Molecular Analysis of a Plasmid-encoded Phenol Hydroxylase from Pseudomonas CF600

Shingler, Victoria; Franklin, F. Christopher H.; Tsuda, Masashi; Holroyd, D.; Bagdasarian, Michael

doi:10.1099/00221287-135-5-1083

Cited by 70 publications

(93 citation statements)

References 21 publications

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“…The Pseudomonas strains used were phenol-catabolizing Pseudomonas sp. strain CF600 (17), Pseudomonas putida KT2440 (hsdR) (5), and P. putida PRS2015 (catB) (21).…”

Section: Materuils and Methodsmentioning

confidence: 99%

“…la). Transcription of the dmp operon is positively regulated by the dmpR gene product, resulting in expression of the specialized catabolic enzymes only when the substrates are present (16,17).…”

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confidence: 99%

“…strain CF600 confers the ability to grow on phenol, on cresols (monomethyl phenols), or on 3,4-dimethylphenol as the sole source of carbon and energy (17). The dmp system is composed of the closely linked but divergently transcribed dmpR gene and the dmp operon that encodes all the enzymes required for catabolism of the substrates to central metabolites (16,18) (see Fig.…”

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confidence: 99%

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Sensing of aromatic compounds by the DmpR transcriptional activator of phenol-catabolizing Pseudomonas sp. strain CF600

1994

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The dmp operon of the pVI150 catabolic plasmid of Pseudomonas sp. strain CF600 encodes the enzymes involved in the catabolism of phenol and methylphenols. The regulator of this dmp pathway, DmpR, is a member of the NtrC family of transcriptional activators and controls transcription of the dmp operon in response to aromatic effector compounds (V. Shingler, M. Bartilson, and T. Moore, J. Bacteriol. 175:1596Bacteriol. 175: -1604Bacteriol. 175: , 1993. Using a lux gene fusion reporter system, in which the DmpR-regulated operon promoter controls the expression of luciferase activity, we have shown in the study reported here that DmpR is activated by, but responds differentially to, the presence of a wide range of aromatic compounds. In many microbial regulatory systems, including some members of the NtrC family, the response to environmental fluctuations involves information transfer from surface sensory proteins to transcriptional regulators. However, DmpR-mediated activation of phenol metabolism in response to aromatic compounds occurs in the absence of a specific sensory protein. We used hybrids between DmpR and XylR, a structurally related regulator of toluene and xylene metabolism, to demonstrate that it is the amino-terminal domains of these regulators that determine the specificity of transcriptional activation. The results suggest that it is the direct interaction of aromatic compounds with the DmpR and XylR proteins that regulates their transcriptional promoting activity.

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“…The Pseudomonas strains used were phenol-catabolizing Pseudomonas sp. strain CF600 (17), Pseudomonas putida KT2440 (hsdR) (5), and P. putida PRS2015 (catB) (21).…”

Section: Materuils and Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Sensing of aromatic compounds by the DmpR transcriptional activator of phenol-catabolizing Pseudomonas sp. strain CF600

1994

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“…strain CF600 harboring the IncP-2 catabolic megaplasmid pVI150 can efficiently grow on phenol, monomethylated phenols, and 3,4-dimethylphenol as sole sources of carbon and energy (40). The 15 structural genes for the enzymes of the catabolic pathway are located within the plasmid-encoded dmp operon (Fig.…”

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confidence: 99%

Growth phase-dependent transcription of the sigma(54)-dependent Po promoter controlling the Pseudomonas-derived (methyl)phenol dmp operon of pVI150

1996

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Transcription from Pseudomonas-derived ؊24, ؊12 Po promoter of the pVI150-encoded dmp operon is mediated by the 54 -dependent DmpR activator in response to the presence of aromatic pathway substrates in the medium. However, global regulatory mechanisms are superimposed on this regulatory system so that the specific response to aromatic effectors is absent in cultures until the stationary phase is reached. Here we genetically dissect the system to show that the growth phase response is faithfully mimicked by a minimal system composed of the dmpR regulatory gene and the Po promoter regulatory region and can be reproduced in heterologous Escherichia coli. Using this system, we show that the growth phase-dependent DmpR-mediated response to aromatic compounds is limited to fast-growing cultures. Thus, during exponential growth of cultures in minimal media containing different carbon sources, the response to aromatics is immediate, while the response is suppressed in cultures grown on rich media until the exponential-to-stationary phase transition. Elements known to be involved in the DmpR-mediated transcription from Po were analyzed for the ability to influence the growth phase response. Most dramatically, overexpression of DmpR was shown to completely abolish the growth phase response, suggesting that a negatively acting factor may mediate this level of regulation. The possible mechanism of action and integration of the specific regulation of the dmp operonencoded catabolic enzymes with the physiological status of the bacteria are discussed.Pseudomonas sp. strain CF600 harboring the IncP-2 catabolic megaplasmid pVI150 can efficiently grow on phenol, monomethylated phenols, and 3,4-dimethylphenol as sole sources of carbon and energy (40). The 15 structural genes for the enzymes of the catabolic pathway are located within the plasmid-encoded dmp operon (Fig. 1A) (43). Complete mineralization of pathway substrates is achieved by hydroxylation of the phenolic ring to form catechol, followed by a conversion to Krebs cycle intermediates pyruvate and acetyl coenzyme A via the sequential steps of the meta-cleavage pathway (reviewed in reference 35). Transcription of the dmp operon from the operon promoter, Po, is tightly regulated by the divergently transcribed dmpR gene product so that the enzymes of the pathway are expressed only in the presence of pathway substrates or structural analogs ( Fig. 1) (33,39,41).DmpR belongs to the prokaryotic enhancer-binding family of 54 -dependent regulators, which function to positively control transcription from Ϫ12, Ϫ24 promoters recognized by RNA polymerase utilizing the alternative sigma factor, 54

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“…PhcT was not involved in the gratuitous expression. Strain R5 thus possesses a more elaborate mechanism for regulating the mPH operon expression than has been found in other bacteria.The expression of a bacterial catabolic pathway for aromatic compounds is often controlled by one or more transcriptional regulatory proteins (21), and sometimes, one transcriptional regulator controls the expression of another transcriptional regulator (24).The expression of multicomponent phenol hydroxylase (mPH) (9,11,12,19,20,36,37) is generally thought to be controlled by a regulator of the XylR/DmpR subclass within the NtrC-type family of transcriptional regulators, resulting in the expression of phenol-metabolizing enzymes only in the presence of the pathway substrate or its structural analog (2,12,14,18,19,22,26,(30)(31)(32)(33)37). The regulators of this subclass are activated by direct interaction with an effector molecule which is normally the substrate for the catabolic pathway the regulators control (29).…”

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An AraC/XylS Family Member at a High Level in a Hierarchy of Regulators for Phenol-Metabolizing Enzymes inComamonas testosteroniR5

et al. 2002

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Comamonas testosteroni strain R5 expresses a higher level of phenol-oxygenating activity than any other bacterial strain so far characterized. The expression of the operon encoding multicomponent phenol hydroxylase (mPH), which is responsible for the phenol-oxygenating activity, is controlled by two transcriptional regulators, PhcS and PhcR, in strain R5. In this study, we identified a third transcriptional regulator for the mPH operon (PhcT) that belongs to the AraC/XylS family. While the disruption of phcT in strain R5 significantly reduced the expression of the mPH operon, it did not eliminate the expression. However, the disruption of phcT in strain R5 increased the expression of phcR. The phenol-oxygenating activity was abolished by the disruption of phcR, indicating that PhcT alone was not sufficient to activate the expression of the mPH operon. The disruption of phcS has been shown in our previous study to confer the ability of strain R5 to express the mPH operon in the absence of the genuine substrate for mPH. PhcT was not involved in the gratuitous expression. Strain R5 thus possesses a more elaborate mechanism for regulating the mPH operon expression than has been found in other bacteria.The expression of a bacterial catabolic pathway for aromatic compounds is often controlled by one or more transcriptional regulatory proteins (21), and sometimes, one transcriptional regulator controls the expression of another transcriptional regulator (24).The expression of multicomponent phenol hydroxylase (mPH) (9,11,12,19,20,36,37) is generally thought to be controlled by a regulator of the XylR/DmpR subclass within the NtrC-type family of transcriptional regulators, resulting in the expression of phenol-metabolizing enzymes only in the presence of the pathway substrate or its structural analog (2,12,14,18,19,22,26,(30)(31)(32)(33)37). The regulators of this subclass are activated by direct interaction with an effector molecule which is normally the substrate for the catabolic pathway the regulators control (29).Comamonas testosteroni R5 has been shown to exhibit an exceptionally high level of activity for phenol oxygenation (42). We have cloned a DNA fragment encoding mPH (phcKLM-NOP) and its cognate transcriptional activator (phcR) of the XylR/DmpR subclass from strain R5 (37). This work (37) and an electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (our unpublished data) indicated that the high activity of strain R5 was due to the high level of mPH expression, leading us to investigate its transcriptional mechanism. PhcR caused the expression of the mPH operon even in the absence of the genuine substrate for mPH, but this gratuitous expression was repressed by a member of the GntR family of transcriptional regulators named PhcS (38). This GntR family member for regulating the mPH operon has also been identified for C. testosteroni TA441. For strain TA441, the regulator named AphS repressed the transcription of the mPH operon even in the presence of phenol, which pre...

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Molecular Analysis of a Plasmid-encoded Phenol Hydroxylase from Pseudomonas CF600

Cited by 70 publications

References 21 publications

Sensing of aromatic compounds by the DmpR transcriptional activator of phenol-catabolizing Pseudomonas sp. strain CF600

Sensing of aromatic compounds by the DmpR transcriptional activator of phenol-catabolizing Pseudomonas sp. strain CF600

Growth phase-dependent transcription of the sigma(54)-dependent Po promoter controlling the Pseudomonas-derived (methyl)phenol dmp operon of pVI150

An AraC/XylS Family Member at a High Level in a Hierarchy of Regulators for Phenol-Metabolizing Enzymes inComamonas testosteroniR5

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