Project description:The efficacy of inoculation of single pure bacterial cultures into complex microbiomes, for example, in order to achieve increased pollutant degradation rates in contaminated material (i.e., bioaugmentation), has been frustrated by insufficient knowledge on the behaviour of the inoculated bacteria under the specific abiotic and biotic boundary conditions. Here we present a comprehensive analysis of global gene expression of the bacterium Sphingomonas wittichii RW1 in contaminated sand, compared to regular suspended batch growth in liquid culture. RW1 is a well-known bacterium capable of mineralizing polycyclic aromatic hydrocarbons such as dioxins, dibenzofurans and other chlorinated congeners. We tested the reactions of the cells both during the immediate transition phase from liquid culture to sand with or without dibenzofuran, as well during growth and stationary phase in sand. Cells during transition resemble going through stationary phase, showing evidence of stress responses and nutrient scavenging, and even of major adjustments in their primary metabolism if they were not pre-cultured on the same contaminant as found in the soil. Cells growing and surviving in soil show very different signatures as in liquid or in liquid culture exposed to chemicals inducing drought stress, and we obtain evidence for numerous soil-specific expressed genes. We conclude that studies focusing on inoculation efficacy should test behavior under conditions as closely as possible mimicking the intended microbiome conditions. We were interested to study the global reactions of bacteria with biodegradative properties under near-environmental as compared to laboratory culture conditions. We compared here the genome-wide responses of RW1 between regular laboratory batch growth on the aromatic substrates DBF and salicylate with growth in sandy soil with or without the same aromatic compounds. We analysed the cellular reactions immediately after introduction into the sand, during exponential growth and at stationary phase, all in carefully controlled and replicated experimental conditions.

Project description:The efficacy of inoculation of single pure bacterial cultures into complex microbiomes, for example, in order to achieve increased pollutant degradation rates in contaminated material (i.e., bioaugmentation), has been frustrated by insufficient knowledge on the behaviour of the inoculated bacteria under the specific abiotic and biotic boundary conditions. Here we present a comprehensive analysis of global gene expression of the bacterium Sphingomonas wittichii RW1 in contaminated sand, compared to regular suspended batch growth in liquid culture. RW1 is a well-known bacterium capable of mineralizing polycyclic aromatic hydrocarbons such as dioxins, dibenzofurans and other chlorinated congeners. We tested the reactions of the cells both during the immediate transition phase from liquid culture to sand with or without dibenzofuran, as well during growth and stationary phase in sand. Cells during transition resemble going through stationary phase, showing evidence of stress responses and nutrient scavenging, and even of major adjustments in their primary metabolism if they were not pre-cultured on the same contaminant as found in the soil. Cells growing and surviving in soil show very different signatures as in liquid or in liquid culture exposed to chemicals inducing drought stress, and we obtain evidence for numerous soil-specific expressed genes. We conclude that studies focusing on inoculation efficacy should test behavior under conditions as closely as possible mimicking the intended microbiome conditions We were interested to study the global reactions of bacteria with biodegradative properties under near-environmental as compared to laboratory culture conditions. we compared here the genome-wide responses of RW1 between regular laboratory batch growth on the aromatic substrates DBF and salicylate with growth in sandy soil with or without the same aromatic compounds. We analysed the cellular reactions immediately after introduction into the sand, during lag phase, all in carefully controlled and replicated experimental conditions.

Project description: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 nonadapted 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 quantitative reverse transcription-PCR of four selected 3-phenylpropanoid genes revealed a response threshold of 30 to 50 nM for p-coumarate and 1 to 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 to EbA5321 [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 wild-type cells than in an unmarked in-frame ΔpprR mutant. In trans-complementation of pprR into the ΔpprR background restored wild-type-like transcript levels. When adapted to p-coumarate, the three genotypes had relative transcript abundances similar to those of ebA5316-21 despite a significantly longer lag phase of the pprR-complemented mutant (∼100-fold higher pprR transcript level than the wild type). Notably, transcript levels of ebA5316-21 were ∼10- to 100-fold higher in p-coumarate- than succinate- or benzoate-adapted cells across all three genotypes. This indicates the additional involvement of an unknown transcriptional regulator. Furthermore, physiological, transcriptional, and (aromatic) acyl-coenzyme A ester intermediate analyses of the wild type and ΔpprR mutant grown with binary substrate mixtures suggest a mode of catabolite repression of superior order to PprR.IMPORTANCE Lignin is a ubiquitous heterobiopolymer built from a suite of 3-phenylpropanoid subunits. It accounts for more than 30% of the global plant dry material, and lignin-related compounds are 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 of 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.

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Aromatoleum aromaticum EbN1

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