Generation of Novel Bacterial Regulatory Proteins That Detect Priority Pollutant Phenols

Abstract

The genetic systems of bacteria that have the ability to use organic pollutants as carbon and energy sources can be adapted to create bacterial biosensors for the detection of industrial pollution. The creation of bacterial biosensors is hampered by a lack of information about the genetic systems that control production of bacterial enzymes that metabolize pollutants. We have attempted to overcome this problem through modification of DmpR, a regulatory protein for the phenol degradation pathway of Pseudomonas sp. strain CF600. The phenol detection capacity of DmpR was altered by using mutagenic PCR targeted to the DmpR sensor domain. DmpR mutants were identified that both increased sensitivity to the phenolic effectors of wild-type DmpR and increased the range of molecules detected. The phenol detection characteristics of seven DmpR mutants were demonstrated through their ability to activate transcription of a lacZ reporter gene. Effectors of the DmpR derivatives included phenol, 2-chlorophenol, 2,4-dichlorophenol, 4-chloro-3-methylphenol, 2,4-dimethylphenol, 2-nitrophenol, and 4-nitrophenol.

Role of the DmpR-Mediated Regulatory Circuit in Bacterial Biodegradation Properties in Methylphenol-Amended Soils

“…A number of different molecular genetic strategies have previously been used to isolate mutations within the A domain of DmpR that positively or negatively modulate the response to aromatic effectors. These include positive genetic selection systems and random PCR mutagenesis to generate mutants with enhanced sensitivity and/or novel sensory capacity (31,42,52), genetic selection of second-site suppressors of constitutively active mutations of DmpR (26), and DNA shuffling between the A domain of DmpR and that of the toluene-xylene response regulator XylR (44). The locations of the mutations identified in this study relative to previously identified mutations are shown in Fig.…”

“…Not all effectors are equally efficient in activating DmpR, and modulation of the A-C domain inhibitory interaction through mutations in either the A or C domain can alter the magnitude of the response to a given effector (26,31). In addition, mutations within the signal reception A domain can expand the range of compounds that can activate DmpR to promote transcription (31,42,52). As illustrated in Fig.…”

“…The mutations DmpR-R109C, DmpR-D116N, and DmpR-E135K show the broadest spectrum of response to para-substituted compounds, which includes the ability to respond to the priority-list pollutants 2,4-dimethylphenol, 2,4-dichlorophenol, and 4-nitrophenol. Thus, this class of mutants have potential utility in whole-cell biosensor applications for pollution monitoring (44,52).…”

“…Locations of the A-domain dmpR mutations identified in the present study (upper arrowheads) in relation to previously identified A-domain mutations (lower arrowheads). Open arrowheads indicate mutations that positively affect the response to aromatic compounds as identified by enhanced responses to an effector(s) of wild-type DmpR and/or expanded specificity range (31,42,52). Grey and black arrowheads indicate mutations that negatively affect the aromatic response of DmpR.…”

Content of phenolic compounds in soils originating from two long-term fertilization experiments

“…They are also included in the composition of plant root exudates, and are components of fl ower and fruit dyes (Krupa and Latocha 2007). The main sources of these compounds are industry and agricultural activity (Wise and Kuske 2000, Thakur et al 2013, Wang et al 2015.…”