fold and 26-fold, respectively. 177

Significant increase in expression of hped was observed after 30 min and reached its highest 178

fold change (46-fold) 120 min after the 100 µM effector pulse. Taken together, expression 179

started earliest and reached highest level, the closer a targeted gene was located to the 180

presumptive transcriptional start of its appendant operon and the higher the concentration of 181

the pulsed effector was. 182

Irrespective of gene position and effector concentration, however, transcript formation 183

could only be observed down to 10 nM p-hydroxyacetophenone. The absence of expression 184

at lower tested concentrations of p-hydroxyacetophenone (1 nM and 0.1 nM) complemented 185

the microHPLC-based limit of detection for p-hydroxyacetophenone and narrowed the 186

response threshold down to between 10 nM and 1 nM effector. 187

188

189

DISCUSSION 190

Data on naturally occurring amounts of monocyclic aromatic compounds are scarcer 191

than one might expect. Nonetheless, several studies show that monocyclic aromatic 192

compounds utilized by "A. aromaticum" EbN1 occur on a global scale at highly varying 193

concentrations in diverse environments. In surface sediments from the highly contaminated 194

Randle Reef, Lake Ontario (Canada), concentrations of mixed m- and p-cresols ranged from 195

81.4 to 147.9 µmol/kg dw (42). In freshwater sediments of the Potomac River (USA), phenol 196

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and p-cresol were detected at concentrations of 0.98 nM and 92.9 nM, respectively (43). 197

Freshwater samples from the Xi River in China showed profound, season-dependent 198

concentration variations of phenol (19.3 nM to 48.2 µM) and p-cresol (3.1 nM to 9.6 µM) (44). 199

In a US-wide census of water resources, samples were collected from 139 streams across 200

30 states, among which acetophenone, phenol and p-cresol were detected at mean 201

concentrations of 1.25, 7.44 and 0.46 nM, respectively (45). For seven boreal lake sediments 202

in Sweden it was shown that p-hydroxyacetophenone makes up 22–32% of all hydroxyl 203

phenols (46). In marine sediments, p-hydroxyacetophenone occurs in depths from 30 m to 204

90 m at concentrations of around 1.7 mmol/kg organic carbon (47). 205

Chemoreception allows bacteria to constantly monitor the chemical composition of 206

their proximate environment and to adapt their nutritional and behavioral strategy 207

accordingly. Proteins involved in chemoreception have been studied intensively with respect 208

to domain architecture, specificity and affinity (dissociation constant, Kd) of ligand-sensor 209

interaction, as well as signal transduction (e.g., 25), while comparatively little is known about 210

the sensors’ threshold of responsiveness viz. limit of detection. A well-known field of 211

chemoreception is bacterial quorum sensing (QS) that monitors and responds to the 212

accumulation of extracellular signals reflecting cell density and/or community composition 213

(48, 49). The phototrophic purple nonsulfur bacterium Rhodopseudomonas palustris 214

produced the QS signal p-coumaroyl-homoserine lactone (pC-HSL) employing the synthase 215

RpaI. Transcription of the rpaI gene is positively controlled by the regulator RpaR in the 216

presence of 250 nM pC-HSL (50). A whole-cell sensing system for autoinducer-2 (AI-2) 217

based on Vibrio harveyi strain BB170 showed a limit of detection of 25 nM AI-2 (51). Binding 218

affinities (Kd values) of chemoreceptors for organic substrates, as mostly determined by 219

isothermal titration calorimetry, apparently reside in the lower µM range: 0.46 µM phenol and 220

4.12 µM catechol for the transcriptional regulator MopR (NtrC family) from Acinetobacter 221

calcoaceticus (34), 39 µM citrate for McpQ from Pseudomonas putida KT2440 (52), 35.6 µM 222

serine and 99.4 µM aspartate for Tsr and Tar, respectively, of Escherichia coli (53), and 58.6 223

µM pyruvate for the sensor histidine kinase BtsS of E.coli (54). 224

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Against the background of the above described knowledge on in situ concentrations 225

of aromatic compounds and the binding affinities of chemoreceptors, the here elucidated 226

response threshold (1–10 nM) for p-hydroxyacetophenone in "A. aromaticum" EbN1 sheds 227

new light on the lower limit of responsiveness towards aromatic growth substrates. From an 228

environmental point of view, it is interesting to approximate, which in situ concentration of a 229

given substrate suffices to transcriptionally turn on its degradation pathway. Such a threshold 230

of responsiveness has implications for the type of habitat and/or environmental condition that 231

provide(s) a given bacterium with nutritional opportunities for survival and eventually for 232

proliferation. Conversely, it may approximate a threshold concentration below which an in 233

principle utilizable compound may escape biodegradation, become instead an enduring 234

constituent of dissolved organic matter and get ultimately preserved in the geosphere. 235

236

237

MATERIALS AND METHODS 238

Bacterial strain and cultivation conditions. "Aromatoleum aromaticum" EbN1 has 239

been subcultured and stored in our laboratory since its isolation (11). Anaerobic cultivation of 240

"A. aromaticum" EbN1 was performed at 28°C in defined, bicarbonate-buffered, and 241

ascorbate-reduced mineral medium with the electron acceptor nitrate, as previously 242

described (11). Organic substrates (benzoate and p-hydroxyacetophenone) were provided 243

from sterile aqueous stock solutions. For the purpose of best possible reproducibility, each 244

growth experiment was started from the same batch of glycerol stocks of "A. aromaticum" 245

EbN1 grown with benzoate (4 mM) complying to the following sequence of cultivation steps: 246

(i) A dilution series (101 to 106), likewise with benzoate (4 mM) as sole source of carbon 247

and energy, was inoculated from a glycerol stock and incubated for 4 days. (ii) The 1st pre-248

culture supplied with 2 mM benzoate (80 ml culture volume in 100-ml flat glass bottles sealed 249

with butyl rubber stoppers) was inoculated with 5% (vol/vol) of the 106 dilution and incubated 250

for 3 days. (iii) The 2nd pre-culture was carried out under the same conditions, inoculated 251

with 5% (vol/vol) of the 1st pre-culture and incubated for 17 h. (iv) The triplicate main cultures 252

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(see following paragraph on "Growth experiments") with 1 mM benzoate (400 ml culture 253

volume in 500-ml flat glass bottles sealed with butyl rubber stoppers) were inoculated with 254

2% (vol/vol) of the 2nd pre-culture. All chemicals were of analytical grade. 255

Growth experiments. The responsiveness of "A. aromaticum" EbN1 to varying 256

concentrations of p-hydroxyacetophenone was studied with non-adapted cells. In each 257

experiment, benzoate (1 mM) was initially provided as the growth-limiting sole source of 258

organic carbon and energy. Upon its highly reproducible complete depletion after 17.5 h of 259

incubation, a distinct pulse of p-hydroxyacetophenone was given at one of the seven tested 260

concentrations (500 µM, 100 µM, 1 µM, 100 nM, 50 nM, 30 nM, and 10 nM). Throughout the 261

entire incubation time (approx. 24 h), 3 ml samples of the culture broth were retrieved by 262

means of sterile, N2-flushed syringes. An aliquot of 1 ml was used for monitoring growth by 263

measuring the optical density at 660 nm (OD660). The remaining 2 ml were immediately 264

centrifuged (20,000 g, 10 min, 4°C) and the supernatant was stored at 20°C for 265

subsequent determination of substrate depletion by micro high-performance-liquid-266

chromatography (microHPLC). Per test condition, three replicate cultures were performed. 267

Cultivation and cell harvesting for transcript profiling. Cultivation was performed 268

as described in above paragraph "Growth experiments", with the additional concentrations of 269

1 nM and 0.1 nM p-hydroxyacetophenone. At each sampling point, 5 ml culture broth was 270

withdrawn from each of the 3 replicate cultures per tested concentration of p-271

hydroxyacetophenone. Samples were retrieved with sterile, N2-flushed syringes and 272

immediately added to 10 ml of RNAprotect Bacterial Reagent (Qiagen, Hilden, Germany), 273

mixed rigorously, incubated for 5 min at room temperature and centrifuged (4,000 g, 10 274

min, 4°C). Pellets were resuspended in 0.5 ml RNAprotect Bacterial Reagent and 275

transferred into 2 ml microcentrifuge tubes and centrifuged (20,000 g, 10 min, room 276

temperature). Supernatants were discarded and pellets were shock frozen in liquid N2 and 277

stored at 80°C until further analyses. Sampling time points were 5 min prior to addition of p-278

hydroxyacetophenone (control) followed by 5, 15, 20, 60 and 120 min after addition; in 279

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selected cases also after 240 min (100 µM, 10 µM and no p-hydroxyacetophenone) and 480 280

min (100 µM and no p-hydroxyacetophenone). 281

Quantitation of aromatic compounds by microHPLC. Quantitative determination of 282

the depletion profiles of benzoate and p-hydroxyacetophenone was achieved with a newly 283

developed method employing a microHPLC (UltiMate 3000; ThermoFischer, Germering, 284

Germany). The system was equipped with a Thermo Accucore column (C18, 150 1 mm, 2.6 285

µm bead size; ThermoFischer) and a RS Diode Array Detector (ThermoFischer); it was 286

operated at 40°C with a flow rate of 100 µl/min. The 20 min-gradient composed of the eluent 287

A (5% (vol/vol) acetonitrile in H2O with 0.01% (vol/vol) H3PO4 (85%)) and eluent B (90% 288

(vol/vol) acetonitrile in H2O with 0.01% (vol/vol) H3PO4 (85%)) was as follows: 2.5 min 289

constant at 3% B, 4 min linear ramping to 65% B, 1 min linear ramping to 99% B, 1.5 min 290

constant at 99% B, 2 min linear ramping to 3% B, and finally 9 min constant at 3% B. 291

Benzoate was detected at 229 nm with a retention time of 9.32 min and a dynamic range 292

from 50 nM to 50 µM. p-Hydroxyacetophenone was detected at 275 nm with a retention time 293

of 6.99 min and a dynamic range from 5 nM to 50 µM. A representative chromatogram and 294

the respective calibration curves are provided in Figs. S1 and S2. 295

Preparation of total RNA. Total RNA was prepared from all three biological 296

replicates per sampling time point for every experiment (in total 153 preparations), using 297

saturated acidic phenol (60°C) essentially as previously described (55, 56). In brief, each cell 298

pellet was treated twice with hot acidic phenol and after centrifugation the aqueous phase 299

was transferred into a 2 ml 5PRIME Phase Lock Gel tube (Quantabio, Beverly, MA, USA). 300

One volume of phenol/chloroform/isoamylalcohol (25:24:1) was added and the tube was 301

gently inverted for 5 min. After centrifugation (20,000 g, 5 min, room temperature), nucleic 302

acids were precipitated using ice-cold ethanol (96%) during incubation for 30 min at 80°C. 303

After centrifugation (20,000 g, 30 min, 4°C), the pellet was washed with ice-cold ethanol 304

(75%) and centrifuged again (20,000 g, 15 min, 4°C). The pellet was dried and 305

resuspended in RNase-free water. Subsequently, every sample was subjected to DNase I 306

(RNase-free; Qiagen) digestion. Complete removal of DNA was confirmed by PCR using 307

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genomic DNA of "A. aromaticum" EbN1 as a positive control. RNA quality was controlled by 308

the ExperionTM StdSens RNA Chip (Bio-Rad, Hercules, CA, USA) operated in an ExperionTM 309

Automated Electrophoresis Station (Bio-Rad). RNA concentration was determined using a 310

TrayCell (Hellma Analytics, Müllheim, Germany) operated in a spectrophotometer (UV-311

1800; Shimadzu, Duisburg, Germany). Total RNA was stored in aliquots at 80°C. All 312

chemicals used for preparation of total RNA were of molecular-biology grade. 313

Transcript profiling by qRT-PCR. Specific primers (Table 1) for the five target genes 314

were designed using the Primer3 software package (version 0.4.0; www.primer3.org). cDNA 315

generation and real-time PCR was performed with three technical replicates per RNA 316

preparation using 50 ng of total RNA, the Brilliant III Ultra-Fast SYBR Green QRT-PCR 317

master mix (Agilent, Santa Clara, CA, USA) and the CFX96 Real-time system (Bio-Rad); in 318

total 9 measurements were conducted per analyzed time point. The one-tube RT real-time 319

PCR reaction was carried out as follows: one cycle of reverse transcription for 10 min at 320

50°C and one cycle of PCR initiation for 3 min at 95°C, followed by 40 cycles of 10 s 321

denaturation at 95°C, 30 s of annealing (primer-specific) and 30 s of extension at 60°C, 322

succeeded by real-time detection for 5 s. The gene-specific annealing temperatures were: 323

acsA, 60°C; hped, 60°C; pchF, 54.5°C; ebA335, 60°C; and ebA326, 57.5°C. The specificity 324

of accumulated products was verified by melting curve analysis, ranging from 60°C to 90°C 325

in steps of 0.5°C. The RNA preparation from the samples retrieved 5 min prior to addition of 326

p-hydroxyacetophenone was used as reference, while all samples retrieved at a later time 327

point during incubation represented the test states. The differences in transcript abundance 328

were calculated according to the following equation (57): 329

330

𝑟𝑎𝑡𝑖𝑜 = 𝐸Δ𝐶𝑇(𝑟𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒−𝑡𝑒𝑠𝑡)

331

Primer-specific efficiencies (E) of the PCR reaction for each primer pair were determined as 332

previously reported (58). 333

334

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335

SUPPLEMENTAL MATERIAL 336

Supplemental material for this manuscript may be found at yyyyyyyy 337

SUPPLEMENTAL FILE 1, PDF file, 0.8 MB. 338

339

340

ACKNOWLEDGEMENTS 341

We are grateful to Christina Hinrichs for technical assistance. Author contributions: 342

JV, LW and RR conceived the study; JV, SS and MK performed the cultivation experiments; 343

SS conducted the (micro)HPLC analyses; JV and SS did the RNA work; JV, LW and RR 344

wrote the manuscript. This study was supported by the Deutsche Forschungsgemeinschaft 345

(DFG) within the framework of the research training group "Molecular basis of sensory 346

biology" (GRK 1885). 347

348

349

REFERENCES 350

351

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508

Legends to figures 509

510

Fig 1 Scheme of the proposed transcriptional regulation of the anaerobic degradation of p-511

ethylphenol in the denitrifying bacterium "Aromatoleum aromaticum" EbN1. The highly specific 512

regulation of the peripheral degradation route by the substrate p-ethylphenol and its conversion 513

intermediate p-hydroxyacetophenone is proposed to be mediated by the predicted 54

-dependent 514

sensor/regulator EtpR. The two aromatic compounds are suggested to be recognized by the sensory 515

domain of the EtpR protein, which thereafter activates the transcription of the genes for the catabolism 516

as well as a presumptive associated efflux system. For practical reasons the sensitivity of the system 517

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was investigated with p-hydroxyacetophenone across a concentration range from 100 µM down to 0.1 518

nM, by determining its depletion profiles (Fig. 2) and formation of involved transcripts (Fig. 3). Genes 519

marked in red were selected for transcript profiling. Compound names: 1, p-ethylphenol; 2, 1-(4-520

hydroxyphenyl)ethanol; 3, p-hydroxyacetophenone; 4, presumptive p-hydroxybenzoylacetate; 5, p-521

hydroxybenzoyl-CoA; 6, benzoyl-CoA. Enzyme names: PchCF, predicted p-ethylphenol 522

methylenehydroxylase; Hped, 1-(4-hydroxyphenyl)ethanol dehydrogenase; AcsA, predicted acetyl-523

CoA synthetase; EbA332/5/6/7, presumptive solvent efflux system. UBS, putative upstream-binding 524

sequence of EtpR. 525

526

Fig 2 Growth experiments testing the responsiveness of non-adapted cells of "A. aromaticum" EbN1 527

to p-hydroxyacetophenone. Growth was supported by the primary substrate benzoate (added at 528

limiting 1 mM), which was reproducibly depleted after 17.5 h of incubation. Immediately thereafter, p-529

hydroxyacetophenone was added in a single pulse at varying concentrations, responsiveness to which 530

was assessed by determining its depletion via microHPLC. (A) and (B) display the experiments with 531

provision of 100 µM and 10 nM of p-hydroxyacetophenone, respectively; all experiments were 532

conducted with three biological replicates. The inserted blow-ups show at higher resolution the time-533

point of addition of p-hydroxyacetophenone as well as those for sampling for subsequent targeted 534

transcript profiling. (C) Correlation between the nine tested p-hydroxyacetophenone concentrations 535

and the determined depletion rates; growth curves and compound depletion profiles for the seven p-536

hydroxyacetophenone concentrations not shown in (A) and (B) are provided in Fig. S5. 537

538

Fig 3 Time-resolved, quantitative transcript profiles of "A. aromaticum" EbN1 in response to different 539

extracellular effector (p-hydroxyacetophenone) concentrations. The selected transcripts represent 540

genes (see also Fig. 1) involved in the catabolism of p-hydroxyacetophenone (pchF, hped and acsA) 541

and its presumptive efflux (ebA335 and ebA326). Transcript abundance was determined by qRT-PCR 542

with the time point 5 minutes prior to p-hydroxyacetophenone addition serving as reference. Each data 543

point is based on 3 biological replicates, for each of which 3 technical replicates were analyzed. 544

Growth cultures providing samples for the nine studied p-hydroxyacetophenone concentrations as well 545

as for the control (no addition of effector) are shown in detail in Fig. S6, and fold-changes of transcript 546

abundance in Table S1. 547

548

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549

550

551

552

TABLE 1 Gene-specific primer pairs used for expression analysis of target genes by means of qRT-PCR 553

Primera Target gene Nucleotide sequence (5' to 3') Product length (bp) PCR-efficiency

b

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

554 a F, forward primer; R, reverse primer. 555

b Mean value of all performed qRT-PCR experiments. 556

557

558

559

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