Cross-Regulation between a Novel Two-Component Signal Transduction System for Catabolism of Toluene in Pseudomonas mendocina and the TodST System from Pseudomonas putida

Abstract

The tmoABCDEF genes encode the toluene-4-monooxygenase from Pseudomonas mendocina KR1. Upstream from the tmoA gene an open reading frame, tmoX, encoding a protein 83% identical to TodX (todX being the initial gene in the todXFC1C2BADEGIH operon from Pseudomonas putida DOT-T1E) was found. The tmoX gene is also the initial gene in the tmoXABCDEF gene cluster. The transcription initiation point from the tmoX promoter was mapped, and the sequence upstream revealed striking identity with the promoter of the tod operon of P. putida. The tod operon is regulated by a two-component signal transduction system encoded by the todST genes. Two novel genes from P. mendocina KR1, tmoST, were rescued by complementation of a P. putida DOT-T1E todST knockout mutant, whose gene products shared about 85% identity with TodS-TodT. We show that transcription from P_tmoX and P_todX can be mediated by TmoS-TmoT or TodS-TodT, in the presence of toluene, revealing cross-regulation between these two catabolic pathways.

The first step in the metabolism of toluene in Pseudomonas mendocina KR1 is carried out by toluene-4-monooxygenase (T4MO), encoded by the tmoABCDEF gene cluster (38, 39), which is responsible for the oxidation of toluene to p-cresol (35, 36). Two other independently regulated gene clusters are known to be required for the metabolism of toluene in P. mendocina KR1 (37). One encodes p-cresol methylhydroxylase and p-hydroxybenzaldehyde dehydrogenase, which catalyze the successive oxidation of the methyl group of p-cresol to the corresponding acid, p-hydroxybenzoate; the other encodes p-hydroxybenzoate hydroxylase for forming protocatechuate, which is channeled to the β-ketoadipate route through an ortho-cleavage pathway (12). Although T4MO activity is known to be inducible by toluene, chlorinated solvents, and alkanes (6, 19), no information regarding the regulators involved in the expression of the tmo genes was available when this work was envisaged. To learn about the transcriptional control of the tmo genes, we first sequenced the region upstream of the tmoABCDEF genes in pMC4 (2) by the dideoxy sequencing method with the ABI Prism dRhodamine terminator kit (Applied Biosystems). A 1,362-nucleotide-long open reading frame 27 bp upstream of tmoA was found, whose deduced amino acid sequence shared significant homology with outer membrane proteins encoded in different toluene degradation operons from a number of gram-negative bacteria that use toluene as the sole C source (i.e., 83% identity with TodX of Pseudomonas putida F1 [34] and DOT-T1E [23] and 48% identity with TbuX of Ralstonia pickettii [13]). The initial gene in the tmo cluster was called tmoX.

Identification of the transcription initiation point of the tmoX promoter and time course induction in response to different effectors.

To analyze the expression of the tmo genes, total RNA was isolated from P. mendocina KR1 grown on citrate in the presence and in the absence of toluene or p-cresol, the substrate and the hydroxylated product of the T4MO, respectively. A 23-mer oligonucleotide (5′-CGGTACTTACTATATCCGGCCCG-3′) complementary to tmoX mRNA was labeled with [γ-³²P]ATP, and T4 polynucleotide kinase was used for primer extension analysis (33). About 10⁵ cpm of the 5′-end-labeled primer was hybridized to 20 μg of total RNA, and primer extensions were done with avian myeloblastosis virus reverse transcriptase as described previously (22). Results are shown in Fig. 1. The basal expression from tmoX was negligible in cells growing on citrate (Fig. 1A), and the level of expression increased about 20-fold in response to toluene supplied in the gas phase 30 min after exposure to the compound; thereafter, the expression level was maintained. With p-cresol as an effector, a high level of expression was found 30 min after addition; from then on the signal decreased significantly, due to exhaustion of p-cresol in the culture medium (data not shown). Similar results as those described above were observed when cells were grown with glucose as the carbon source instead of citrate (data not shown). These results suggested that glucose does not exert any kind of catabolite repression on tmoX gene expression, unlike other catabolic pathways for the degradation of aromatic hydrocarbons that are subject to carbon control (4, 18, 29, 30). The cDNA product resulting from primer extension was 289 bases long, which allowed us to map the transcription initiation point from the tmoX gene at a C located 63 bp upstream from the A of the ATG start codon (Fig. 1B). The sequence around −10 resembled that of the −10 regions recognized by RNA polymerase with sigma-70, yet no region with similarity to a −35 box was recognized. Instead, between −24 and −45 the P_tmoX promoter sequence is rich in A's, which may be related to the flexibility of this DNA segment (24). Further upstream, P_tmoX exhibits a pseudopalindrome located at −100 to −113. Strikingly, this segment was found to be almost identical to the Tod box where TodT binds in the P_todX promoter (16), although the pseudopalindrome in P_tmoX is 1 nucleotide shorter. In fact, the tmoX and todX promoter regions were found to be highly similar (Fig. 1B shows the alignment among the todX promoter regions of P. putida F1 and DOT-T1E strains and the P. mendocina KR1 tmoX promoter). The major difference between the promoters was at positions −24 to −30, where the tmoX promoter showed a T-rich region, whereas the the todX promoter presented CG-rich positions.

FIG. 1.

Identification and sequence characteristics of the P. mendocina tmoX promoter. (A) Time course transcription induction from the tmoX promoter. P. mendocina KR1 cells were grown on 10 mM citrate-supplemented M9 minimal medium at 30°C up to a turbidity at 660 nm of 0.8. The culture was split into nine aliquots of 20 ml. Three of them were kept as a control without any additions. A 1 mM concentration of p-cresol was added to three others, and toluene was supplied via the gas phase to the last three aliquots. Each sample was used to extract total RNA 30, 60, and 180 min after the effector was added, and primer extension was carried out as described previously (33). (B) Alignment of todX and tmoX promoters. The alignment was carried out with the Clustal W 1.8 computer program at the Baylor College of Medicine Human Genome Sequencing Center. The transcription initiation point is indicated by gray-boxed black letters, the −10 region is in gray-boxed white letters, and Shine-Dalgarno sequences (27) are in white-boxed black letters. The first ATG is shown at the end of the sequences, and the pseudopalindromic tod boxes are indicated with arrowheads. Identities are shown by black-boxed white letters.

Isolation and characterization of P. mendocina KR1 tmoST genes.

The similarities between the tmoX and todX promoters prompted us to hypothesize that the expression from P_tmoX could be mediated by a set of regulators similar to the TodS-TodT two-component system that controls expression from P_todX (16, 23). In a first approach, the todST genes of P. putida DOT-T1E in pMIR66 were used as a probe against P. mendocina KR1 and P. putida KT2440 chromosomal DNA. In Southern hybridization assays under low-stringency conditions (50°C without formamide), we found a 5-kb HindIII fragment of P. mendocina that hybridized with the todST probe, whereas with P. putida KT2440 no hybridization band was found (data not shown). We reasoned that the P. mendocina genes homologous to todST could complement a P. putida todST mutant unable to grow on toluene as the sole C source. This mutant was generated by gene replacement of the wild-type todST genes in the chromosome of P. putida DOT-T1E for the todS′::Km::′todT mutation contained in pT1-ST1Km (Table 1) via homologous recombination (26). To test the above hypothesis, we constructed a P. mendocina KR1 gene bank in the pLAFR3 cosmid vector (11, 26). The gene bank was mobilized to the DOT-T1E todST mutant, and a transconjugant able to grow on toluene and resistant to tetracycline was selected. The cosmid isolated from the transconjugant, called pMIR51, showed a 5-kb HindIII hybridization band against the todS and todT gene probes. The 5-kb HindIII fragment was subcloned in pBBR1MCS-5 to generate pMAX47-2. Two open reading frames whose translated products yielded a 973- and a 220-amino-acid-long polypeptide, respectively, were found. Their translated sequences were compared with all the entries in the nonredundant database as described in the BLAST program (1). The proteins showed the highest identity with TodS and TodT, a two-component signal transduction system of distinct P. putida strains (83 and 85% identity, respectively) (accession no. AF180147, Y18245, and U72354). Henceforth, the genes cloned in pMAX47-2 will be called tmoS and tmoT. This plasmid was transferred to the P. putida DOT-T1E todST mutant, which reacquired the ability to grow on toluene as the sole C source. Consequently, based on function and sequence, the P. mendocina KR1 TmoS and TmoT regulators belong to the family of two-component regulatory systems and are able to replace TodS and TodT in activating the tod operon.

TABLE 1.

Bacterial strains or plasmids used in this study^a

Bacterial strain or plasmid(s)	Relevant characteristics	Reference or source
P. putida DOT-T1E	Prototroph, Tol+ (tod pathway)	25
P. putida KT2440	Prototroph, Tol−	10
P. mendocina KR1	Prototroph, Tol+ (tmo pathway)	35
P. putida DOT-T1E todST	DOT-T1E todST-minus derivative, Tol−	This study
pBBR1MCS-5	GmroriTRK2	15
pHP45Ω-Km	Kmr Apr Ω/Km interposon	8
pKNG101	SmrmobRK2 oriR6K sacBR	14
pLAFR3	Tcr, derivative from pLAFR1 cosmid modified to include MCS and the Plac promoter fused to the lac α fragment	11
pMAX47-2	Gmr, 5-kb HindIII fragment of pMIR51 containing tmoST genes inserted in pBBR1MCS-5	This study
pMC4	Smr, P. mendocina KR1 tmo-pcu gene cluster cloned at HindIII/BamHI site in pGV1120	A. A. Gatenby
pMIR32	Apr, 7.5-kb BamHI fragment of pMC4 containing tmoXABCDEF genes inserted in pUC19	This study
pMIR36	Apr, 1.2-kb BamHI/ApaLI fragment of pMIR32 containing tmoX promoter inserted at BamHI/HindIII site of pUC19	This study
pMIR38	TcroriRK2 PtmoX::′lacZ as 0.8-kb KpnI/PstI fragment of pMIR36 inserted in pMP220	This study
pMIR51	Tcr, chimeric cosmid of P. mendocina KR1 library bearing the tmoST genes	This study
pMIR66	Gmr, 4-kb HindIII/BamHI fragment of pT1-155 containing the todST genes inserted in pBBR1MCS-5	This study
pMIR70	Apr, 352-bp-length PCR amplification product containing the todX promoter cloned in pGEM-T	This study
pMIR77	TcroriRK2 PtodX::′lacZ as 0.4-kb EcoRI/PstI insert of pMIR70 inserted in pMP220	This study
pMP220	Tcr ′lacZ oriRK2	28
pT1-125	Apr, ∼16-kb BamHI fragment containing the tod operon inserted in pUC18	23
pT1-155	Apr, 4.3-kb XhoI/HindIII fragment of pT1-125 containing the todST genes inserted at SalI/HindIII site of pUC18	This study
pT1-155STKm	Apr Kmr, deletion of 2.7-kb HpaI/EcoRV fragment of pT1-155 and insertion of Ω/Km cassette from pHP45Ω-Km	This study
pT1-ST1Km	Smr KmrtodS′::Km::′todT as 3.8-kb BamHI fragment of T1-155STKm inserted in pKNG101	This study
pUC18, pUC19	Apr, MCS, Plac fused to the α peptide of lacZ	32

^aTol⁺ Tol⁻, growth in toluene as the sole C source and unable to grow in toluene, respectively. Antibiotics were supplied at the indicated concentrations (micrograms per milliliter): ampicillin (Ap), 100; chloramphenicol (Cm), 30; gentamicin (Gm), 10 for Escherichia coli and 100 for Pseudomonas sp. strains; kanamycin (Km), 25; streptomycin (Sm), 50; and tetracycline (Tc), 10. E. coli strains were usually grown at 37°C in LB medium, whereas Pseudomonas sp. strains were grown at 30°C in LB medium or in M9 minimal medium with glucose (25 mM) or citrate (10 mM) as the C source, as previously described (23). MCS, multiple cloning site.

Expression of the transcriptional fusions P_tmoX::′lacZ and P_todX::′lacZ in Pseudomonas.

Transcriptional fusions of the tmoX and todX promoters were constructed by using the wide-range pMP220 reporter plasmid based on ′lacZ (28). For the generation of P_tmoX::′lacZ, the 0.8-kb KpnI-PstI fragment of pMIR36 containing the tmoX promoter was fused to ′lacZ, yielding plasmid pMIR38. This plasmid was introduced by electrotransformation (7) into different Pseudomonas sp. hosts to study the expression of the tmoX promoter in different genetic backgrounds, i.e., P. mendocina KR1 (tmoST⁺), P. putida DOT-T1E (todST⁺), and hosts deficient in both TmoS-TmoT and TodS-TodT such as P. putida KT2440 and P. putida DOT-T1E todST. β-Galactosidase activities were measured in P. mendocina KR1 cells growing in Luria-Bertani (LB) medium in the absence and in the presence of toluene. We found that throughout the growth curve the expression level from the P_tmoX promoter was negligible in the absence of toluene (Fig. 2); in contrast in the presence of the aromatic hydrocarbon β-galactosidase activity increased steadily up to 800 U after 12 h of incubation (Fig. 2). The delay of about 4 h observed in the induction could be a consequence of several circumstances, e.g., the limiting amount of regulators under these experimental conditions, since the delay was shortened as the regulators were supplied in pMAX47-2 (data not shown). As expected, no activity was reported from the tmoX promoter in P. putida KT2440 and the P. putida todST mutant strain, since these strains lack a homologue to the todST and tmoST genes (data not shown). As mentioned above, plasmid pMAX47-2 bearing the tmoST genes was able to confer the ability to grow on toluene on the P. putida DOT-T1E todST mutant strain. We therefore expected this plasmid to restore inducibility from the P_tmoX promoter. To test this hypothesis, β-galactosidase activity was determined in P. putida DOT-T1E todST(pMIR38)(pMAX-47-2) both in the absence and in the presence of toluene. High levels of β-galactosidase activity were detected (>5,000 U in the exponential growth phase) with toluene (Table 2). Similar results were obtained with P. putida KT2440 as the host for plasmids pMIR38 and pMAX47-2.

FIG. 2.

Expression from the tmoX promoter in P. mendocina KR1. Bacteria cells were electrotransformed with plasmid pMIR38 (P_tmoX::′lacZ). P. mendocina KR1(pMIR38) was grown overnight on LB medium supplied with tetracycline, and cells were diluted 100-fold in the same medium with toluene in the gas phase (circles) or without toluene (triangles). Growth was determined as turbidity of the culture (open symbols), and specific β-galactosidase activities (Miller units) (solid symbols) were monitored (21). Cells had been permeabilized with mixed alkyltrimethylammonium bromide at 4°C for 20 min before the β-galactosidase assays were done. Experiments were repeated with three cultures, and results from a typical experiment from a single culture are presented.

TABLE 2.

β-Galactosidase activities expressed from P_tmoX::′lacZ and P_todX::′lacZ in P. mendocina KR1 and P. putida strains^a

Strain	Plasmid(s)	β-Gal activity (Miller units)
		LB		M9 + glucose
		− Toluene	+ Toluene	− Toluene	+ Toluene
KR1	pMIR38	1	600	1	1,330
DOT-T1E	pMIR77	ND	ND	1	590
DOT-T1E todST	pMIR38, pMAX47-2	5	5,350	2	2,550
DOT-T1E todST	pMIR38, pMIR66	4	4,110	4	3,880
DOT-T1E todST	pMIR77, pMAX47-2	4	4,710	3	2,600
DOT-T1E todST	pMIR77, pMIR66	4	4,550	2	2,250

^aPlasmids pMIR38 and pMIR77 contain the P_tmoX::′lacZ and P_todX::′lacZ transcriptional fusions, respectively; while plasmids pMAX47-2 and pMIR66 encode the TmoS-TmoT and TodS-TodT proteins, respectively. Cultures were grown to the mid-exponential growth phase on LB or minimal M9 medium supplemented with 25 mM glucose without toluene or with 3 mM toluene. β-Galactosidase (β-Gal) activity was determined as described in the legend to Fig. 2. Strains KR1 and DOT-T1E bearing the vector plasmids pMP220 and pBBR1MCS-5 showed no activity; strain DOT-T1E todST showed no activity with plasmid pMIR38 or pMIR77, as expected. ND, not determined.

We also tested whether the todST genes induced expression from P_tmoX. This assay was done with the P. putida DOT-T1E todST mutant strain bearing pMIR38 and pMIR66. As above, β-galactosidase activity was high when the cells were grown in the presence of toluene, with levels above 4,000 U, whereas in the absence of the aromatic hydrocarbon the activity was negligible. The assays that we reported were also done with the same strains grown on M9 minimal medium with glucose as the C source, both in the absence and in the presence of toluene. In the presence of toluene, the level of β-galactosidase from the P_tmoX::′lacZ promoter increased almost 1,000-fold (Table 2), while in the absence of the effector expression was negligible. The above results clearly show that expression from P_tmoX can be efficiently induced by either the TmoS-TmoT or the TodS-TodT pair of regulators.

To generate the P_todX::′lacZ fusion, the promoter region was amplified by PCR with plasmid pT1-4 as a template (23) and the following two primers: 5′-CGGAATTCGGTCTGAGGTTTTCATCGAC-3′ and 5′-CGGGATCCAATTACAATCCTTCCACATCTTA-3′. The 352-bp PCR product was cloned in pGEM-T (Promega) to produce pMIR70. The EcoRI-PstI fragment containing the todX promoter was fused to ′lacZ at the same sites in the polylinker of pMP220, resulting in plasmid pMIR77. β-Galactosidase activity was measured in P. putida DOT-T1E(pMIR77) growing on M9 minimal medium with glucose as the C source in both the presence and the absence of toluene, as described above. In the absence of toluene, the expression level from the P_todX promoter was insignificant, but in its presence activity was almost 600 U. As expected, in the P. putida DOT-T1E todST(pMIR77) mutant strain expression from P_todX was negligible (data not shown); however, when the strain was transformed with pMIR66 the expression from P_todX was significantly enhanced in response to the presence of toluene (Table 2). The induction level was about 1,000-fold both in LB rich medium and in M9 minimal medium. Results similar to those reported above were found in this host background when the tmoST genes in plasmid pMAX47-2 were the regulators instead of the todST genes (Table 2). Therefore, this result also indicates that the P_todX promoter can be induced by the regulators TmoS-TmoT of the T4MO pathway besides its own set of regulators, TodS-TodT.

Cross-regulation in catabolic pathways for aromatic compounds.

The above set of results established a case of cross-activation in the two-component signal transduction system that controls the expression of two different catabolic pathways for toluene metabolism. These results can be interpreted as evidence that the same regulatory system was recruited independently to control two different catabolic pathways for the aerobic degradation of toluene. In Pseudomonas sp. strain Y2 the pathway for styrene degradation is under the control of the StyS and StyR proteins (31), which are 85% identical to the TodS and TodT-TmoS and TmoT proteins. Given that TodS also recognizes styrene as an effector and that, upon activation of the TodT protein by TodS with styrene, transcription from the P_todX promoter takes place (23), it seems that this kind of regulator has been recruited on several occasions to control different catabolic pathways. Although this work is, to our knowledge, the first communication of two-component system-based regulators that are interchangeable in the control of catabolic pathways for hydrocarbon degradation, it should be mentioned that Lehay et al. (17) demonstrated cross-regulation of TmbR and TbuT with the benzene monooxygenase and toluene-2-monooxygenase operons from two Burkholderia species. Along the same lines, Fernández et al. (9) demonstrated cross-activation of the xylene monooxygenase and phenol hydroxylase pathways of P. putida by the XylR and DmpR regulators. In the two latter cases, the corresponding regulators belong to the NtrC family of positive transcriptional regulators (3). Therefore, it seems that the same sets of regulators have been recruited several times to control different catabolic pathways, perhaps because the diversity required for the regulators to perform their functions was less strict.

pcu/pobA and tmoST genes form a cluster in P. mendocina KR1.

The oxidation of toluene to Krebs cycle intermediates in P. putida DOT-T1E and F1 by the Tod pathway is effected by a set of enzymes all of which are expressed as a single transcriptional unit, as surmised from the fact that the genes that encode toluene dioxygenase and the subsequent meta-cleavage of 3-methylcatechol are linked (16, 20, 23). The todST genes are found 212 nucleotides downstream of the catabolic operon. These genes encode the regulators (20, 23), indicating that the catabolic and regulatory operons are closely clustered. In contrast, in P. mendocina KR1 the tmoST genes are not located in the vicinity of the tmoXABCDEF gene cluster encoding T4MO that oxidizes toluene into p-cresol. The metabolism of p-cresol in P. mendocina requires at least two gene clusters: the pcu gene products, responsible for the oxidation of p-cresol into p-hydroxybenzoate (Fig. 3), and the pobA and pobR genes, the former encoding p-hydroxybenzoate hydroxylase and the latter encoding its regulator (2). When we analyzed the DNA sequences surrounding tmoST (Fig. 3), we found that the pobA1R1 genes were adjacent to tmoST. The ATG start codon of tmoS and the stop codon of pobA1 are separated by 714 bp. The pobR1 and pobA1 genes are transcribed divergently, as in Acinetobacter species (5), and the pcu genes for the utilization of p-cresol are flanking pobR1. The pcu gene cluster contains five genes, pcuR and pcuCAXB (2). pcuR encodes the regulator of the pcuCBXA genes (M. Olson and A. A. Gatenby, unpublished results). Therefore, it seems that to date the metabolism of toluene to protocatechuate in P. mendocina KR1 involves two sets of clusters, tmoXABCDEF on the one hand and pcu, pobRA, and tmoST on the other. The nature of the gene products in the region between the tmo and the pcu genes is unknown at present.

FIG. 3.

Oxidation of toluene to protocatechuate by P. mendocina KR1 and scheme of the cluster of tmo/pcu/pob genes needed for this biotransformation in this strain. tmoXABCDEF, T4MO genes; c, putative cytochrome c gene; pcuRCAXB, p-cresol utilization genes; pobR1 and pobA1, regulator and p-hydroxybenzoate hydroxylase, respectively; tmoST, two-component signal transduction system; p-HBOH, p-hydroxybenzylalcohol; p-HBHO, p-hydroxybenzylaldehyde; p-HBA, p-hydroxybenzoate; PCA, protocatechuate.

In short, we show that transcription of the P_tmoX promoter that directs the synthesis of the tmoXABCDEF gene cluster for T4MO is induced by a two-component system made up of TmoS and TmoT, homologous to TodS and TodT, respectively. The tmoS and tmoT genes are not linked to the tmoXABCDEF genes but to a set of genes required for the metabolism of p-cresol, the product resulting from toluene oxidation by T4MO.

Nucleotide sequence accession number. The nucleotide sequence of the tmoX gene was deposited in the GenBank database under accession number AF506285. The sequence of the pMAX47-2 insert was deposited in the GenBank database under accession number AY052500.