The Crc global regulator inhibits the Pseudomonas putida pWW0 toluene/xylene assimilation pathway by repressing the translation of regulatory and structural genes

Abstract

In Pseudomonas putida, the expression of the pWW0 plasmid genes for the toluene/xylene assimilation pathway (the TOL pathway) is subject to complex regulation in response to environmental and physiological signals. This includes strong inhibition via catabolite repression, elicited by the carbon sources that the cells prefer to hydrocarbons. The Crc protein, a global regulator that controls carbon flow in pseudomonads, has an important role in this inhibition. Crc is a translational repressor that regulates the TOL genes, but how it does this has remained unknown. This study reports that Crc binds to sites located at the translation initiation regions of the mRNAs coding for XylR and XylS, two specific transcription activators of the TOL genes. Unexpectedly, eight additional Crc binding sites were found overlapping the translation initiation sites of genes coding for several enzymes of the pathway, all encoded within two polycistronic mRNAs. Evidence is provided supporting the idea that these sites are functional. This implies that Crc can differentially modulate the expression of particular genes within polycistronic mRNAs. It is proposed that Crc controls TOL genes in two ways. First, Crc inhibits the translation of the XylR and XylS regulators, thereby reducing the transcription of all TOL pathway genes. Second, Crc inhibits the translation of specific structural genes of the pathway, acting mainly on proteins involved in the first steps of toluene assimilation. This ensures a rapid inhibitory response that reduces the expression of the toluene/xylene degradation proteins when preferred carbon sources become available.

FIGURE 1.

Genetic map and transcriptional regulation of the TOL pathway. A, the enzymes encoded in the upper operon sequentially transform toluene or xylenes into benzoate or methylbenzoates, respectively. These are then transformed into acetate and pyruvate by the enzymes encoded in the meta operon. B, transcriptional regulation of the upper and meta operons. Structural genes (in gray), the regulatory genes xylS and xylR (in white), and the Pu, Pm, P_S1, P_S2, P_R1, and P_R2 promoters, are indicated. The activating (+) or repressing (−) effects of XylR and XylS are specified. Activation of Pu and P_S1 by the XylR regulator requires the presence of an effector such as toluene, xylenes, or 3-methylbenzyl alcohol. To activate Pm, XylS must either bind to an aromatic effector (benzoate or methylbenzoates) or be expressed at high levels.

FIGURE 2.

Effect of Crc on the mRNA levels of the TOL pathway genes. Strain KT2442(pWW0) and its isogenic crc-deficient derivative KT2442C(pWW0) were grown in LB medium in the absence or presence of 3MBA. At mid-exponential phase, cells were collected, and the mRNA levels of the indicated genes were determined by real-time reverse transcription-PCR. The bars show the mRNA levels observed in induced cultures (+3MBA) relative to non-induced cultures (−3MBA) for the wild type (wt) strain (dark bars) and for the crc-deficient derivative (light gray bars). The S.E. is indicated.

FIGURE 3.

Influence of Crc on the levels of the XylR transcriptional activator. Strain KT2442(pWW0) and its isogenic crc-deficient derivative KT2442C(pWW0) were grown in LB medium in the absence or presence of 3MBA. At mid-exponential phase, cells were collected, and the total proteins were resolved by SDS-PAGE. Proteins were transferred to a polyvinylidene difluoride membrane, and the presence of the XylR protein was probed with a recombinant antibody. The lower panel shows the gel stained with Coomassie Blue, and the upper panel shows the Western blot. Equal amounts of proteins were loaded in all wells of the PAGE gel. Lane C corresponds to a protein extract of P. putida strain KT2442 lacking the pWW0 plasmid. The size of the proteins used as markers is indicated on the left (M_r). wt, wild type.

FIGURE 4.

Binding of Crc to RNA fragments including the leader regions of the mRNAs originating at the Pu, Pm, P_S1, and P_R1 promoters. The ability of Crc (106, 212, 425, 850, or 1,700 nm) to bind to the indicated radioactively labeled RNA fragments was determined by band-shift assays in the presence of 1 μg of tRNA. The free RNA and the retarded band corresponding to the Crc-RNA complex are indicated as F and C, respectively. The presumed Crc target on each RNA is indicated in boldface. The AUG start codons of xylU (Pu and PuΔ15 RNAs), xylX (Pm RNA), xylS (P_S1 RNA), and xylR (P_R1 RNA) are boxed. The putative Shine-Dalgarno sequences are underlined. The transcription initiation sites corresponding to the P_S2 and P_R2 promoters are indicated by circled letters.

FIGURE 5.

Binding of Crc to RNA oligonucleotides containing the translation initiation regions of internal genes of the upper and meta polycistronic mRNAs. The RNA sequences shown correspond to the translation initiation regions of the indicated genes. The predicted Crc binding site within each gene is shown in boldface. The AUG start codon is shaded. The sequences boxed by a rectangle correspond to the 26-nt RNA oligonucleotides used to test Crc binding in band-shift assays. The ability of Crc (106, 212, 425, 850, or 1,700 nm) to bind to the radioactively labeled RNA oligonucleotides corresponding to the indicated genes was determined by band-shift assays in the presence of 1 μg of tRNA. The free RNA and the retarded band corresponding to the Crc-RNA complex are indicated as F and C, respectively.

FIGURE 6.

Effect of Crc on the activity of key enzymes of the TOL pathway. Strain KT2442(pWW0) and its isogenic crc-deficient derivative KT2442C(pWW0) were grown in LB medium in the presence or absence of 3MBA. At mid-exponential phase, cells were collected and disrupted by sonication. The specific activity of the enzymes BADH (A), BZDH (B), and C23O (C) was determined in the cell extracts as indicated under “Experimental Procedures.” D shows the repression exerted by Crc on the induction of BADH, BZDH, and C23O, expressed as the ratio of enzyme activity observed under induced conditions (+3MBA) for the crc-deficient strain versus that observed for the wild type (wt) strain. mUnits, milliunits; −3MBA, non-induced conditions. The S.E. is indicated.

FIGURE 7.

Crc-mediated regulation of the TOL pathway genes. A, the structural genes (in gray), the regulatory genes xylS and xylR (in white), and the Pu, Pm, P_S1, P_S2, P_R1, and P_R2 promoters of the TOL pathway are shown at the top of the figure. Black arrowheads indicate the direction of transcription. The mRNAs corresponding to the upper and meta operons and to the xylS and xylR genes are indicated by dotted arrows. The Crc binding sites at these mRNAs are indicated by filled arrowheads (strong sites) or open arrowheads (weak sites). The open triangles over promoters Pu, Pm, and P_S1 indicate the indirect repressing effect of Crc on these promoters, derived from the direct effect of Crc on translation of the XylR activator. B, proteins involved in toluene uptake and assimilation. Proteins or genes directly regulated by Crc are shown in boldface, whereas those not directly affected by Crc are indicated in Roman text. XO, xylene monooxygenase; TO, toluate/benzoate dioxygenase; DHCDH, dihydroxycyclohexadiene carboxylate dehydrogenase; AZDH, acetaldehyde dehydrogenase; CW, cell wall.