Anaerobic degradation of p-cresol (4-methylphenol) by the denitrifying betaproteobacterium Aromatoleum aromaticum EbN1 is regulated with high substrate specificity, presumed to be mediated by the predicted σ54-dependent two-component system PcrSR. An unmarked, in-frame ΔpcrSR deletion mutant showed reduced expression of the genes cmh (21-fold) and hbd (8-fold) that encode the two enzymes for initial oxidation of p-cresol to p-hydroxybenzoate compared to their expression in the wild type. The expression of cmh and hbd was restored by in trans complementation with pcrSR in the ΔpcrSR background to even higher levels than in the wild type. This is likely due to ∼200-/∼30-fold more transcripts of pcrSR in the complemented mutant. The in vivo responsiveness of A. aromaticum EbN1 to p-cresol was studied in benzoate-limited anaerobic cultures by the addition of p-cresol at various concentrations (from 100 μM down to 0.1 nM). Time-resolved transcript profiling by quantitative reverse transcription-PCR (qRT-PCR) revealed that the lowest p-cresol concentrations just affording cmh and hbd expression (response threshold) ranged between 1 and 10 nM, which is even more sensitive than the respective odor receptors of insects. A similar response threshold was determined for another alkylphenol, p-ethylphenol, which strain EbN1 anaerobically degrades via a different route and senses by the σ54-dependent one-component system EtpR. Based on these data and theoretical considerations, p-cresol or p-ethylphenol added as a single pulse (10 nM) requires less than a fraction of a second to reach equilibrium between intra- and extracellular space (∼20 molecules per cell), with an estimated Kd (dissociation constant) of <100 nM alkylphenol (p-cresol or p-ethylphenol) for its respective sensory protein (PcrS or EtpR). IMPORTANCE Alkylphenols (like p-cresol and p-ethylphenol) represent bulk chemicals for industrial syntheses. Besides massive local damage events, large-scale micropollution is likewise of environmental and health concern. Next to understanding how such pollutants can be degraded by microorganisms, it is also relevant to determine the microorganisms’ lower threshold of responsiveness. Aromatoleum aromaticum EbN1 is a specialist in anaerobic degradation of aromatic compounds, employing a complex and substrate-specifically regulated catabolic network. The present study aims at verifying the predicted role of the PcrSR system in sensing p-cresol and at determining the threshold of responsiveness for alkylphenols. The findings have implications for the enigmatic persistence of dissolved organic matter (escape from biodegradation) and for the lower limits of aromatic compounds required for bacterial growth.

The betaproteobacterial degradation specialist Aromatoleum aromaticum EbN1T utilizes several plant-derived 3-phenylpropanoids coupled to denitrification. In vivo responsiveness of A. aromaticum EbN1T was studied by exposing non-adapted cells to distinct pulses (spanning 100 μM to 0.1 nM) of 3-phenylpropanoate, cinnamate, 3-(4-hydroxyphenyl)propanoate, or p-coumarate. Time-resolved, targeted transcript analyses via qRT-PCR of four selected 3-phenylpropanoid genes revealed a response threshold of 30–50 nM for p-coumarate and 1–10 nM for the other three tested 3-phenylpropanoids. At these concentrations, transmembrane effector equilibration is attained by passive diffusion rather than active uptake via the ABC transporter presumably serving the studied 3-phenylpropanoids as well as benzoate. Highly substrate-specific enzyme formation (EbA5316–21) for the shared peripheral degradation pathway putatively involves the predicted TetR-type transcriptional repressor PprR. Accordingly, relative transcript abundances of ebA5316–21 are lower in succinate- and benzoate-grown wildtype cells compared to an unmarked in-frame ΔpprR mutant. In trans complementation of pprR into the ΔpprR background restored wildtype-like transcript levels. When adapted to p-coumarate, the three genotypes had similar relative transcript abundances of ebA5316–21, despite a significantly longer lag-phase of the pprR-complemented mutant (∼100-fold higher pprR transcript level than wildtype). Notably, transcript levels of ebA5316–21 were ∼10–100-fold higher in p-coumarate- versus succinate- or benzoate-adapted cells across all three genotypes. This possibly indicates the additional involvement of a yet unknown transcriptional regulator. Furthermore, physiological, transcriptional and (aromatic) acyl-CoA ester intermediate analyses of wildtype and ΔpprR mutant grown with binary substrate mixtures suggest a mode of catabolite repression of superior order to PprR. IMPORTANCE Lignin is a ubiquitous hetero-biopolymer built from of a suite of 3-phenylpropanoid subunits. It not only accounts for more than 30% of the global plant dry material, but lignin-related compounds are also increasingly released into the environment from anthropogenic sources, i.e., by wastewater effluents from the paper and pulp industry. Hence, following biological or industrial decomplexation of lignin, vast amounts of structurally diverse 3-phenylpropanoids enter terrestrial and aquatic habitats, where they serve as substrates for microbial degradation. This raises the question what signaling systems environmental bacteria employ to detect these nutritionally attractive compounds and to adjust their catabolism accordingly. Moreover, determining in vivo response thresholds of an anaerobic degradation specialist such as A. aromaticum EbN1T for these aromatic compounds provides insights into the environmental fate of the latter, i.e., when they could escape biodegradation due to too low ambient concentrations.

Phaeobacter inhibens DSM 17395 is a heterotrophic member of the ubiquitous, marine Roseobacter group and specialized in the aerobic utilization of carbohydrates and amino acids via pathways widespread among roseobacters. The in vivo responsiveness of P. inhibens DSM 17395 was studied with non-adapted cells (succinate-grown), which were exposed to a single pulse (100-0.01 µM) each of N-acetylglucosamine, mannitol, xylose, leucine, phenylalanine or tryptophan (effectors). Responsiveness was then determined by time-resolved transcript analyses (qRT-PCR) of 'degradation' and 'uptake' genes selected based on previously reported substrate-specific proteome profiles. The transcriptional response thresholds were: 50-100 nM for nagK (N-acetylglucosamine kinase), paaA (ring 1,2-phenylacetyl-CoA epoxidase), and kynA (tryptophan 2,3-dioxygenase), 10-50 nM for xylA (xylose isomerase), and around 10 nM for mtlK (mannitol 2-dehydrogenase). A threshold for leucine could not be determined due to the elevated intrinsic presence of leucine in the exometabolome of succinate-grown cells (no effector addition). Notably, the response thresholds for presumptive carbohydrate-binding proteins of ABC-transporters were in the same range or even lower: 10-100 nM for c27930 (N-acetylglucosamine) and even below 10 nM for c13210 (mannitol) and xylF (xylose). These results shed new light on the sensory/regulatory sensitivity of a well-studied roseobacter for recognizing potential substrates at low ambient concentrations and on the concentration threshold below which these might escape biodegradation ('emergent recalcitrance' concept of DOM persistence).

The betaproteobacterial genus <i>Aromatoleum</i> comprises facultative denitrifiers specialized in the anaerobic degradation of recalcitrant organic compounds (aromatic and terpenoid). This study reports on the complete and manually annotated genomes of <i>Ar. petrolei</i> ToN1^T (5.41 Mbp) and <i>Ar. bremense</i> PbN1^T (4.38 Mbp), which cover the phylogenetic breadth of the genus <i>Aromatoleum</i> together with previously genome sequenced <i>Ar. aromaticum</i> EbN1^T [Rabus et al., Arch Microbiol. 2005 Jan;183(1):27–36]. The gene clusters for the anaerobic degradation of aromatic and terpenoid (strain ToN1^T only) compounds are scattered across the genomes of strains ToN1^T and PbN1^T. The richness in mobile genetic elements is shared with other <i>Aromatoleum</i> spp., substantiating that horizontal gene transfer should have been a major driver in shaping the genomes of this genus. The composite catabolic network of strains ToN1^T and PbN1^T comprises 88 proteins, the coding genes of which occupy 86.1 and 76.4 kbp (1.59 and 1.75%) of the respective genome. The strain-specific gene clusters for anaerobic degradation of ethyl-/propylbenzene (strain PbN1^T) and toluene/monoterpenes (strain ToN1^T) share high similarity with their counterparts in <i>Ar. aromaticum</i> strains EbN1^T and pCyN1, respectively. Glucose is degraded via the ED-pathway in strain ToN1^T, while gluconeogenesis proceeds via the reverse EMP-pathway in strains ToN1^T, PbN1^T, and EbN1^T. The diazotrophic, endophytic lifestyle of closest related genus <i>Azoarcus</i> is known to be associated with nitrogenase and type-6 secretion system (T6SS). By contrast, strains ToN1^T, PbN1^T, and EbN1^T lack <i>nif</i> genes for nitrogenase (including cofactor synthesis and enzyme maturation). Moreover, strains PbN1^T and EbN1^T do not possess <i>tss</i> genes for T6SS, while strain ToN1^T does and facultative endophytic “<i>Aromatoleum</i>” sp. CIB is known to even have both. These findings underpin the functional heterogeneity among <i>Aromatoleum</i> members, correlating with the high plasticity of their genomes.

In all living organisms, adenosine triphosphate (ATP) and NAD(H) represent universal molecular currencies for energy and redox state, respectively, and are thus widely applicable molecular proxies for an organism’s viability and activity. To this end, corresponding luciferase-based assays in combination with a microplate reader were established with the marine model bacterium <i>Phaeobacter inhibens</i> DSM 17395 (<i>Escherichia coli</i> K12 served as reference). Grey multiwell plates best balanced sensitivity and crosstalk, and optimal incubation times were 5 min and 30 min for the ATP and NAD(H) assay, respectively, together allowing limits of detection of 0.042, 0.470 and 0.710 nM for ATP, NAD⁺, and NADH, respectively. Quenching of bacterial cell samples involved Tris-EDTA-DTAB and bicarbonate base-DTAB for ATP and NAD(H) assays, respectively. The ATP and NAD(H) yields determined for <i>P. inhibens</i> DSM 17395 at ¼ OD_max were found to reside well within the range previously reported for <i>E. coli</i> and other bacteria, e.g., 3.28 µmol ATP (g cells_dry)⁻¹. Thus, the here described methods for luciferase-based determination of ATP/NAD(H) pools open a promising approach to investigate energy and redox states in marine (environmental) bacteria.