Post on 27-Oct-2020
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Towards the Response Threshold for p-Hydroxyacetophenone 1
in the Denitrifying Bacterium "Aromatoleum aromaticum" EbN1 2
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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
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Running title: Responsiveness towards p-hydroxyacetophenone 11
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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
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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
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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
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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
1. Umezawa T. 2010. The cinnamate/monolignol pathway. Phytochem Rev 9:1-17. 352 2. Overton E, Wade T, Radovíc J, Meyer B, Miles MS, Larter S. 2016. Chemical composition of 353
Macondo and other crude oils and compositional alterations during oil spills. Oceanography 354 29:50-63. 355
3. Bolden AL, Kwiatkowski CF, Colborn T. 2015. New look at BTEX: are ambient levels a 356 problem? Environ Sci Technol 49:5261-76. 357
4. Widdel F, Rabus R. 2001. Anaerobic biodegradation of saturated and aromatic hydrocarbons. 358 Curr Opin Biotechnol 12:259-76. 359
5. Carmona M, Zamarro MT, Blázquez B, Durante-Rodríguez G, Juárez JF, Valderrama JA, 360 Barragán MJ, García JL, Díaz E. 2009. Anaerobic catabolism of aromatic compounds: a genetic 361 and genomic view. Microbiol Mol Biol Rev 73:71-133. 362
6. Fuchs G, Boll M, Heider J. 2011. Microbial degradation of aromatic compounds - from one 363 strategy to four. Nat Rev Microbiol 9:803-16. 364
7. Decker K, Jungermann K, Thauer RK. 1970. Energy production in anaerobic organisms. Angew 365 Chem Internat Edit 9:138-58. 366
8. Zumft WG. 1997. Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 367 61:533-616. 368
9. Shapleigh JP. 2013. Denitrifying prokaryotes, p 405-25, The Prokaryotes doi:10.1007/978-3-369 642-30141-4_71. 370
on April 12, 2021 by guest
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ownloaded from
14
10. Fowler D, Coyle M, Skiba U, Sutton MA, Cape JN, Reis S, Sheppard LJ, Jenkins A, Grizzetti B, 371 Galloway JN, Vitousek P, Leach A, Bouwman AF, Butterbach-Bahl K, Dentener F, Stevenson D, 372 Amann M, Voss M. 2013. The global nitrogen cycle in the twenty-first century. Phil Trans R 373 Soc B 368:20130164. 374
11. Rabus R, Widdel F. 1995. Anaerobic degradation of ethylbenzene and other aromatic 375 hydrocarbons by new denitrifying bacteria. Arch Microbiol 163:96-103. 376
12. Rabus R, Trautwein K, Wöhlbrand L. 2014. Towards habitat-oriented systems biology of 377 "Aromatoleum aromaticum" EbN1: chemical sensing, catabolic network modulation and 378 growth control in anaerobic aromatic compound degradation. Appl Microbiol Biotechnol 379 98:3371-88. 380
13. Rabus R, Kube M, Heider J, Beck A, Heitmann K, Widdel F, Reinhardt R. 2005. The genome 381 sequence of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1. Arch 382 Microbiol 183:27-36. 383
14. Kühner S, Wöhlbrand L, Fritz I, Wruck W, Hultschig C, Hufnagel P, Kube M, Reinhardt R, Rabus 384 R. 2005. Substrate-dependent regulation of anaerobic degradation pathways for toluene and 385 ethylbenzene in a denitrifying bacterium, strain EbN1. J Bacteriol 187:1493-503. 386
15. Wöhlbrand L, Kallerhoff B, Lange D, Hufnagel P, Thiermann J, Reinhardt R, Rabus R. 2007. 387 Functional proteomic view of metabolic regulation in "Aromatoleum aromaticum" strain 388 EbN1. Proteomics 7:2222-2239. 389
16. Wöhlbrand L, Wilkes H, Halder T, Rabus R. 2008. Anaerobic degradation of p-ethylphenol by 390 "Aromatoleum aromaticum" strain EbN1: pathway, regulation, and involved proteins. J 391 Bacteriol 190:5699-709. 392
17. Trautwein K, Wilkes H, Rabus R. 2012. Proteogenomic evidence for -oxidation of plant-393 derived 3-phenylpropanoids in "Aromatoleum aromaticum" EbN1. Proteomics 12:1402-13. 394
18. Schühle K, Nies J, Heider J. 2016. An indoleacetate-CoA ligase and a phenylsuccinyl-CoA 395 transferase involved in anaerobic metabolism of auxin. Environ Microbiol 18:3120-32. 396
19. Ebenau-Jehle C, Mergelsberg M, Fischer S, Brüls T, Jehmlich N, von Bergen M, Boll M. 2017. 397 An unusual strategy for the anoxic biodegradation of phthalate. ISME J 11:224-36. 398
20. Büsing I, Höffken HW, Breuer M, Wöhlbrand L, Hauer B, Rabus R. 2015. Molecular genetic 399 and crystal structural analysis of 1-(4-hydroxyphenyl)-ethanol dehydrogenase from 400 'Aromatoleum aromaticum' EbN1. J Mol Microbiol Biotechnol 25:327-39. 401
21. Muhr E, Schühle K, Clermont L, Sünwoldt K, Kleinsorge D, Seyhan D, Kahnt J, Schall I, Cordero 402 PR, Schmitt G, Heider J. 2015. Enzymes of anaerobic ethylbenzene and p-ethylphenol 403 catabolism in 'Aromatoleum aromaticum': differentiation and differential induction. Arch 404 Microbiol 197:1051-62. 405
22. Büsing I, Kant M, Dörries M, Wöhlbrand L, Rabus R. 2015. The predicted 54-dependent 406 regulator EtpR is essential for expression of genes for anaerobic p-ethylphenol and p-407 hydroxyacetophenone degradation in "Aromatoleum aromaticum" EbN1. BMC Microbiol 408 15:251. 409
23. Tropel D, van der Meer JR. 2004. Bacterial transcriptional regulators for degradation 410 pathways of aromatic compounds. Microbiol Mol Biol Rev 68:474-500. 411
24. Fernandez-López R, Ruiz R, de la Cruz F, Moncalián G. 2015. Transcription factor-based 412 biosensors enlightened by the analyte. Front Microbiol 6:648. 413
25. Ortega A, Zhulin IB, Krell T. 2017. Sensory repertoire of bacterial chemoreceptors. Microbiol 414 Mol Biol Rev 81:e00033-17. 415
26. Ulrich LE, Koonin EV, Zhulin IB. 2005. One-component systems dominate signal transduction 416 in prokaryotes. Trends Microbiol 13:52-6. 417
27. Marijuán PC, Navarro J, del Moral R. 2010. On prokaryotic intelligence: strategies for sensing 418 the environment. BioSyst 99:94-103. 419
28. Lacal J, Guazzaroni ME, Gutiérrez-del-Arroyo P, Busch A, Vélez M, Krell T, Ramos JL. 2008. 420 Two levels of cooperativeness in the binding of TodT to the tod operon promoter. J Mol Biol 421 384:1037-47. 422
on April 12, 2021 by guest
http://aem.asm
.org/D
ownloaded from
15
29. Pérez-Pantoja D, Kim J, Silva-Rocha R, de Lorenzo V. 2015. The differential response of the 423 Pben promoter of Pseudomonas putida mt-2 to BenR and XylS prevents metabolic conflicts in 424 m-xylene biodegradation. Environ Microbiol 17:64-75. 425
30. Marqués S, Ramos JL. 1993. Transcriptional control of the Pseudomonas putida TOL plasmid 426 catabolic pathways. Mol Microbiol 9:923-9. 427
31. Devos D, Garmendia J, de Lorenzo V, Valencia A. 2002. Deciphering the action of aromatic 428 effectors on the prokaryotic enhancer-binding protein XylR: a structural model of its N-429 terminal domain. Environ Microbiol 4:29-41. 430
32. Craven SH, Ezezika OC, Haddad S, Hall RA, Momany C, Neidle EL. 2009. Inducer responses of 431 BenM, a LysR-type transcriptional regulator from Acinetobacter baylyi ADP1. Mol Microbiol 432 72:881-94. 433
33. Vogne C, Bisht H, Arias S, Fraile S, Lal R, van der Meer JR. 2011. Characterisation of the 434 putative effector interaction site of the regulatory HbpR protein from Pseudomonas azelaica 435 by site-directed mutagenesis. PLoS One 6:e16539. 436
34. Ray S, Gunzburg MJ, Wilce M, Panjikar S, Anand R. 2016. Structural basis of selective 437 aromatic pollutant sensing by the effector binding domain of MopR, an NtrC family 438 transcriptional regulator. ACS Chem Biol 11:2357-65. 439
35. Buck M, Cannon W. 1992. Specific binding of the transcription factor sigma-54 to promoter 440 DNA. Nature 358:422-4. 441
36. Shingler V. 2011. Signal sensory systems that impact 54 -dependent transcription. FEMS 442 Microbiol Rev 35:425-40. 443
37. Ramos JL, Krell T, Daniels C, Segura A, Duque E. 2009. Responses of Pseudomonas to small 444 toxic molecules by a mosaic of domains. Curr Opin Microbiol 12:215-20. 445
38. Ng LC, Poh CL, Shingler V. 1995. Aromatic effector activation of the NtrC-like transcriptional 446 regulator PhhR limits the catabolic potential of the (methyl)phenol degradative pathway it 447 controls. J Bacteriol 177:1485-90. 448
39. Pal R, Bala S, Dadhwal M, Kumar M, Dhingra G, Prakash O, Prabagaran SR, Shivaji S, Cullum J, 449 Holliger C, Lal R. 2005. Hexachlorocyclohexane-degrading bacterial strains Sphingomonas 450 paucimobilis B90A, UT26 and Sp+, having similar lin genes, represent three distinct species, 451 Sphingobium indicum sp. nov., Sphingobium japonicum sp. nov. and Sphingobium francense 452 sp. nov., and reclassification of [Sphingomonas] chungbukensis as Sphingobium 453 chungbukense comb. nov. Int J Syst Evol Microbiol 55:1965-72. 454
40. Suar M, van der Meer JR, Lawlor K, Holliger C, Lal R. 2004. Dynamics of multiple lin gene 455 expression in Sphingomonas paucimobilis B90A in response to different 456 hexachlorocyclohexane isomers. Appl Environ Microbiol 70:6650-6. 457
41. Werlen C, Jaspers MCM, van der Meer JR. 2004. Measurement of biologically available 458 naphthalene in gas and aqueous phases by use of a Pseudomonas putida biosensor. Appl 459 Environ Microbiol 70:43-51. 460
42. Poerschmann J, Parsi Z, Gorecki T. 2008. Non-discriminating flash pyrolysis and 461 thermochemolysis of heavily contaminated sediments from the Hamilton Harbor (Canada). J 462 Chromatogr A 1186:211-21. 463
43. Kolpin DW, Blazer VS, Gray JL, Focazio MJ, Young JA, Alvarez DA, Iwanowicz LR, Foreman WT, 464 Furlong ET, Speiran GK, Zaugg SD, Hubbard LE, Meyer MT, Sandstrom MW, Barber LB. 2013. 465 Chemical contaminants in water and sediment near fish nesting sites in the Potomac River 466 basin: determining potential exposures to smallmouth bass (Micropterus dolomieu). Sci Total 467 Environ 443:700-16. 468
44. Li B, Hu X, Liu R, Zeng P, Song Y. 2015. Occurrence and distribution of phthalic acid esters and 469 phenols in Hun River Watersheds. Environmental Earth Sciences 73:5095-5106. 470
45. Kolpin DA, Furlong ET, Meyer MT, Thurman EM, Zaugg SD, Barber LB, Buxton HT. 2002. 471 Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 472 1999-2000: a national reconnaissance. Environ Sci Technol 36:1202-11. 473
on April 12, 2021 by guest
http://aem.asm
.org/D
ownloaded from
16
46. Chmiel HE, Niggemann J, Kokic J, Ferland M-È, Dittmar T, Sobek S. 2015. Uncoupled organic 474 matter burial and quality in boreal lake sediments over the Holocene. Journal of Geophysical 475 Research: Biogeosciences 120:1751-1763. 476
47. Hedges JI, Clark WA, Cowie GL. 1988. Organic matter sources to the water column and 477 surficial sediments of a marine bay. Limnol Oceanogr 33:1116-36. 478
48. Papenfort K, Bassler BL. 2016. Quorum sensing signal-response systems in Gram-negative 479 bacteria. Nat Rev Microbiol 14:576-88. 480
49. Whiteley M, Diggle SP, Greenberg EP. 2017. Progress in and promise of bacterial quorum 481 sensing research. Nature 551:313-320. 482
50. Hirakawa H, Oda Y, Phattarasukol S, Armour CD, Castle JC, Raymond CK, Lappala CR, Schaefer 483 AL, Harwood CS, Greenberg EP. 2011. Activity of the Rhodopseudomonas palustris p-484 coumaroyl-homoserine lactone-responsive transcription factor RpaR. J Bacteriol 193:2598-485 607. 486
51. Raut N, Pasini P, Daunert S. 2013. Deciphering bacterial universal language by detecting the 487 quorum sensing signal, autoinducer-2, with a whole-cell sensing system. Anal Chem 85:9604-488 9. 489
52. Martin-Mora D, Reyes-Darias JA, Ortega A, Corral-Lugo A, Matilla MA, Krell T. 2016. McpQ is 490 a specific citrate chemoreceptor that responds preferentially to citrate/metal ion complexes. 491 Environ Microbiol 18:3284-3295. 492
53. Tajima H, Imada K, Sakuma M, Hattori F, Nara T, Kamo N, Homma M, Kawagishi I. 2011. 493 Ligand specificity determined by differentially arranged common ligand-binding residues in 494 bacterial amino acid chemoreceptors Tsr and Tar. J Biol Chem 286:42200-10. 495
54. Behr S, Kristoficova I, Witting M, Breland EJ, Eberly AR, Sachs C, Schmitt-Kopplin P, 496 Hadjifrangiskou M, Jung K. 2017. Identification of a high-affinity pyruvate receptor in 497 Escherichia coli. Sci Rep 7:1388. 498
55. Oelmüller U, Krüger N, Steinbüchel A, Friedrich CG. 1990. Isolation of prokaryotic RNA and 499 detection of specific mRNA with biotinylated probes. J Microbiol Meth 11. 500
56. Lahme S, Trautwein K, Strijkstra A, Dörries M, Wöhlbrand L, Rabus R. 2014. Benzoate 501 mediates the simultaneous repression of anaerobic 4-methylbenzoate and succinate 502 utilization in Magnetospirillum sp. strain pMbN1. BMC Microbiol 14:269. 503
57. Pfaffl MW. 2001. A new mathematical model for relative quantification in real-time RT-PCR. 504 Nucleic Acids Res 29:2002-7. 505
58. Ramakers C, Ruijter JM, Deprez RHL, Moorman AFM. 2003. Assumption-free analysis of 506 quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62-6. 507
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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