Role of the ptsN gene product in catabolite repression of the Pseudomonas putida TOL toluene degradation pathway in chemostat cultures

Abstract

The Pseudomonas putida KT2440 TOL upper pathway is repressed under nonlimiting conditions in cells growing in chemostat with succinate as a carbon source. We show that the ptsN gene product IIA(Ntr) participates in this repression. Crc, involved in yeast extract-dependent repression in batch cultures, did not influence expression when cells were growing in a chemostat with succinate at maximum rate.

FIG. 1.

pWW0 TOL plasmid upper-pathway regulation. The diagram shows stimulation by the XylR protein of the two TOL pathway σ⁵⁴-dependent promoters, Pu and P_S1. The TOL upper-pathway and regulatory genes xylS and xylR are shown as gray arrows. R is any substituent group in the aromatic ring. Elliptical and square boxes indicate the inactive and active forms of XylR, respectively. Plus and minus signs indicate transcription activation and repression by XylR, respectively. Black small boxes represent XylR binding sites to activate Pu and P_S1 (UASs). Promoters are outlined as flags.

FIG. 2.

Repression levels of Pu and P_S1 promoters from P. putida (pWW0) and its ptsN and crc mutants. Cells were grown in continuous culture, at D = 0.05 h⁻¹ (carbon limitation) and D = 0.85 h⁻¹ (carbon excess), when o-xylene was added in the gas phase during 5 min. The presence of messengers was analyzed using reverse primer extension of equal amounts (10 or 20 μg) of total RNA and the corresponding labeled specific oligonucleotide (2). Samples were run in urea sequencing gels, which were exposed to a phosphor screen (Fuji Photo Film Co. Ltd.) for 5 to 12 h. Phosphor screens were scanned with a phosphorimaging instrument (Molecular Imager FX; Bio-Rad), and data were quantified with Quantity One software (Bio-Rad). The graph shows the mRNA levels of Pu (A) and P_S1 (B) promoters in the three strains at D = 0.85 h⁻¹ (repression conditions) as a percentage of the levels observed in each strain at a low growth rate (D = 0.05 h⁻¹, full induction levels). (C) Representative urea-polyacrylamide gel electrophoresis results corresponding to mRNA-derived cDNA from the two promoters at each growth rate are shown. wt, wild type.

FIG. 3.

Level of mRNA derived from the P_R1 and P_R2 promoters from P. putida (pWW0) and its ptsN and crc mutants grown in continuous culture in response to o-xylene at D = 0.05 h⁻¹ (carbon limitation) and D = 0.85 h⁻¹ (carbon excess). The presence of messengers was analyzed by reverse primer extension of equal amounts (10 or 20 μg) of total RNA with the corresponding labeled specific oligonucleotide (2). Samples were run in urea sequencing gels, which were exposed to a phosphor screen (Fuji Photo Film Co. Ltd.) for 5 to 12 h. Phosphor screens were scanned with a phosphorimaging instrument (Molecular Imager FX; Bio-Rad). Data were quantified with Quantity One software (Bio-Rad). (A) The diagram at the top of the figure represents the xylR/xylS intergenic region (15). (B) The graph shows the mRNA levels of P_R1 and P_R2 promoters in the three strains at D = 0.85 h⁻¹ as a percentage of the levels observed in each strain at a low growth rate (D = 0.05 h⁻¹). (C) Representative urea-polyacrylamide gel electrophoresis results corresponding to mRNA-derived cDNA from the two promoters at each growth rate are shown. wt, wild type.

FIG. 4.

Effect of glucose on Pu expression. β-galactosidase activity (in Miller units [MU]) was measured in o-xylene-induced batch cultures of P. putida strain KT2440 and its ptsN and crc mutants carrying the pS10 plasmid with Pu fused to the lacZ gene and the regulator gene xylR (2) in the presence or absence of glucose.