In vivo and in vitro effects of (p)ppGpp on the sigma(54) promoter Pu of the TOL plasmid of Pseudomonas putida

Abstract

The connection between the physiological control of the sigma(54)-dependent Pu promoter of the TOL plasmid pWW0 of Pseudomonas putida and the stringent response mediated by the alarmone (p)ppGpp has been examined in vivo an in vitro. To this end, the key regulatory elements of the system were faithfully reproduced in an Escherichia coli strain and assayed as lacZ fusions in various genetic backgrounds lacking (p)ppGpp or overexpressing relA. Neither the responsiveness of Pu to 3-methyl benzylalcohol mediated by its cognate activator XylR nor the down-regulation of the promoter by rapid growth were affected in relA/spoT strains to an extent which could account for the known physiological control that governs this promoter. Overexpression of the relA gene [predicted to increase intracellullar (p)ppGpp levels] did, however, cause a significant gain in Pu activity. Since such a gain might be the result of indirect effects, we resorted to an in vitro transcription system to assay directly the effect of ppGpp on the transcriptional machinery. Although we did observe a significant increase in Pu performance through a range of sigma(54)-RNAP concentrations, such an increase never exceeded twofold. The difference between these results and the behavior of the related Po promoter of the phenol degradation plasmid pVI150 could be traced to the different promoter sequences, which may dictate the type of metabolic signals recruited for the physiological control of sigma(54)-systems.

FIG. 1

Physiological control of the Pu promoter of P. putida reproduced in an E. coli host. E. coli strain CC118 harboring plasmid pMCP1 (xylR⁺/Pu-lacZ), which encodes the wild-type XylR protein, was grown at 30°C in LB rich medium (A), M9 minimal medium with 0.2% succinate (panel B), or M9-succinate medium with 0.2% Casamino Acids (C). At an early growth stage, the cultures were exposed to saturating vapors of the XylR effector 3-methyl benzylalcohol (3mBA), and the accumulation of β-galactosidase activity (expressed in Miller units) was monitored during subsequent growth. The enzymatic activities shown are the average of duplicate samples as explained in the text. Note the repressive effect of Casamino Acids during exponential growth. The organization of the reporter system encoded by pMCP2 is sketched (not to scale) at the top of the figure.

FIG. 2

Pu performance in vivo in the absence of (p)ppGpp or with an excess of the alarmone. (A) The wild-type strain E. coli MG1655 and its ppGpp⁰ isogenic derivative E. coli CF1693 (relA spoT) harboring the reporter plasmid pMCP2 (xylRΔA⁺/Pu-lacZ) were grown in LB medium at 30°C and assayed for β-galactosidase activity (expressed in Miller units). The growth rates of the two strains were indistinguishable. There was a moderate decrease in accumulation of the reporter product in the relA spoT host. The reporter system encoded by pMCP2, bearing the constitutive xylR alele xylRΔA, is sketched at the top of the figure. (B) The wild-type strain E. coli MG1655 was cotransformed with pMCP2 (xylRΔA⁺/Pu-lacZ) and pCNB0209R (expressing a His-tagged RelA product under the control of a lacI/Plac system) (see the text). The cotransformant was grown in LB medium, and the accumulation of β-galactosidase was monitored throughout growth after the addition of 0.1 mM IPTG. The expression levels of the RelA product were revealed by the protein blot, shown at the top of the figure, which was probed with an anti-poly His monoclonal antibody (the last lane corresponds to advanced stationary-phase cells not plotted in the graph). The significant increase in Pu activity upon relA overexpression is evident.

FIG. 3

Effect of ppGpp on the transcriptional activity of Pu in vitro. (A) Results of multiple-round transcription reactions with 5 nM Pu-containing template pEZ10, 100 nM XylRΔA, 25 nM IHF, 25 nM core RNAP, 100 nM ς⁵⁴, and increasing concentrations of purified ppGpp as indicated. Under these conditions, pEZ10 produces an mRNA of 394 nucleotides. The levels of transcript found in each case are plotted below the autoradiograph of the gel. The baseline value starts at 50 arbitrary units. (B) Control with the placUV5 promoter. The transcription reaction mixtures contained 5 nM supercoiled pTE103-PlacUV5 template mixed with 25 nM E. coli ς⁷⁰ RNAP holoenzyme and different amounts of ppGpp. In this system, PlacUV5 produces an mRNA of 313 nucleotides.

FIG. 4

Comparison of the effects of the ppGpp on the transcriptional activity of Pu and Po in vitro. The result of a typical multiple-round transcription experiment run in parallel with 5 nM Pu-containing template pEZ10 or Po-containing template pTE103-Po is shown at the top of the figure. Assays were carried out under standard conditions (100 nM XylRΔA, 25 nM IHF, 25 nM core RNAP, 100 nM ς⁵⁴), and 350 nM purified ppGpp was added or omitted as indicated. Note the different sizes of the transcripts arising from Pu (394 nucleotides) or Po (311 nucleotides) due to the different distances from the promoter to the T7 terminator in the vector plasmid (see Materials and Methods). The average stimulation of Pu and Po activity by ppGpp on the basis of four independent experiments is represented below the autoradiograph.

FIG. 5

Effect of ppGpp on activation of Pu with increasing concentrations of ς⁵⁴-RNAP. The upper part of the figure shows the result of multiple-round transcription reactions containing 5 nM Pu-containing plasmid template pEZ10. To this was added 100 nM XylRΔA, 25 nM IHF, and increasing concentrations of ς⁵⁴-RNAP with or without 300 nM ppGpp as indicated. The intensity of the bands was quantified in a Bio-Rad Molecular Imager FX system and plotted in arbitrary units as a function of RNAP concentration (shown in the lower part of the figure). The effect of ppGpp on the transcriptional output appears to be constant throughout the entire range of enzyme concentrations used, and therefore it does not appear to stimulate binding of the promoter by the ς⁵⁴-RNAP (see the text for an explanation).