Cloning and Characterization of linR, Involved in Regulation of the Downstream Pathway for {gamma}-Hexachlorocyclohexane Degradation in Sphingomonas paucimobilis UT26

In Sphingomonas paucimobilis UT26, LinD and LinE activities,which are responsible for the degradation of ${gamma}$ -hexachlorocyclohexane,are inducibly expressed in the presence of their substrates,2,5-dichlorohydroquinone (2,5-DCHQ) and chlorohydroquinone (CHQ).The nucleotide sequence of the 1-kb upstream region of the linEgene was determined, and an open reading frame (ORF) was foundin divergent orientation from linE. Because the putative proteinproduct of the ORF showed similarity to the LysR-type transcriptionalregulator (LTTR) family, we named it linR. The fragment containingthe putative LTTR recognition sequence (a palindromic TN₁₁Asequence), which exists immediately upstream of linE, was ligatedwith the reporter gene lacZ and was inserted into the plasmidexpressing LinR under the control of the lac promoter. Whenthe resultant plasmid was introduced into Escherichia coli,the LacZ activity rose in the presence of 2,5-DCHQ and CHQ.RNA slot blot analysis for the total RNAs of UT26 and UT102,which has an insertional mutation in linR, revealed that theexpression of the linD and linE genes was induced in the presenceof 2,5-DCHQ, CHQ, and hydroquinone in UT26 but not in UT102.These results indicated that the linR gene is directly involvedin the inducible expression of the linD and linE genes.

INTRODUCTION

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Introduction

${gamma}$ -Hexachlorocyclohexane ( ${gamma}$ -HCH; also called ${gamma}$ -BHC or lindane) isa haloorganic insecticide which has been used worldwide. Sphingomonaspaucimobilis UT26 can utilize ${gamma}$ -HCH as a sole source of carbonand energy (4). The degradation pathway of ${gamma}$ -HCH in UT26 consistsof upstream and downstream pathways, which are shown in Fig.1 (11). In the upstream pathway, ${gamma}$ -HCH is transformed to 2,5-dichlorohydroquinone(2,5-DCHQ), which is further degraded in the downstream pathway.In the previous studies, we isolated five genes (linA, linB,linC, linD, and linE) involved in these pathways and characterizedtheir protein products (3, 7, 8, 12, 13). The linA, linB, andlinC genes, for the upstream pathway, exist separately on theUT26 genome and are constitutively expressed (4, 12, 13). Onthe other hand, the linD and linE genes, for the downstreampathway, are located near each other and are inducibly expressedin the presence of their substrates (7, 8). They may constitutean operon (7). LinE is a novel meta-cleavage dioxygenase whichcleaves aromatic rings with two hydroxy groups at para positions.We also demonstrated that PcpA from a pentachlorophenol-degradingbacterium, Sphingomonas chlorophenolica ATCC 39723, has activitysimilar to that of LinE (14). These results directly demonstrateda new type of ring cleavage pathway for aromatic compounds,the "hydroquinone (HQ) pathway." Although reports of the HQpathway have been limited, we consider it one of the major degradationpathways for aromatic compounds whose regulation system forgene expression has been established. However, the regulationsystem for the genes of the HQ pathway in UT26 is still unknown.In this study, we cloned and characterized a regulatory geneencoding a transcriptional regulator for linD and linE.

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FIG. 1. Proposed assimilation pathway of ${gamma}$ -HCH in S. paucimobilis UT26. Compounds: 1, ${gamma}$ -HCH; 2, ${gamma}$ -pentachlorocyclohexene; 3, 1,3,4,6-tetrachloro-1,4-cyclohexadiene; 4, 1,2,4-trichlorobenzene; 5, 2,4,5-trichloro-2,5-cyclohexadiene-1-ol; 6, 2,5-dichlorophenol; 7, 2,5-dichloro-2,5-cyclohexadiene-1,4-diol; 8, 2,5-DCHQ; 9, CHQ; 10, HQ; 11, acylchloride; 12, ${gamma}$ -hydroxymuconic semialdehyde; 13, maleylacetate; 14, ß-ketoadipate.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains, plasmids, and culture conditions.
The bacterial strains and plasmids used in this study are listedin Table 1. Sphingomonas strains and Escherichia coli were grownon Luria broth (6). The cultures were incubated at 30°Cfor Sphingomonas strains and at 37°C for E. coli. Antibioticswere used at final concentrations of 50 µg/ml for ampicillinand kanamycin and 25 µg/ml for nalidixic acid.

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TABLE 1. Bacterial strains and plasmids used in this study

Isolation of DNA.
Plasmid DNA of E. coli was isolated by the alkaline lysis methodof Maniatis et al. (6). Total DNAs from Sphingomonas strainswere isolated as described previously (9).

Southern blot analysis.
Southern blot analysis was performed with the ECL (enhancedchemiluminescence) gene detection system (Amersham, ArlingtonHeights, Ill.) according to the protocol provided.

Nucleotide sequence determination.
The nucleotide sequences of a 1.0-kb PstI-SmaI fragment of plasmidpLR1 and the fragments amplified by PCR were determined by thedideoxy chain termination method with the LI-COR (Lincoln, Neb.)model 4000L DNA-sequencing system.

Construction of plasmids for promoter activity assay.
Various lengths of the region upstream of linE followed by the5' end of the linE gene were amplified by PCR and fused to thelacZ gene in the same frame on the plasmid pMC1403 (18) to formplasmids pMEU1, pMEU2, and pMEU3. The primers used to amplifythe region upstream of linE are LEUP-1 (5'-GCCGAATTCTCGTGCAGCGGCGCTGA-3'),LEUP-2 (5'-GCGGGATCCAGTTGCATCATGATCGCTC-3'), LEUP-3 (5'-GCCGAATTCTATATTCACAATCTG-3'),and LEUP-4 (5'-GCCGAATTCTATGAAGGTCCGC-3'). To amplify the fragmentsfor pMEU1, pMEU2, and pMEU3, primers LEUP-4, LEUP-3, and LEUP-2,respectively, were used in combination with LEUP-1. The resultantplasmids can express the LinE-LacZ fusion protein if the upstreamregion has promoter activity. To form pMEU1R, pMEU2R, and pMEU3R,the PvuII-PvuII fragment containing P_lac-linR was isolated frompLR1 and inserted into the BalI site of each plasmid. To makethe BalI sites easily digested, each plasmid was purified fromE. coli JM110 (dcm mutant).

ß-Galactosidase assay.
E. coli harboring each plasmid was incubated with isopropyl-ß-D-thiogalactopyranoside(1 mM). The culture was divided, and each substrate was added(10 µM final concentration). After incubation for 2 h,the cells were harvested and resuspended in 1 ml of Z buffer(60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, 50 mM2-mercaptoethanol). The cells were disrupted by sonication (Sonifier250; Branson, Danbury, Conn.) and centrifuged at 12,000 x g.The supernatant was used for the measurement of ß-galactosidaseactivity. The ß-galactosidase activity of the cellextract was measured as follows. One hundred microliters of13.3 mM o-nitrophenyl-ß-D-galactopyranoside was addedto 400 µl of cell extract. After incubation at 30°C,250 µl of Na₂CO₃ solution (1 M) was added to stop thereaction, and the absorbance at 420 nm was measured. The proteinconcentration of the cell extract was determined by using aprotein assay kit (Bio-Rad Laboratories, Richmond, Calif.) withbovine serum albumin as a standard. One unit of ß-galactosidaseactivity was defined as the amount of enzyme that would hydrolyze1 nmol of o-nitrophenyl-ß-D-galactopyranoside in 1min at 30°C.

RNA slot blot analysis.
Total RNAs of UT26 and UT102 were isolated as described previously(8). 2,5-DCHQ, chlorohydroquinone (CHQ), and HQ were used asinducers at final concentrations of 10 µM. Six microgramsof each RNA was blotted onto the nylon membrane with a PR600slot blot manifold (Amersham, Arlington Heights, Ill.). Northernblot hybridization was performed as described previously (8).

Nucleotide sequence accession number.
The nucleotide sequence data reported in this paper were registeredin the DDBJ nucleotide sequence database under accession no.AB021863.

RESULTS AND DISCUSSION

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Results and Discussion

Cloning and sequencing of the linR gene.
S. paucimobilis UT26 inducibly expresses LinD and LinE activitiesin the presence of 2,5-DCHQ, substrate for LinD (8) and CHQ,substrate for both LinD and LinE (7, 8). On the other hand,UT102, which is one of the Tn5-induced mutants of UT26, showsfaint LinD and LinE activities with or without their substrates(7, 10). It can be considered that this phenotype of UT102 iscaused by a mutation in a regulatory gene for the expressionof linD and linE. As UT102 has an insertional mutation otherthan Tn5 in the upstream region of the linE gene (K. Miyauchi,Y. Nagata, and M. Takagi, unpublished data), we determined thenucleotide sequence of this region. The nucleotide sequenceof a 1.0-kb PstI-SmaI fragment was determined, and we foundan open reading frame (ORF) of 909 bp in this region (Fig. 2).Because this ORF showed similarity to regulatory proteins (seebelow) and UT102 has an insertion sequence in the N terminusof the ORF (Fig. 2) (Miyauchi et al., unpublished), we designatedthe ORF linR. The linR gene appears to be divergently transcribedfrom linE and to use GTG as a start codon. The G+C content ofthe linR gene is 61.3%, which is similar to those of other lingenes (7, 8, 12, 13) except for linA (53.9%) (3). The deducedmolecular mass of LinR is 33.6 kDa.

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FIG. 2. Restriction map of linR and its flanking region. The arrow indicates the position of the insertion mutation in UT102. Abbreviations for restriction enzymes: B, BamHI; E, EcoRI; P, PstI; S, SacI; Sm, SmaI.

Homology search analysis of LinR.
A FASTA homology search revealed that LinR shows similarityto LysR-type transcriptional regulators (LTTRs) (16). The proteinswhich showed high similarity to LinR are as follows: OhbR (29%identity; 69% similarity), a LysR-like protein in an operonfor ortho-halobenzoate degradation in Pseudomonas aeruginosastrain JB2 (AF087482); NahR (31% identity; 68% similarity),a regulatory protein for naphthalene degradation genes in plasmidNAH7 of Pseudomonas putida (J04233); NagR (32% identity; 66%similarity), the regulator of the nag operon in Ralstonia sp.strain U2 plasmid pWWU2 (AF036940-2); NahR (31% identity; 68%similarity), a regulatory protein for naphthalene degradationgenes in Pseudomonas stutzeri AN10 (AF039534-4); and SyrM1 (32%identity; 66% similarity), a LysR-like protein in Rhizobiumsp. strain NGR234 (AE000091-2). The LTTR family is one of theubiquitous transcriptional regulators in prokaryotes (16), andthe transcription of the genes responsible for the degradationof aromatic compounds is often regulated by LTTRs (2). Likeknown LTTRs, LinR also contains a putative helix-turn-helixstructure (S₂₂VSAAARELDLPQPTASHGLARLRKALGDPL₅₂) at its N terminus,which is proposed to be responsible for DNA binding. Like manyLTTRs, LinR does not have a glycine residue in the middle ofthe helix-turn-helix (D₃₁ in LinR), which is conserved amongclassic helix-turn-helix structures (16), while OhbR, two NahRs,and SyrM have it. In the immediate upstream region of the linEgene, there is a consensus sequence for LTTR binding (ATTCACAATCTGAAT),which is a palindromic TN₁₁A sequence (beginning and end underlined)(16) (Fig. 3).

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FIG. 3. Construction of plasmids pMEU1R, pMEU2R, and pMEU3R. The putative LinR-binding site and the start codons of linR and linE are indicated.

In vivo analysis of the function of LinR.
To characterize the function of LinR in the expression of linE,we constructed the plasmids pMEU1R, pMEU2R, and pMEU3R (Fig.3). We also constructed pMEU1, pMEU2, and pMEU3, which do nothave the linR gene, as a control for each plasmid. E. coli harboringpMEU1, pMEU2, or pMEU3 showed only a faint LacZ activity underevery condition we tested (data not shown). E. coli harboringeither pMEU1R or pMEU2R showed an increase in LacZ activityin the presence of 2,5-DCHQ and CHQ (23- to 36-fold), whileE. coli harboring pMEU3R did not (Fig. 4). These results indicatedthat the linR gene and the palindromic TN₁₁A sequence whichis located upstream of linE are necessary for the upregulationof linE expression. It also seemed that the TN₁₁A sequence isrequired to repress the expression of the linE gene when thesubstrates are not present.

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FIG. 4. ß-Galactosidase (ß-gal) activities of the cell extracts of E. coli harboring pMEU1R, pMEU2R, or pMEU3R. Standard errors calculated from three independent experiments are shown.

Substrate specificity of LinR.
The substrate specificity of LinR was studied with the E. coliin vivo system described above. In addition to 2,5-DCHQ andCHQ, only 2,6-DCHQ gave upregulation activity to E. coli harboringpMEU1R (Table 2). This result indicated that LinR specificallyrecognizes mono- or di-CHQs among the substrates we tested.

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TABLE 2. Substrate specificity of LinR

RNA slot blot analysis.
To confirm the function of the linR gene for the expressionof linE and linD, RNA slot blot analysis was performed withtotal RNAs of UT26 and UT102, which is a linR mutant. Figure5 shows that linD and linE were inducibly expressed in the presenceof 2,5-DCHQ, CHQ, and HQ in UT26, while almost no expressionof linD and linE was observed in UT102 even in the presenceof these substrates. The linA gene, which is constitutivelyexpressed regardless of the presence of the substrates (4),was used as a control. This result indicated that HQ workedas an inducer for linD and linE expression in UT26 (Fig. 5)in addition to 2,5-DCHQ and CHQ, while it did not in the E.coli in vivo system (Fig. 4 and Table 2), suggesting a differencebetween the permeabilities of HQ in Sphingomonas and E. coli.The possibility that the intermetabolite of HQ works as an inducerin UT26 could not be excluded. However, we can conclude thatCHQs are direct substrates of LinR at least, because E. colidoes not have activity for conversion of CHQs. Like LinR, theinducer of NahR for the expression of naphthalene-degradativegenes is an aromatic compound, salicylate (17), while that ofCatR for the expression of catechol-degradative genes is cis,cis-muconate,which is the ring cleavage product of catechol (15). The BenMprotein, which regulates the expression of the ben operon inAcinetobacter sp. strain ADP1, responds to both aromatic compoundsand ring cleavage products, i.e., benzoate and cis,cis-muconate(1). It would be very interesting to know why these substratesare selected as the inducers for the regulation system of eachdegradation pathway.

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FIG. 5. RNA slot blot analysis of total RNAs of UT26 (lanes 1 to 4) and UT102 (lanes 5 to 8) incubated with 2,5-DCHQ (lanes 1 and 5), CHQ (lanes 2 and 6), HQ (lanes 3 and 7), and ethanol (lanes 4 and 8). The probes used are also shown.

HQ pathway.
The results described above indicate that the linR gene is necessaryfor the inducible expression of linE and linD. Because (C)HQsare specific substrates not only for LinD and LinE but alsofor LinR (Fig. 4 and 5 and Table 2), the genes encoding thesethree proteins seem to constitute a specific system for thedegradation of (C)HQs, while the gene expression of some dehalogenasesfor xenobiotic haloalkanes is constitutive (5). The existenceof a well-established degradation system strongly suggests thatthe HQ pathway is one of the major pathways for the degradationof aromatic compounds. linD and linE are considered to forman operon for the following reasons, although we could not detectthe band corresponding to the predicted length for the putativelinE-linD operon by Northern blot analysis (data not shown).First, the linD and linE genes are located close to each other.Second, the expression of linD is induced simultaneously withthat of linE (Fig. 5). Third, we could not identify an obviousterminator sequence between the linE and linD genes. We aretrying to identify other genes which belong to this operon.

ACKNOWLEDGMENTS

This work was supported in part by a Grant-in-Aid for ScientificResearch from the Ministry of Education, Science, Sports, andCulture of Japan.

This work was performed using the facilities of the BiotechnologyResearch Center, The University of Tokyo.

FOOTNOTES

* Corresponding author. Mailing address: Department of Bioengineering, Kamitomioka, Nagaoka, Niigata 940-2188, Japan. Phone: 81-258-47-9427. Fax: 81-258-47-9450. E-mail: kmiya{at}vos.nagaokaut.ac.jp.

REFERENCES

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