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

Abstract

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

The alarmone (p)ppGpp mediates physiological‐responsive control at the σ54‐dependent Po promoter

“…Previous data had shown that, in LB or minimal media containing casamino acids (CAA) as the sole carbon and energy source, transcription from the Po promoter was delayed until the exponential to stationary phase transition, whereas in minimal media containing glucose or organic acids, the transcriptional response from Po was immediate (Sze et al, 1996). These results suggest that the presence of amino acids in the media may be responsible for the delayed response, causing silencing of DmpR-mediated transcription until they had been depleted.…”

“…However, we have previously eliminated this possibility using a null rpoS mutant E. coli strain (Sze et al, 1996). Another obvious indirect mechanism by which the (p)ppGpp signal could act would be at the level of expression of known positive players in the DmpR-Po regulatory circuit.…”

“…However, the physiologically regulated response from Po can be mimicked in E. coli using plasmid pVI466, which carries dmpR in its native configuration relative to Po-luxAB (see Fig. 3; Sze et al, 1996). Therefore, we turned to E. coli (p)ppGppdeficient strains to address this possibility.…”

“…DmpR expression is constant over the growth curve. However, in rich media, expression of the dmp operon in the presence of effector aromatics is suppressed until the exponential to stationary phase transition (Sze et al, 1996). This level of regulation can be mimicked in both E. coli and Pseudomonas putida by a minimal system comprising the dmpR regulatory gene, the Po promoter and the DmpR binding sites.…”

“…Having achieved their active form, the regulators are thought to activate transcription through a common mechanism involving looping out of the intervening DNA between the regulator-bound upstream activator sites (UAS) and the cognate 54 -RNAP-bound promoter (reviewed by Kustu et al, 1991). In some 54 -dependent systems, including the dmp system used here (Sze et al, 1996), this process has been suggested as being assisted by binding of the DNA bending protein integration host factor (IHF), whereas in others, binding of the HU protein or intrinsic bends have been implicated (see Pé rez-Martin et al, 1994; Pé rez-Martin and de Lorenzo, 1995 and references therein). Holoenzyme RNAP using the 54 factor (E 54 ), unlike Austin and Dixon, 1992; for a review, see Morett and Segovia, 1993).…”