Integration of signals through Crc and PtsN in catabolite repression of Pseudomonas putida TOL plasmid pWW0

Abstract

Toluene degradation in Pseudomonas putida KT2440 pWW0 plasmid is subjected to catabolite repression. Pu and P(S1) promoters of the pWW0 TOL plasmid are down-regulated in vivo during exponential growth in rich medium. In cells growing on minimal medium, yeast extract (YE) addition mimics exponential-phase rich medium repression of these promoters. We have constructed and tested mutants in a series of global regulators described in Pseudomonas. We describe that a mutant in crc (catabolite repression control) partially relieves YE repression. Macroarray experiments show that crc transcription is strongly increased in the presence of YE, inversely correlated with TOL pathway expression. On the other hand, we have found that induced levels of expression from Pu and P(S) in the presence of YE are partially derepressed in a ptsN mutant of P. putida. PtsN but not Crc seems to directly interfere with XylR activation at target promoters. The effect of the double mutation in ptsN and crc is not the sum of the effects of each independent mutation and suggests that both regulators are elements of a common regulatory pathway. Basal expression levels from these promoters in the absence of inducer are still XylR dependent and are also repressed in the presence of yeast extract. Neither crc nor ptsN could relieve this repression.

FIG. 1.

TOL pathway regulatory network. The thick horizontal line depicts the TOL region including the upper and meta-cleavage pathways and the two regulatory genes XylR and XylS. Elliptical boxes indicate the inactive form of the regulatory proteins. Rectangular boxes indicate the active form of the regulatory proteins. Thin lines represent the connections between regulatory proteins and promoters, where a plus sign indicates transcription activation and a minus sign indicates inhibition of transcription. The dotted line indicates transcription activation of overproduced XylS in the absence of effector. The sigma factor(s) involved in transcription initiation is indicated above each promoter. Aromatic substrates of the pathways acting as effectors of the regulatory proteins are indicated. The regulatory circuits are explained in the text. R indicates possible substituent groups of the ring.

FIG. 2.

Yeast-extract-mediated repression of mRNA expression from Pu (A) and P_S1 (B) promoters in P. putida KT2440 (pWW0). Cells grown overnight in M9 minimal medium with glucose as the carbon source were diluted to an optical density at 660 nm of 0.2. When the cultures reached exponential growth, they were divided into four fractions, three of which were supplemented with o-xylene (o-xyl), 1% (wt/vol) yeast extract, or both. The fourth fraction was left unsupplemented as a control. Cultures were incubated at 30°C for 30 min, and samples were collected for primer extension mRNA analysis. cDNA bands corresponding to each promoter (134 nucleotides for Pu and 206 for P_S1 [bottom]) were quantified and compared.

FIG. 3.

Yeast-extract-mediated repression of mRNA expression from Pu (A) and P_S1 (B) promoters in mutants P. putida KT2440/ptsN (pWW0), P. putida KT2440/crc (pWW0), and P. putida KT2440/ptsNcrc (pWW0) compared to the control strain P. putida KT2440 (pWW0). Cell growth, sampling, and analysis done were as described in the legend to Fig. 2.

FIG. 4.

Expression from P_R1 (A and C) and P_R2 (B and D) promoters in mutants P. putida KT2440/ptsN (pWW0) and P. putida KT2440/crc (pWW0) compared to the control strain P. putida KT2440 (pWW0) in the presence (+) or absence (−) of effector (o-xylene [o-xyl]) and repressor agent (yeast extract). Cell growth, sampling, and analysis were done as described in the legend to Fig. 2, except that oligonucleotides complementary to xylR were used. cDNA bands corresponding to each P_R promoter (208 n for P_R1 and 180 n for P_R2 [bottom]) were quantified and compared. IHF, integration host factor; wt, wild type.

FIG. 5.

Effect of the presence of yeast extract on the mRNA levels of genes involved in the control of the expression in TOL plasmid catabolic operons. Genes in the DNA macroarray used to determine the mRNA levels are displayed in the panels. P. putida KT2440 (pWW0) cells were grown on M9 minimal medium with glucose as the carbon source. When the cultures reached exponential growth, they were divided into two fractions, both of them supplemented with o-xylene in the gas phase, and to one of them, 1% (wt/vol) yeast extract was added. Cultures were incubated at 30°C for 30 min, and samples were collected for mRNA isolation. Radioactively labeled cDNA was synthesized and hybridized to the DNA macroarray as described in Materials and Methods. IHF, integration host factor.