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Journal of Bacteriology, May 2007, p. 3660-3664, Vol. 189, No. 9
0021-9193/07/$08.00+0     doi:10.1128/JB.01662-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

HpdR Is a Transcriptional Activator of Sinorhizobium meliloti hpdA, Which Encodes a Herbicide-Targeted 4-Hydroxyphenylpyruvate Dioxygenase

Suvit Loprasert,^1* Wirongrong Whangsuk,¹ James M. Dubbs,¹ Ratiboot Sallabhan,¹ Kumpanart Somsongkul,¹ and Skorn Mongkolsuk^1,2

Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok 10210, Thailand,¹ Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand²

Received 27 October 2006/ Accepted 19 February 2007

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Sinorhizobium meliloti hpdA, which encodes the herbicide target 4-hydroxyphenylpyruvate dioxygenase, is positively regulated by HpdR. Gel mobility shift and DNase I footprinting analyses revealed that HpdR binds to a region that spans two conserved direct-repeat sequences within the hpdR-hpdA intergenic space. HpdR-dependent hpdA transcription occurs in the presence of 4-hydroxyphenylpyruvate, tyrosine, and phenylalanine, as well as during starvation.

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Tyrosine catabolism begins with the conversion of L-tyrosine to 4-hydroxyphenylpyruvate (HPP), followed by the HPP dioxygenase (HPPD)-catalyzed conversion to homogentisate. Homogentisate is then further degraded and incorporated into the Krebs cycle. HPPD is found in all types of organisms and has both agricultural and therapeutic significance. In plants, this pathway has an anabolic branch from homogentisate that forms essential isoprenoid redox cofactors such as plastoquinone and tocopherol (14). This has been the basis for the development of very effective herbicides (i.e., triketones and isoxasoles) that are currently used commercially (15). In humans, deficiencies in specific enzymes of the tyrosine catabolism pathway give rise to a number of severe metabolic disorders (6). In fact, HPPD-inhibiting herbicides can act as therapeutic agents for a number of lethal congenital defects in tyrosine catabolism by preventing the accumulation of toxic metabolites (19). The gene encoding HPPD (hpdA) has already been identified in several species, including humans (17), Arabidopsis thaliana (7), Pseudomonas spp. (16), and Streptomyces avermitilis (5), but the mechanism of hpdA regulation remains totally unknown.

Sinorhizobium meliloti is a gram-negative soil bacterium that is best known for forming nitrogen-fixing symbiotic relationships with legumes (2) and uses HPPD to catabolize tyrosine as a nitrogen source (12). No information is available about the physiological and genetic responses of soil bacteria to herbicides. The fact that S. meliloti is an agriculturally important soil organism that is normally exposed to herbicides makes it a good model system for the study of the regulation of this herbicide-targeted enzyme (HPPD).

The gene encoding HPPD in S. meliloti Rm1021, SMc03211 (hpdA), was amplified by PCR and ligated into NcoI/BamHI-digested pETBlue2 (Novagen), using engineered NcoI and XhoI sites, to yield pET-hpdA carrying hpdA fused to six C-terminal His residues. Isopropyl-ß-D-thiogalactopyranoside-induced overexpression of recombinant HpdA-His₆ in Escherichia coli DE3 resulted in the production of an oxidized brown pigment (ochronotic pigment) of polymerized homogentisate that has previously been observed in E. coli strains overexpressing S. avermitilis HpdA (5) (data not shown). Pigment formation was inhibited by growth of the expression strain in medium containing isoxaflutole (5.5 mM), a herbicide that inhibits HpdA, demonstrating that S. meliloti HpdA-His₆ is functionally active (data not shown).

Northern blots were generated using total S. meliloti Rm1021 RNA extracted from exponential phase cells, as previously described (13), that had received a 30-min exposure to HPP and were then probed with an hpdA gene-specific internal probe. It was found that a 1.3-kb hpdA monocistronic transcript was induced in the presence of HPP (Fig. 1A, WT). To aid in further regulatory studies, an hpdA promoter-lacZ transcriptional fusion plasmid was constructed by ligation of a PCR fragment containing engineered EcoRI and BamHI sites at either end into EcoRI/BglII-digested pICH20 (11) to yield pICHA1. This promoter fragment, spanning a region from 350 bp upstream of the hpdA translation start to 66 bp within the hpdA coding sequence, was then excised from pICHA1 with HindIII and ligated into the HindIII site of the lacZ fusion vector, pMP220 (20), to yield the hpdA promoter-lacZ transcriptional fusion plasmid, pMPA1 (Fig. 2A). Similar induction studies using S. meliloti strain Rm1021 carrying pMPA1 confirmed the Northern blot analyses in that hpdA promoter activity was induced by HPP and the amino acids tyrosine and phenylalanine (Fig. 1B and data not shown). Induction of hpdA promoter activity was also observed upon incubation in minimal medium (155 mM Na₂HPO₄, 44 mM KH₂PO₄, 17 mM NaCl, 37.4 mM N4₄Cl, 11.7 mM sucrose, 1 mM MgSO₄, 0.25 mM CaCl₂, and 4 mM biotin) lacking either a carbon or nitrogen source (Fig. 1C). This is similar to the expression pattern that has been reported for S. meliloti hmgA, which encodes homogentisate dioxygenase and which catalyzes the next step in the tyrosine degradation pathway after HpdA (12).

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FIG. 1. HpdR-dependent regulation of S. meliloti hpdA expression in response to tyrosine and HPP. (A) An autoradiogram of a Northern blot of total RNA extracted from wild-type S. meliloti, the S. meliloti hpdR insertion mutant (hpdR), and the S. meliloti hpdR insertion mutant strain complemented with a functional hpdR (hpdR/hpdR+) and probed with a 32P-labeled internal fragment of S. meliloti hpdA. Cultures were either induced for 30 min with 5 mM HPP (H) or uninduced (U) prior to RNA extraction. (B) Results of ß-galactosidase assays of wild-type S. meliloti and hpdR and hpdR/hpdR+ strains carrying the hpdA promoter-lacZ fusion plasmid pMPA1 are shown. Cultures were either uninduced or induced for 30 min with 5 mM HPP or tyrosine prior to the assay. (C) HpdR-dependent regulation of hpdA expression in response to nutrient starvation. The results of ß-galactosidase assays of wild-type S. meliloti and the hpdR strain carrying the hpdA promoter-lacZ fusion plasmid pMPA1 are shown. Cultures were either uninduced or incubated in M-9 minimal medium lacking either a carbon (sucrose) or a nitrogen (NH4Cl) source for 30 min prior to the assay. Error bars show standard deviations. WT, wild type.

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FIG. 2. Deletion mapping of the S. meliloti hpdA promoter. (A) Diagram of the various hpdA promoter-lacZ transcriptional fusion constructs used. The numbers represent the location of the hpdA translation start (+1) and the amount of upstream sequence contained in each fusion plasmid. The ability of each fusion to be induced in the presence of HPP or tyrosine is indicated. The dotted box delimits a region important for HpdA expression. (B) Results of ß-galactosidase assays of extracts of wild-type S. meliloti strains carrying the various hpdA promoter-lacZ fusion plasmids shown in Fig. 3A. Cultures were either uninduced or induced for 30 min with 5 mM HPP or tyrosine prior to the assay. Error bars show standard deviations.

A divergently transcribed open reading frame, SMc03210, was identified 130 bp upstream of hpdA. A protein database search using BLASTP (18) and the deduced amino acid sequence encoded by SMc03210 revealed that its gene product is similar to the leucine-responsive regulatory protein (Lrp) family of transcription regulators (37% identity to Lrp of E. coli). In E. coli, Lrp is a global regulator of genes involved in modulating a variety of metabolic functions, including the metabolism of amino acids and pilus synthesis, and can act as either a positive or negative regulator that responds to the level of leucine (3). Therefore, it seemed likely that SMc03210, from here on referred to as hpdR, was a regulator of hpdA. In order to confirm this, a 196-bp PstI/NruI hpdR internal fragment was inserted into PstI/SmaI-digested pKNOCK-Gm (1). The resulting construct was electroporated into S. meliloti Rm1021 to generate the gentamicin-resistant insertion mutant strain, S. meliloti hpdR. This strain no longer produced the 1.3-kb hpdA mRNA in response to HPP exposure (Fig. 1A, lanes hpdR). S. meliloti hpdR carrying pMPA1 did not induce lacZ expression in the presence of HPP or tyrosine (Fig. 1B) or in response to carbon or nitrogen starvation (Fig. 1C).

Complementation studies were performed by inserting a functional hpdR gene into the chromosome of S. meliloti hpdR using a mini-Tn5 construct. This was accomplished by inserting a 701-bp PCR-generated fragment, spanning a region starting 14 bp downstream of the hpdR stop codon to 198 bp upstream of the hpdR translation start, into plasmid pUTTn5-Ery (9) to generate pUTn-hpdR. Conjugal transfer of pUTn-hpdR into S. meliloti hpdR, followed by erythromycin selection, yielded S. meliloti hpdR/hpdR⁺, which carries the mutant hpdR allele as well as a functional chromosomal copy of hpdR at a second site. HPP-inducible production of the 1.3-kb hpdA message was restored in Northern blots of the S. meliloti hpdR/hpdR⁺ strain's total RNA (Fig. 1A), as was HPP- and tyrosine-inducible expression of ß-galactosidase in S. meliloti hpdR/hpdR⁺ carrying pMPA1 (Fig. 1B). Thus, the expression studies clearly indicated that HpdR is a positive regulator of hpdA expression that responds to HPP, tyrosine, and phenylalanine and both carbon and nitrogen starvation.

Efforts were then focused on the delineation of the hpdA promoter, as well as on localizing the HpdR binding sites. As a first step toward accomplishing this, additional hpdA promoter-lacZ transcriptional fusions containing 132 bp (pMPAA), 70 bp (pMPAB), and 38 bp (pMPAC) of the sequence 5' to the hpdA translation start were constructed (Fig. 2A). The hpdA promoter-lacZ fusions were constructed in a manner similar to that described for pMPA1, with the exception that the EcoRI/BamHI-digested PCR fragments were ligated directly into EcoRI/BamHI-digested pMP220. The results of ß-galactosidase assays using the S. meliloti strains carrying pMPA1, pMPAA, pMPAB, and pMPAC indicated that the regulatory sequence(s) responsible for the HPP- and tyrosine-inducible expression of hpdA was localized between bp –70 (pMPAB) and bp –132 (pMPAA) upstream of the hpdA start codon (Fig. 2A and B).

Examination of the sequence between positions –132 (pMPAA) and the hpdA start codon revealed the presence of two conserved repeat sequences, denoted S1 (between positions –82 and –97) and S2 (between positions –43 and –58). The fact that the lacZ fusion plasmid, pMPAB, lacked the complete upstream direct repeat (S1) suggested that S1, and possibly S2, were involved in hpdA regulation and might bind HpdR. In order to determine this, gel mobility shift assays using purified recombinant S. meliloti HpdR-His₆ and ³²P-labeled PCR fragments spanning various regions of the hpdA upstream sequence (Fig. 3A) were performed to determine if HpdR binds to the direct repeats (S1 and S2) within the hpdA promoter region (9). Fusion of the carboxy terminus of HpdR to six histidine residues was accomplished by PCR amplification of an S. meliloti hpdR fragment containing engineered NcoI and XhoI sites overlapping the ATG start codon and the terminal glycine codon (GGC). Digestion of this fragment with NcoI/XhoI was followed by ligation into NcoI/BamHI-digested pETBlue2 (Novagen) to yield pET-hpdR. Recombinant HpdR-His₆ was purified from soluble extracts of the isopropyl-ß-D-thiogalactopyranoside-induced E. coli DE3 strain carrying pET-hpdR by Ni affinity chromatography using the manufacturer's protocols (QIAGEN). HpdR-His₆ was found to bind to ³²P-labeled PCR fragment A, spanning the region from bp +78 to bp –171 relative to the hpdA translation start (Fig. 3B, lane 2), thus demonstrating that HpdR directly regulates hpdA. Further mobility shift analyses clearly indicated that HpdR binds to the region containing the S1 and S2 repeats. HpdR did not bind to probe B (bp +78 to bp –40) (Fig. 3B, lane 4); however, probes C and D, containing S1 and S2, respectively, both bound HpdR (Fig. 3A and B, lanes 6 and 10). The fact that fragment D required a 3-fold-higher concentration of HpdR-His₆ to detect binding suggests that HpdR has a lower affinity for S2 (Fig. 3B, compare lanes 6, 8, and 10). Alternatively, additional 5' flanking sequence may be necessary for efficient binding to S2 and/or HpdR binding to S1 may facilitate subsequent binding to S2.

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FIG. 3. Binding of HpdR to the S. meliloti hpdA promoter region. (A) A map of the hpdA intergenic region is shown along with the PCR fragments, A through D, used as probes in the gel mobility shift assays. The numbers indicate the positions of the ends of each probe relative to the hpdA translation start. The dark boxes indicate the positions of the S1 and S2 direct repeat sequences. The ability of HpdR to bind to each probe is also indicated (+, –, or ±). (B) A compilation of autoradiograms of gel mobility shift assays performed using recombinant S. meliloti HpdR-His6 and the 32P-labeled PCR fragments shown in Fig. 3A. The probe used is shown at the top of each panel. The odd lanes contain gel shift reaction mixtures with no protein added. Lanes 2, 6, and 8 contain reaction mixtures to which HpdR-His6 was added to a final concentration of 0.16 mg/ml. The reaction mixtures whose results are shown in lanes 4 and 10 contained 0.48 mg/ml HpdR-His6. D(low), low concentration of HpdR-His6 (0.16 mg/ml); D(high), high concentration of HpdR-His6 (0.48 mg/ml).

HpdR binding to both S1 and S2 was confirmed in DNase I footprint analyses (Fig. 4A). Footprinting was performed as previously described (8). HpdR protected the hpdA promoter from approximately bp –43 to bp –133 upstream of the hpdA codon (Fig. 4A and B). A number of DNase I-hypersensitive sites were present within this region, suggesting that HpdR induced changes in the promoter conformation. Addition of the inducer, HPP, to HpdR footprint reactions did not result in any gross changes to the DNase I footprint. However, several DNase I-hypersensitive sites were lost in the presence of HPP, while increased protection of a T residue at position –56 (top strand) and a G residue at position –133 (bottom strand) was observed, indicating that HPP induces changes in the HpdR/hpdA promoter complex. In Pseudomonas putida, the Lrp family regulator of branch-chained ketoacid metabolism, BkdR, showed similar binding characteristics in DNase I footprint assays when bound to the bkdR-bkdA1 intergenic region that included alterations in hypersensitive-site intensities in the presence of inducers (10).

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FIG. 4. DNase I footprint of HpdR bound to the HpdA promoter. (A) Autoradiograms of DNase I protection assays of HpdR using an hpdR-hpdA intergenic probe, labeled on either the top or bottom strand, in the presence or absence of the inducer, HPP. Single end-labeled probes were generated by PCR amplification of the S1- and S2-spanning region in pICHA1, in which one of the primers had been end labeled using T4 polynucleotide kinase and [-32P]ATP. 32P-dideoxy sequencing ladders, generated using the same oligonucleotides as those used to amplify the probe, are included as sizing standards. The brackets indicate the positions of the conserved S1 and S2 repeats. The vertical lines delimit the extents of the protected regions. The arrowheads denote DNase I-hypersensitive sites, while the dots indicate bands that display increased protection in the presence of HPP. (B) Sequence of the hpdR-hpdA intergenic region summarizing the DNase I footprint data. The DNase I-protected region is shaded, and the horizontal lines indicate the S1 and S2 repeats. The arrowheads identify DNase I-hypersensitive sites, while the dots indicate bands that show increased protection in the presence of HPP.

Given the importance of the region containing the S1 and S2 repeats in HpdR-mediated regulation of hpdA, the hpdA promoter regions in other bacteria were examined for the presence of similar sequence elements (Fig. 5). Direct repeat sequences similar to S1 and S2 were found within the intergenic region between hpdA and hpdR in Mesorhizobium loti MAFF303099 (two copies), Bradyrhizobium japonicum USDA110 (two copies), Rhodopseudomonas palustris CGA009 (two copies), Silicibacter pomeroyi DSS-3 (two copies), Burkholderia pseudomallei 1710b (two copies), Bordetella bronchiseptica RB50 (two copies), Burkholderia thailandensis E264 (three copies), Roseobacter denitrificans OCH114 (three copies), Novosphingobium aromaticivorans DSM12444 (three copies), and Paracoccus denitrificans PD1222 (two copies). The putative HpdR binding sites of these organisms were aligned and an HpdR consensus binding sequence, (C/G)(A/T)TGC-N₇-CTGC, was identified (Fig. 5). This sequence bears little resemblance to the consensus Lrp binding site, (C/T)AGXA(A/T)ATT(T/A)TWCT(G/A) (where W = G, T, or A and X = C, A, or T) determined by Brinkman et al. (3).

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FIG. 5. Comparison of HpdR binding site repeat sequences. A comparison of the S. meliloti hpdA promoter S1 and S2 repeat sequences with repeats present in the hpdA promoters of; Mesorhizobium loti MAFF303099 (M1 and M2), Bradyrhizobium japonicum USDA110 (BJ1 and BJ2), Rhodopseudomonas palustris CGA009 (R1 and R2), Silicibacter pomeroyi DSS-3 (SP1 and SP2), Burkholderia pseudomallei 1710b (BP1 and BP2), Bordetella bronchiseptica RB50 (BB1 and BB2), Burkholderia thailandensis E264 (BT1, BT2, and BT3), Roseobacter denitrificans OCH114 (RD1, RD2, and RD3), Novosphingobium aromaticivorans DSM12444 (NA1, NA2, and NA3), and Paracoccus denitrificans PD1222 (PD1 and PD2). A consensus sequence in which the height of each letter is proportional to the frequency of the residue at that position is shown at the bottom (4). Numbers on the right are sequence lengths, in base pairs.

Taken together, the data presented here clearly demonstrate that HpdR positively regulates hpdA expression through direct binding to the HpdA promoter within a region containing two conserved direct repeat sequences. This is the first identification of a regulator for the herbicide target hpdA.

 
We thank P. Munpiyamit for the photograph preparation and W. Rongmuang for technical assistance.

This research was supported by grants from the Chulabhorn Research Institute and an advanced research scholar grant, BRG4880002, from the Thailand Research Fund to S.L.

 
* Corresponding author. Mailing address: Laboratory of Biotechnology, Chulabhorn Research Institute, Vibhavadee-Rangsit Highway, Bangkok 10210, Thailand. Phone: (662) 574-0622. Fax: (662) 574-2027. E-mail: suvit{at}cri.or.th

Published ahead of print on 2 March 2007.

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Journal of Bacteriology, May 2007, p. 3660-3664, Vol. 189, No. 9
0021-9193/07/$08.00+0     doi:10.1128/JB.01662-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Loprasert, S. Articles by Mongkolsuk, S. Search for Related Content PubMed PubMed Citation Articles by Loprasert, S. Articles by Mongkolsuk, S.