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Journal of Bacteriology, June 2008, p. 4210-4217, Vol. 190, No. 12
0021-9193/08/$08.00+0     doi:10.1128/JB.00061-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Transcriptional Regulation of the Nitrile Hydratase Gene Cluster in Pseudomonas chlororaphis B23

Toshihide Sakashita, Yoshiteru Hashimoto, Ken-Ichi Oinuma, and Michihiko Kobayashi^*

Institute of Applied Biochemistry, and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan

Received 13 January 2008/ Accepted 2 April 2008

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An enormous amount of nitrile hydratase (NHase) is inducibly produced by Pseudomonas chlororaphis B23 after addition of methacrylamide as the sole nitrogen source to a medium. The expression pattern of the P. chlororaphis B23 NHase gene cluster in response to addition of methacrylamide to the medium was investigated. Recently, we reported that the NHase gene cluster comprises seven genes (oxdA, amiA, nhpA, nhpB, nhpC, nhpS, and acsA). Sequence analysis of the 1.5-kb region upstream of the oxdA gene revealed the presence of a 936-bp open reading frame (designated nhpR), which should encode a protein with a molecular mass of 35,098. The deduced amino acid sequence of the nhpR product showed similarity to the sequences of transcriptional regulators belonging to the XylS/AraC family. Although the transcription of the eight genes (nhpR, oxdA, amiA, nhpABC, nhpS, and acsA) in the NHase gene cluster was induced significantly in the P. chlororaphis B23 wild-type strain after addition of methacrylamide to the medium, transcription of these genes in the nhpR disruptant was not induced, demonstrating that nhpR codes for a positive transcriptional regulator in the NHase gene cluster. A reverse transcription-PCR experiment revealed that five genes (oxdA, amiA, nhpA, nhpB, and nhpC) are cotranscribed, as are two other genes (nhpS and acsA). The transcription start sites for nhpR, oxdA, nhpA, and nhpS were mapped by primer extension analysis, and putative –12 and –24 ⁵⁴-type promoter binding sites were identified. NhpR was found to be the first transcriptional regulator of NHase belonging to the XylS/AraC family.

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We have extensively studied the biological metabolism of toxic compounds which have a triple bond between their carbon and nitrogen atoms, such as nitriles (RCN) (22) and isonitriles (RNC) (5, 8, 9). The microbial degradation of nitriles proceeds through two different enzymatic pathways (21): (i) nitrilase catalyzes the hydrolysis of nitriles into acids [RC(O)OH] and ammonia (19, 25), and (ii) nitrile hydratase (NHase) catalyzes the hydration of nitriles to amides [RC(O)NH₂] (1, 24), which are subsequently hydrolyzed to acids and ammonia by amidase (18). These enzymes have received much attention in applied fields (13, 14, 48), as well as academic fields (2, 4, 36, 38). One of the fruits of our application-oriented nitrile studies is the current industrial production of acrylamide and nicotinamide using the NHase of an actinomycete, Rhodococcus rhodochrous J1 (22, 46). Acrylamide is one of the most important commodities, and it is in great demand (200,000 tons per year worldwide) as a flocculant, a component of synthetic fibers, a soil conditioner, and a petroleum recovery agent. The industrial production of acrylamide using the ferric NHase of Rhodococcus sp. strain N-774, the first-generation strain, started in 1985 (12, 45). This biotransformation of acrylonitrile to acrylamide by a microbial NHase was the first application of biotechnology in the petrochemical industry and also the first successful example of the introduction of an industrial bioconversion process for the manufacture of a commodity chemical. On the other hand, the NHase of Pseudomonas chlororaphis B23 (33), which was previously used as a catalyst for acrylamide manufacture (42, 46), is now used for the production of 5-cyanovaleramide, a herbicide intermediate, at the industrial level (10). Strong induction of NHase formation was observed after addition of methacrylamide as the sole nitrogen source (47). A substrate of an enzyme often acts as an inducer; however, the reaction product of NHase, methacrylamide, serves as a strong inducer of the NHase of P. chlororaphis B23. Because the amount of NHase produced comprises more than 50% of the total soluble proteins in P. chlororaphis B23 when this strain is cultured in the presence of methacrylamide (34), there must be a very interesting regulation mechanism for enzyme expression. We previously cloned the NHase gene from this strain and discovered the existence of a gene cluster that includes the aldoxime dehydratase (29, 37), NHase, and amidase (35) genes. Very recently, we also discovered that the acyl coenzyme A (acyl-CoA) synthetase gene (acsA) is an additional member of this gene cluster and that aldoxime dehydratase, NHase, amidase, and acyl-CoA synthetase sequentially cooperate with one another in vivo for utilization butyraldoxime as a carbon and nitrogen source (11). Although we have clarified the overall "nitrile pathway" (aldoxime nitrile amide acid acyl-CoA) at the protein and gene levels, the mechanism that regulates NHase gene cluster expression in P. chlororaphis B23 has not been clarified.

In this study, we investigated transcriptional regulation of the NHase gene cluster in P. chlororaphis B23. We found that nhpR located in the region upstream of the aldoxime dehydratase gene encodes a transcriptional activator that controls NHase gene cluster expression. The proposed transcriptional activation mechanism for the NHase gene cluster provides an explanation for the induction of nitrile degradation and synthesis enzymes in response to addition of methacrylamide to the growth medium.

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Plasmids, strains, and media. Plasmid pPCN4 comprising the 6.5-kb SphI-SalI fragment of pUC19 (35) was used as a probe to clone the region upstream of the aldoxime dehydratase gene in P. chlororaphis B23. Escherichia coli DH10B (Invitrogen, Carlsbad, CA) was used as the host for pUC (49) and pSTV29 (Takara Bio, Tokyo, Japan). E. coli transformants were grown in 2x YT medium (31). The minimum medium for P. chlororaphis B23 was composed of 1% sucrose, 0.5% (NH₄)₂SO₄, 0.05% KH₂PO₄, 0.05% K₂HPO₄, 0.05% MgSO₄·7H₂O, and 0.001% FeSO₄·7H₂O (pH 7.0). Unless otherwise stated, sucrose and (NH₄)₂SO₄ were used as the carbon and nitrogen sources, respectively.

DNA manipulations. Restriction endonucleases, DNA polymerase, and T4 DNA ligase were purchased from Toyobo Co., Ltd. (Osaka, Japan). Nucleotides were sequenced by the dideoxy chain termination method using an ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA). Unless otherwise stated, DNA manipulations were performed essentially as described by Maniatis et al. (31).

Disruption of the chromosomal nhpR gene. To disrupt the nhpR gene on the P. chlororaphis B23 chromosome, a 2.9-kb DNA region containing the complete nhpR gene between the HindIII and XhoI sites was cloned into the HindIII and SalI sites of pSTV29, which is a chloramphenicol-resistant vector and cannot replicate in P. chlororaphis B23. The coding sequence of the kanamycin resistance gene was amplified by PCR with pUC-4K (43) as the template. In order to disrupt the HindIII and XhoI sites in the kanamycin resistance gene, site-directed mutagenesis was carried out by means of an overlap extension PCR protocol without any change in the amino acid sequence (15, 39). The primers used for amplification of the kanamycin resistance gene were Km-F (5'-ATCGGTGCGGGCCTCTTCGC-3'), Km-R (5'-CGAGTGAGTAATCCGTGGGGT-3'), HindIII-F (5'-TCCGGTGAGAATGGCAACAGTTTATGCATT-3'), HindIII-R (5'-TCTGGAAAGAAATGCATAAACTGTTGCCAT-3'), XhoI-F (5'-TGGAATTTAATCGCGGCCTGGAACAAGACG-3'), and XhoI-R (5'-CAACGGGAAACGTCTTGTTCCAGGCCGCGA-3'). The amplified kanamycin resistance gene (containing no HindIII and XhoI sites) was digested with HincII and then inserted into the BalI site in the nhpR gene on pSTV29. The resulting plasmid was introduced into P. chlororaphis B23 by electroporation to generate a P. chlororaphis B23 nhpR disruptant. Transformants were selected on 2x YT agar plates containing kanamycin (50 µg/ml) and the same plates containing chloramphenicol (30 µg/ml). Double crossovers were identified as Km^r and Cm^s. Transformants were subjected to PCR and Southern hybridization analyses in order to examine insertion of the kanamycin resistance gene into the nhpR gene on the P. chlororaphis B23 chromosome.

Enzyme assay. Aldoxime dehydratase activity was measured anaerobically under reduced conditions (standard assay B), as described previously (37). One unit of aldoxime dehydratase activity was defined as the amount of enzyme that catalyzed the formation of 1 µmol butyronitrile/min from butyraldoxime under the standard assay conditions. NHase activity was assayed by the method described previously (33). One unit of NHase activity was defined as the amount of enzyme that catalyzed the formation of 1 µmol propionamide from propionitrile under the standard assay conditions. Protein concentrations were determined as described by Bradford (3). Specific activity is expressed in U/mg of protein.

RNA experiments. For the isolation of RNA from P. chroloraphis B23, cells were cultivated in 10 ml of medium with or without 0.5% methacrylamide at 28°C. Total RNA was isolated with an RNeasy mini kit (Qiagen, Hilden, Germany).

Primer extension analysis. Primer extension experiments were performed using SuperScript III reverse transcriptase (Invitrogen) by the method of Maniatis et al. (31). The primers used for detection of the start sites of the nhpR, oxdA, nhpA, and nhpS genes were 5'-AGTGCCAGCGTTGTCTTTTC-3', 5'-TGTGCAAAGGCCAGGAAGCG-3', 5'-TCGAAGGTGTCGCAGTCGTG-3', and 5'-AGTCGCCGACAACGCTGTCC-3', respectively. The primers were labeled with ³²P at the 5' end with T4 polynucleotide kinase (Takara Bio).

RT-PCR. For cDNA synthesis, total RNA was incubated for 200 min at 50°C with SuperScript III reverse transcriptase (Invitrogen). Control reactions to assess the level of DNA contamination in the RNA samples were carried out without the reverse transcriptase. Primers RT1, RT2, and RT3 were used for the reverse transcription (RT) reaction, and the F1-R1, F2-R2, and F3-R2 primer pairs were used for successive PCR amplification. The sequences of the primers were as follows: RT1, 5'-GCGTGGGGCGTGCCGATAAG-3'; RT2, 5'-ACAGGCAGGCCTTGCGCATC-3'; RT3, 5'-TTGCGCAGAATGAACCGTTG-3'; F1, 5'-CCCATGCATCGACAACGTTC-3'; F2, 5'-AAAATGGAAGCTACCGATAC-3'; F3, 5'-GCTTGCCGACAGACTGGACC-3'; R1, 5'-ATAAGGCCTCGGTGTCAAAG-3'; and R2, 5'-TGGGCAGATGGTCGACAAAC-3'. The thermal cycling conditions were as follows: 1 min at 94°C, followed by 30 cycles of 10 s at 98°C and 15 min at 68°C with TaKaRa LA Taq (Takara Bio).

Real-time PCR. For cDNA synthesis, total RNA was incubated for 50 min at 50°C with SuperScript III reverse transcriptase (Invitrogen) in the presence of random hexamer primers. Control reactions to assess the level of DNA contamination in the RNA samples were carried out without the reverse transcriptase. Real-time PCR was performed using a thermal cycler Dice real-time system (Takara Bio) with SYBR Premix EX Taq (Takara Bio). Transcripts of nhpR, oxdA, amiA, nhpABC, nhpS, and acsA were quantified with the following primers: nhpR-F (5'-ACGAGAAGGTCGAGCAAAGC-3'), nhpR-R (5'-ACACGGCAATGGTCCTCGAC-3'), oxdA-F (5'-ACGCATCTCAAATGCCCACG-3'), oxdA-R (5'-TACTGCACGCCGAGATAACC-3'), amiA-F (5'-ACAGGACATCACCGGGCATC-3'), amiA-R (5'-GTCCACCGAGGCTTCAAACG-3'), nhpA-F (5'-TCAAGAGCAAGGAACTCATC-3'), nhpA-R (5'-TTCCGTCCTTGAGCAGCAGC-3'), nhpB-F (5'-TGCACCGCACCTCAGAGCAG-3'), nhpB-R (5'-CGTCACCCCATAGATCTTTC-3'), nhpC-F (5'-GGACGAATATCGGTTGCAGG-3'), nhpC-R (5'-GCACTCAGCAAACAATGGTC-3'), nhpS-F (5'-GACACAGGAAGTCACCCAAC-3'), nhpS-R (5'-GCAGCGGTTCCATTCACCTC-3'), acsA-F (5'-GATTATCTGCAGAGCGCCAC-3'), and acsA-R (5'-CATGGCCATCCTGCGCTTCG-3'). The transcript level of each gene was normalized to that of the internal control, the ribosomal rpsL gene. The PCR primers used for quantifying rpsL transcripts were rpsL-F (5'-AAACCGTTGGTCAGACGCAC-3') and rpsL-R (5'-ACGTCGTGGCGTATGCACTC-3'). The thermal cycling conditions were as follows: 10 s at 95°C, followed by 45 cycles of 5 s at 95°C and 30 s at 60°C. The data acquisition step was performed at 60°C, and a final melting curve analysis was used to ensure amplification of a single product. This experiment was carried out three times independently, and each RNA sample was analyzed at least in duplicate. All experiments gave similar results.

The oxdA, amiA, and nhpA transcripts were quantified by RT followed by absolute real-time PCR using a thermal cycler Dice real-time system with SYBR Premix EX Taq. For cDNA synthesis, total RNA was incubated for 50 min at 50°C with SuperScript III reverse transcriptase (Invitrogen). Primer nhpC-R was used for the RT reaction, and transcripts of oxdA, amiA, and nhpA were quantified with the following primers: oxdA-F, oxdA-R, amiA2-F (5'-TACCCTCGACCAGGTTTTAG-3'), amiA2-R (5'-TAGGCGTCGAAACTCGGTTG-3'), nhpA-F, and nhpA-R.

Nucleotide sequence accession number. The nucleotide sequence data reported in this paper have been deposited in the DDBJ/GenBank database under accession number AB374936.

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Cloning and nucleotide sequence of the 5' region upstream of the aldoxime dehydratase gene. In previous studies (11, 35, 37), we found that seven genes, which encode aldoxime dehydratase, amidase, the - and β-subunits of NHase, NhpC, NhpS, and acyl-CoA synthetase in that order, are located close to each other. To clarify the overall gene organization involved in the nitrile metabolic pathway of P. chlororaphis B23, gene walking upstream from the position of oxdA was performed. A 5.2-kb PstI-PstI fragment (accession number AB374936) was cloned into pUC18, and the resultant plasmid was designated pPCN11.

We determined the sequence of the region upstream of the oxdA gene and found an open reading frame (ORF) in the orientation opposite that of the structural genes of aldoxime dehydratase, amidase, the - and β-subunits of NHase, NhpC, NhpS, and acyl-CoA synthetase (Fig. 1A). This ORF was 936 nucleotides long and encoded a 311-amino-acid protein (molecular mass, 35,098 Da). The amino acid sequence of the gene product is similar to the amino acid sequences of regulatory proteins of the XylS/AraC family (44), including NitR, which is the positive regulator of nitrile metabolism in R. rhodochrous J1 (25) (26% identity), alkylbenzoate metabolism regulatory protein XylS1 in Pseudomonas putida (7) (23% identity), and the cad gene regulator CadR in Bradyrhizobium sp. strain HW13 (17) (22%) (Fig. 2). Accordingly, the ORF was designated nhpR.

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FIG. 1. (A) Organization of the NHase gene cluster, including nhpR, in the P. chlororaphis B23 genome. Large arrows indicate ORFs, and the translation products and designations of the genes are indicated above the arrows. The oligonucleotides used for the RT-PCR experiment are indicated by small arrows at the bottom. (B) Mapping of the 5' ends of the nhpR, oxdA, nhpA, and nhpS transcripts. Primer extension analysis involving total RNA isolated from the P. chlororaphis B23 wild-type strain cultured in the presence (lane 1) or absence (lane 2) of methacrylamide was carried out. Primer-extended products were electrophoresed in parallel with sequence ladders generated with the same primer. The positions of the transcription start site are indicated by arrows. (C) Analysis of NHase gene cluster transcription by RT-PCR. Lane 1, amplification with RT1, F1, and R1 (5.8 kb); lane 2, amplification with RT2, F1, and R1; lane 3, amplification with RT3, F3, and R2 (2.9 kb); lane 4, amplification with RT3, F2, and R2; lane M, molecular mass markers.

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FIG. 2. Alignment of the amino acid sequence of NhpR from P. chlororaphis B23 with homologous sequences. Highlighting indicates identical sequences, and dashes indicate gaps introduced to maximize the alignment. The helix-turn-helix motif (amino acids 228 to 247 of NhpR) is enclosed in a box. Designations: NitR, a regulator of nitrilase from R. rhodochrous J1 (25); CadR, a regulator of the cad gene from Bradyrhizobium sp. strain HW13 (17); and XylS1, a regulator involved in alkylbenzoate metabolism from P. putida (7).

NhpR is a transcriptional regulator for the NHase gene cluster. Since prokaryotic regulators are frequently located adjacent to the genes under their control (32), we hypothesized that nhpR encoded a regulator of the NHase gene cluster. In order to assess the significance of NhpR-associated changes in the expression of genes involved in the metabolism of nitriles, we constructed an nhpR disruptant of P. chlororaphis B23 as described in Materials and Methods. Both the wild-type strain and the resulting nhpR disruptant grew in minimum medium (Fig. 3A). The wild-type strain was able to grow in minimum medium containing methacrylamide instead of ammonium sulfate as a nitrogen source, and the formation of NHase was highly enhanced after addition of methacrylamide to the medium. Although the wild-type strain culture was found to have a significant, high optical density at 600 nm, the nhpR disruptant did not grow on methacrylamide instead of ammonium sulfate as the sole nitrogen source (Fig. 3B). Considering that the gene products of the NHase gene cluster are responsible for the degradation of methacrylamide, these findings indicated that NhpR should be an activator of the genes in the NHase gene cluster.

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FIG. 3. Growth curves for the P. chlororaphis B23 wild-type strain () and nhpR disruptant () at 28°C in the minimum medium with ammonium sulfate (A) or methacrylamide (B) as the sole nitrogen source. Growth was measured by determining the optical density at 600 nm (OD600).

To confirm the activation of the gene products derived from the NHase gene cluster by NhpR, the nhpR disruptant was further analyzed to determine its ability to produce aldoxime dehydratase and NHase after addition of methacrylamide as an inducer to the minimum medium. Although aldoxime dehydratase activity and NHase activity were detected for the wild-type strain, neither activity was detected for the nhpR disruptant even in the presence of methacrylamide as an inducer (Table 1), suggesting that NhpR is an activator for the expression of the gene products of the NHase gene cluster.

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TABLE 1. OxdA activity and NHase activity in cell extracts of the P. chlororaphis B23 wild-type strain and the nhpR disruptant cultured in the presence or absence of methacrylamide

Determination of the transcription start sites. To find the transcription start site of each gene in the NHase gene cluster, primer extension analysis was carried out using RNA prepared from P. chlororaphis B23 cells grown in the minimum medium with ammonium sulfate or methacrylamide as a nitrogen source. A radiolabeled primer was annealed to the RNA sequence near the 5' end of the coding sequence of each gene in the NHase gene cluster, and the primers were then extended with reverse transcriptase. Three transcription initiation sites were identified in the regions upstream of oxdA, nhpA, and nhpS only with RNA prepared from cells grown in the presence of methacrylamide as the sole nitrogen source in the medium (Fig. 1B). Although a transcription initiation site of nhpR was identified from RNA prepared in the absence of methacrylamide, slight transcriptional activation of nhpR was observed with RNA prepared in the presence of methacrylamide. No initiation site was identified upstream of amiA, nhpB, nhpC, or acsA. A single initiation site for nhpR transcription is located 328 bp upstream from the ATG initiation codon of nhpR; a single initiation site for oxdA transcription is located 233 bp upstream from the ATG initiation codon of oxdA; a single initiation site for nhpA transcription is located 34 bp upstream from the ATG initiation codon of nhpA; and a single initiation site for nhpS transcription is located 276 bp upstream from the ATG initiation codon of nhpS. Around 24 bp upstream of each transcription start site, possible ⁵⁴-dependent sequences, –24(TGGGGTG) and –12(ATCAG) for nhpR, –24(TTCCGCC) and –12(TTTCA) for oxdA, –24(AGACCGG) and –12(TTGCA) for nhpA, and –24(TCATCGA) and –12(TCGCA) for nhpS, were found.

Transcription units of the NHase gene cluster. To establish whether adjacent genes in the NHase gene cluster are cotranscribed, we conducted RT-PCR with RNA prepared from P. chlororaphis B23 grown in the minimum medium with methacrylamide instead of ammonium sulfate as the sole nitrogen source. When the RT primer (RT1) that hybridizes to the 3' end of nhpC and PCR primers F1 (5' of oxdA) and R1 (3' of nhpC) were used, a PCR product of the expected size (5.8 kb) was obtained. In contrast, when the RT primer (RT2) that hybridizes to the 5' end of nhpS and the same PCR primers (F1 and R1) were used, no PCR product was obtained. Furthermore, when the RT primer (RT3) that hybridizes to the 3' end of acsA and PCR primers F3 (5' of nhpS) and R2 (3' of acsA) were used, a PCR product of the expected size (2.9 kb) was obtained, although when the same RT primer (RT3) and PCR primers F2 (3' of nhpC) and R2 (3' of acsA) were used, no PCR product was obtained (Fig. 1C). No amplification product was obtained when reverse transcriptase was omitted from the reaction mixture (data not shown). These results demonstrated that five of the genes (oxdA, amiA, nhpA, nhpB, and nhpC) and two of the genes (nhpS and acsA) are transcribed as two major polycistronic transcriptional units. Considering the transcription start site upstream of nhpA, three genes (nhpABC) would be transcribed as a third polycistronic transcriptional unit.

Gene transcription analysis by quantitative real-time PCR. In order to determine whether the seven genes are under the control of NhpR in the presence of methacrylamide, real-time PCR was performed. RNAs were prepared from the P. chlororaphis B23 wild-type strain and P. chlororaphis B23 nhpR disruptant grown in minimum medium (containing ammonium sulfate as a nitrogen source) with or without methacrylamide as an inducer. As a result, addition of methacrylamide to the medium led to significant increases in the transcription of seven genes (oxdA, amiA, nhpABC, nhpS, and acsA) in the wild-type strain. On the other hand, when the nhpR disruptant was grown in the presence of methacrylamide (with ammonium sulfate as a nitrogen source), no increase in the transcription of the seven genes was observed (Fig. 4). This difference in the real-time PCR data suggests that NhpR triggers the transcription of the seven genes in response to the addition of methacrylamide to the medium. Similar to the transcription of the seven genes, the transcription of the nhpR gene was also inducibly triggered by NhpR with methacrylamide (Fig. 4), demonstrating that nhpR autoactivates its own transcription as well.

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FIG. 4. Analysis of expression of the seven genes in the NHase gene cluster and nhpR by real-time PCR. The expression levels in the P. chlororaphis B23 wild-type strain and nhpR disruptant were determined. Each strain was cultured in the minimum medium with methacrylamide or ammonium sulfate as the nitrogen source. The results were normalized to the transcription of rpsL. Differences for the wild-type strain and the nhpR disruptant with and without methacrylamide were calculated based on the transcription in the wild-type strain without methacrylamide, which was defined as 1.0.

NHase was produced at a high level in P. chlororaphis B23 grown in the medium with methacrylamide. Therefore, we compared the level of mRNA transcribed from the nhpA promoter with the level of mRNA transcribed from the oxdA promoter. To determine the absolute transcription ratio for the oxdA and nhpA promoters, we carried out real-time PCR with RNA prepared from P. chlororaphis B23 cells grown in the minimum medium with methacrylamide as an inducer. After the RT reaction had been performed with the RT primer (nhpC-R) that hybridizes to the 3' end of nhpC, the PCR was conducted to compare the levels of transcription of oxdA, amiA, and nhpA. As a result, the transcription level of oxdA was found to be almost the same as that of amiA. This finding is consistent with the finding that no promoter was identified upstream of amiA and with the finding that oxdA and amiA are cotranscribed as the same mRNA unit. On the other hand, the levels of nhpA transcribed from the two promoters upstream of oxdA and nhpA were about three times higher than the levels of oxdA and amiA transcribed from the promoter upstream of oxdA (data not shown).

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We were interested in the discovery of a regulatory protein that plays a key role in control of the expression of the NHase gene cluster in P. chlororaphis B23 for the following reasons: (i) the induction level of useful NHase is very high (34); and (ii) although a substrate of an inducible enzyme often acts as an inducer in the regulation mechanism, methacrylamide, which is the reaction product of NHase, acts as a strong inducer of NHase in P. chlororaphis B23 (47).

Sequence analysis of the region upstream of the aldoxime dehydratase gene revealed the presence of the nhpR gene exhibiting sequence similarity to a gene encoding a positive regulator belonging to the XylS/AraC family, which includes NitR involved in nitrile metabolism in R. rhodochrous J1 (25). Like NHase in P. chlororaphis B23, the nitrilase in R. rhodochrous J1 is strongly induced after addition of an inducer to the medium (20). In contrast to the finding that methacrylamide (which is a reaction product) induces NHase in P. chlororaphis B23, isovaleronitrile (which is a reaction substrate) induces nitrilase in R. rhodochrous J1. Although nitR is in the region downstream of the nitrilase gene (nitA) in the same direction (25), nhpR is in the region upstream of the NHase gene cluster in the direction opposite that of the structural genes in the NHase gene cluster (Fig. 1A). Very recently, an ORF which exhibits sequence similarity to the ORF encoding an AraC family protein was found to be located in the region upstream of a cluster including the aldoxime dehydratase and NHase genes in Pseudomonas sp. strain K-9 (16). However, the function of this ORF has not been determined. The XylS/AraC family, the members of which are positive regulators involved in the metabolism of carbon sources and in pathogenesis (41), is characterized by sequence similarity within the carboxyl terminus, which is the region containing a helix-turn-helix DNA-binding motif (6, 40). Consistent with the sequence similarity with the XylS/AraC family, analysis of the deduced amino sequence of NhpR revealed that it has a helix-turn-helix DNA-binding motif at its carboxyl terminus (Fig. 2). Within this family, for regulators recognizing chemical signals (inducers), the nonconserved N-terminal region is presumed to be responsible for binding to an activator molecule (6).

In P. chlororaphis B23, the chromosomal nhpR mutation led to an inability to grow in the medium with methacrylamide as the sole nitrogen source (Fig. 3) and to an inability to activate the transcription of the eight genes (nhpR, oxdA, amiA, nhpABC, nhpS, and acsA) in the NHase gene cluster in the minimum medium containing ammonium sulfate as a nitrogen source with methacrylamide as an inducer (Fig. 4). These results indicated that the nhpR gene product is a transcriptional activator of the NHase gene cluster. Consistent with this finding, the nhpR mutation resulted in complete loss of aldoxime dehydratase and NHase activities (Table 1). Because the genes in the NHase gene cluster are close together, it was speculated that the genes in this cluster constitute an operon. To experimentally verify this hypothesis, we determined the transcription start sites for these genes. Using total RNA from P. chlororaphis B23, we performed a primer extension analysis of the region upstream of each gene in the NHase gene cluster. Interestingly, four transcription start sites appeared to be upstream of nhpR, oxdA, nhpA, and nhpS (Fig. 1B). The promoter regions upstream of the transcription start sites of nhpR, oxdA, nhpA, and nhpS contained putative ⁵⁴-dependent sequences, –24(TGGGGTG)/–12(ATCAG), –24(TTCCGCC)/–12(TTTCA), –24(AGACCGG)/–12(TTGCA), and –24(TCATCGA)/–12(TCGCA), respectively. ⁵⁴ is a regulatory factor needed for expression of the genes whose products function in the assimilation of nitrogen. For example, ⁵⁴ is required for transcription of genes whose products are needed for biological nitrogen fixation (e.g., genes encoding amino acid transport components and degradative enzymes) (30). Although the four ⁵⁴-dependent promoters that we identified upstream of nhpR, oxdA, nhpA, and nhpS are not significantly similar to the consensus ⁵⁴-dependent promoter sequences –24(TGGCACG)/–12(TTGCA), it is reasonable to conclude that ⁵⁴-dependent promoter sequences are in the NHase gene cluster because aldoxime dehydratase, amidase, and NHase are involved in nitrile metabolism (aldoxime nitrile amide acid + ammonia) for supplying a nitrogen source (11). To the best of our knowledge, this is the first report that a ⁵⁴-dependent regulatory system is involved in nitrile metabolism.

To understand the putative operon structure, we conducted Northern blot analysis using RNA prepared from P. chlororaphis B23 grown in the minimum medium with methacrylamide as the sole nitrogen source. Hybridization with a probe for each gene resulted in no clear specific signal, probably due to degradation of the mRNA. Therefore, we conducted RT-PCR. As shown in Fig. 1C, we observed amplified 5.8- and 2.9-kb fragments corresponding to the lengths of oxdA-nhpC and nhpS-acsA. This suggested that oxdA-nhpC and nhpS-acsA constitute operons. Together with the two transcriptional start sites in the oxdA-nhpC operon, the nhpABC genes would be transcribed from two promoters. It is very interesting that the transcription of nhpABC from these two promoters is controlled by nhpR in response to the addition of methacrylamide. Because nitriles are generally very toxic due to their cyano functional groups, a high level of NHase expression could be needed for the immediate catabolism of nitriles in P. chlororaphis B23. Furthermore, because the reaction product of NHase acts as a strong inducer of the NHase gene cluster, a large amount of NHase can cause strong induction of the genes in the NHase gene cluster.

Since an enormous amount of NHase (corresponding to more than 50% of the total soluble protein) is produced in P. chlororaphis B23 grown with methacrylamide (34), a strong inducible promoter for the expression of NHase was predicted to be located upstream of nhpA. Contrary to our speculation, it was demonstrated that the level of transcription of the NHase gene was almost three times the levels of transcription of oxdA and amiA, indicating that the transcription ratio for the promoter region upstream of nhpA is about twice that for the promoter region upstream of oxdA. It is presumed that the high level of NHase expression is due to the fact that the regulation of NHase gene expression occurs at the posttranscriptional level or to stabilization of the protein (e.g., through protection from proteolytic degradation).

In this study, we initially demonstrated that nhpR upstream of the NHase gene cluster positively regulates the expression of four enzymes derived from the eight genes (oxdA, amiA, nhpA, nhpB, nhpC, nhpS, acsA,and nhpR) comprising the NHase gene cluster after addition of methacrylamide to the minimum medium as an inducer (Fig. 4 and 5). This regulation mechanism and the organization of the gene cluster in this strain are different from the regulation mechanism and the organization of both gene clusters encoding the NHase (26, 27, 28) and nitrilase (23, 25) in R. rhodochrous J1. We previously reported that four enzymes (aldoxime dehydratase, NHase, amidase, and acyl-CoA synthetase) encoded in the NHase gene cluster are inducibly expressed when P. chlororaphis B23 is grown with butyraldoxime as the sole carbon and nitrogen source (11). Although we investigated the regulatory mechanism involving methacrylamide in this research, the regulatory mechanism involving butyraldoxime is assumed to be the same as that involving methacrylamide since butyraldoxime can be degraded to butyramide (which is a product of the NHase reaction together with methacrylamide) in this strain. Until now, the question of whether NhpR directly binds to sequences upstream of oxdA, nhpA, and nhpS had not been addressed. In order to purify NhpR, we constructed expression plasmids for nhpR. Although E. coli was transformed with each of the resultant plasmids, NhpR expressed in E. coli was produced as inclusion bodies under any conditions (data not shown). Thus, further analyses to obtain an overview of the regulatory system for the nitrile pathway are in progress.

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FIG. 5. Proposed model for transcription of the NHase gene cluster caused by NhpR in P. chlororaphis B23. mRNA transcribed from the cluster is indicated by arrows at the bottom. +, stimulation of transcription.

 
This work was supported in part by the 21st Century COE Program (http://www.tara.tsukuba.ac.jp/coe21/), by a grant-in-aid for scientific research, and by a grant-in-aid for young scientists from the Ministry of Education, Culture, Sports, Science, and Technology.

We thank Hideaki Maseda (The University of Tokushima) for useful discussions.

 
* Corresponding author. Mailing address: Institute of Applied Biochemistry, and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan. Phone: (81)-29-853-4679. Fax: (81)-29-853-4605. E-mail: sec-mmb{at}agbi.tsukuba.ac.jp

Published ahead of print on 11 April 2008.

T.S. and Y.H. contributed equally to this work.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. Asano, Y., Y. Tani, and H. Yamada. 1980. A new enzyme "nitrile hydratase" which degrades acetonitrile in combination with amidase. Agric. Biol. Chem. 44:2251-2252.
2
2. Bartling, D., M. Seedorf, C. R. Schmidt, and W. E. Weiler. 1994. Molecular characterization of two cloned nitrilases from Arabidopsis thaliana: key enzymes in biosynthesis of the plant hormone indole-3-acetic acid. Proc. Natl. Acad. Sci. USA 91:6021-6025.[Abstract/Free Full Text]
3
3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[CrossRef][Medline]
4
4. Endo, I., M. Odaka, and M. Yohda. 1999. An enzyme controlled by light: the molecular mechanism of photoreactivity in nitrile hydratase. Trends Biotechnol. 17:244-248.[CrossRef][Medline]
5
5. Fukatsu, H., Y. Hashimoto, M. Goda, H. Higashibata, and M. Kobayashi. 2004. Amine-synthesizing enzyme, N-substituted formamide deformylase: screening, purification, characterization and gene cloning. Proc. Natl. Acad. Sci. USA 101:13726-13731.[Abstract/Free Full Text]
6
6. Gallegos, M. T., C. Michan, and J. L. Ramos. 1993. The XylS/AraC family of regulators. Nucleic Acids Res. 21:807-810.[Abstract/Free Full Text]
7
7. Gallegos, M. T., A. P. Williams, and L. J. Ramos. 1997. Transcriptional control of the multiple catabolic pathways encoded on the TOL plasmid pWW53 of Pseudomonas putida MT53. J. Bacteriol. 179:5024-5029.[Abstract/Free Full Text]
8
8. Goda, M., Y. Hashimoto, S. Shimizu, and M. Kobayashi. 2001. Discovery of a novel enzyme, isonitrile hydratase, involved in nitrogen-carbon triple bond cleavage. J. Biol. Chem. 276:23480-23485.[Abstract/Free Full Text]
9
9. Goda, M., Y. Hashimoto, M. Takase, S. Herai, Y. Iwahara, H. Higashibata, and M. Kobayashi. 2002. Isonitrile hydratase from Pseudomonas putida N19-2. J. Biol. Chem. 277:45860-45865.[Abstract/Free Full Text]
10
10. Hann, C. E., A. Eisenberg, K. S. Fager, E. N. Perkins, G. F. Gallagher, M. S. Cooper, E. J. Gavagan, B. Stieglitz, M. S. Hennessey, and R. DiCosimo. 1999. 5-Cyanovaleramide production using immobilized Pseudomonas chlororaphis B23. Bioorg. Med. Chem. 7:2239-2245.[CrossRef][Medline]
11
11. Hashimoto, Y., H. Hosaka, K.-I. Oinuma, M. Goda, H. Higashibata, and M. Kobayashi. 2005. Nitrile pathway involving acyl-CoA synthetase: overall metabolic gene organization, and purification and characterization of the enzyme. J. Biol. Chem. 280:8660-8667.[Abstract/Free Full Text]
12
12. Hashimoto, Y., M. Nishiyama, S. Horinouchi, and T. Beppu. 1994. Nitrile hydratase gene from Rhodococcus sp. N-774 requirement for its downstream region for efficient expression. Biosci. Biotechnol. Biochem. 58:1859-1865.[Medline]
13
13. Hashimoto, Y., M. Nishiyama, F. Yu, I. Watanabe, S. Horinouchi, and T. Beppu. 1992. Development of a host-vector system in a Rhodococcus strain and its use for expression of the cloned nitrile hydratase gene cluster. J. Gen. Microbiol. 138:1003-1010.[Abstract/Free Full Text]
14
14. Herai, S., Y. Hashimoto, H. Higashibata, H. Maseda, H. Ikeda, S. Omura, and M. Kobayashi. 2004. Hyper-inducible expression system for streptomycetes. Proc. Natl. Acad. Sci. USA 101:14031-14035.[Abstract/Free Full Text]
15
15. Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. 1989. Site-directed mutagenesis by overlap extension using polymerase chain reaction. Gene 77:51-59.[CrossRef][Medline]
16
16. Kato, Y., and Y. Asano. 2006. Molecular and enzymatic analysis of the "aldoxime-nitrile pathway" in the glutaronitrile degrader Pseudomonas sp. K-9. Appl. Microbiol. Biotechnol. 70:92-101.[CrossRef][Medline]
17
17. Kitagawa, W., S. Takami, K. Miyauchi, E. Masai, Y. Kamagata, T. M. James, and M. Fukuda. 2002. Novel 2,4-dichlorophenoxyacetic acid degradation genes from oligotrophic Bradyrhizobium sp. strain HW13 isolated from a pristine environment. J. Bacteriol. 184:509-518.[Abstract/Free Full Text]
18
18. Kobayashi, M., Y. Fujiwara, M. Goda, H. Komeda, and S. Shimizu. 1997. Identification of active sites in amidase: evolutionary relationship between amide bond- and peptide bond-cleaving enzymes. Proc. Natl. Acad. Sci. USA 94:11986-11991.[Abstract/Free Full Text]
19
19. Kobayashi, M., H. Izui, T. Nagasawa, and H. Yamada. 1993. Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile: cloning of the Alcaligenes gene and site-directed mutagenesis of cysteine residues. Proc. Natl. Acad. Sci. USA 90:247-251.[Abstract/Free Full Text]
20
20. Kobayashi, M., T. Nagasawa, and H. Yamada. 1989. Nitrilase of Rhodococcus rhodochrous J1. Purification and characterization. Eur. J. Biochem. 182:349-356.[Medline]
21
21. Kobayashi, M., T. Nagasawa, and H. Yamada. 1992. Enzymatic synthesis of acrylamide: a success story not yet over. Trends Biotechnol. 10:402-408.[CrossRef][Medline]
22
22. Kobayashi, M., and S. Shimizu. 1998. Metalloenzyme nitrile hydratase: structure, regulation, and application to biotechnology. Nat. Biotechnol. 16:733-736.[CrossRef][Medline]
23
23. Kobayashi, M., and S. Shimizu. 2000. Nitrile hydrolases. Curr. Opin. Chem. Biol. 4:95-102.[CrossRef][Medline]
24
24. Kobayashi, M., T. Suzuki, T. Fujita, M. Masuda, and S. Shimizu. 1995. Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium and Rhizobium. Proc. Natl. Acad. Sci. USA 92:714-718.[Abstract/Free Full Text]
25
25. Komeda, H., Y. Hori, M. Kobayashi, and S. Shimizu. 1996. Transcriptional regulation of the Rhodococcus rhodochrous J1 nitA gene encoding a nitrilase. Proc. Natl. Acad. Sci. USA 93:10572-10577.[Abstract/Free Full Text]
26
26. Komeda, H., M. Kobayashi, and S. Shimizu. 1996. A novel gene cluster including the Rhodococcus rhodochrous J1 nhlBA genes encoding a low molecular mass nitrile hydratase (L-NHase) induced by its reaction product. J. Biol. Chem. 271:15796-15802.[Abstract/Free Full Text]
27
27. Komeda, H., M. Kobayashi, and S. Shimizu. 1996. Characterization of the gene cluster of high-molecular-mass nitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rhodochrous J1. Proc. Natl. Acad. Sci. USA 93:4267-4272.[Abstract/Free Full Text]
28
28. Komeda, H., M. Kobayashi, and S. Shimizu. 1997. A novel transporter in cobalt uptake. Proc. Natl. Acad. Sci. USA 94:36-41.[Abstract/Free Full Text]
29
29. Konishi, K., K. Ishida, K.-I. Oinuma, T. Ohta, Y. Hashimoto, H. Higashibata, T. Kitagawa, and M. Kobayashi. 2004. Identification of crucial histidines involved in carbon-nitrogen triple bond synthesis by aldoxime dehydratase. J. Biol. Chem. 279:47619-47625.[Abstract/Free Full Text]
30
30. Kustu, S., E. Santero, J. Keener, D. Popham, and D. Weiss. 1989. Expression of sigma 54 (ntrA)-dependent genes is probably united by a common mechanism. Microbiol. Rev. 53:367-379.[Free Full Text]
31
31. Maniatis, T., F. E. Fritsch, and J. Sambrook. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
32
32. Moriwaki, N., K. Kimbara, and F. Kawai. 2006. Dual regulation of a polyethylene glycol degradative operon by AraC-type and GalR-type regulators in Sphingopyxis macrogoltabida strain 103. Microbiology 73:452-457.
33
33. Nagasawa, T., H. Nanba, K. Ryuno, K. Takeuchi, and H. Yamada. 1987. Nitrile hydratase of Pseudomonas chlororaphis B23. Purification and characterization. Eur. J. Biochem. 162:691-698.[Medline]
34
34. Nagasawa, T., K. Ryuno, and H. Yamada. 1989. Superiority of Pseudomonas chlororaphis B23 nitrile hydratase as a catalyst for the enzymatic production of acrylamide. Experientia 45:1066-1070.[CrossRef]
35
35. Nishiyama, M., S. Horinouchi, M. Kobayashi, T. Nagasawa, H. Yamada, and T. Beppu. 1991. Cloning and characterization of genes responsible for metabolism of nitrile compounds from Pseudomonas chlororaphis B23. J. Bacteriol. 173:2465-2472.[Abstract/Free Full Text]
36
36. Normanly, J., P. Grisafi, R. G. Fink, and B. Bartel. 1997. Arabidopsis mutants resistant to the auxin effects of indole-3-acetonitrile are defective in the nitrilase encoded by the NIT1 gene. Plant Cell 10:1781-1790.
37
37. Oinuma, K.-I., Y. Hashimoto, K. Konishi, M. Goda, T. Noguchi, H. Higashibata, and M. Kobayashi. 2003. Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas chlororaphis B23. J. Biol. Chem. 278:29600-29608.[Abstract/Free Full Text]
38
38. Pekarsky, Y., M. Campiglio, Z. Siprashvili, T. Druck, Y. Sedkov, S. Tillib, A. Draganescu, P. Wermuth, H. J. Rothman, K. Huebner, M. A. Buchberg, A. Mazo, C. Brenner, and M. C. Croce. 1998. Nitrilase and Fhit homologs are encoded as fusion proteins in Drosophila melanogaster and Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 95:8744-8749.[Abstract/Free Full Text]
39
39. Pogulis, R. J., A. N. Vallejo, and L. R. Pease. 1996. In vitro recombination and mutagenesis by overlap extension PCR methods. Mol. Biol. 57:167-176.
40
40. Ramos, L. J., F. Rojo, L. Zhou, and K. N. Timmis. 1990. A family of positive regulators related to the Pseudomonas putida TOL plasmid XylS and the Escherichia coli AraC activators. Nucleic Acids Res. 18:2149-2152.[Abstract/Free Full Text]
41
41. Rosenberg, E. Y., D. Bertenthal, M. L. Nilles, K. P. Bertrand, and H. Nikaido. 2003. Bile salts and fatty acids induce the expression of the Escherichia coli AcrAB multidrug efflux pump through their interaction with Rob regulatory protein. Mol. Microbiol. 48:1609-1619.[CrossRef][Medline]
42
42. Ryuno, K., T. Nagasawa, and H. Yamada. 1988. Isolation of advantageous mutants of Pseudomonas chlororaphis B23 for the enzymatic production of acrylamide. Agric. Biol. Chem. 52:1813-1816.
43
43. Taylor, L. A., and R. E. Rose. 1988. A correction in the nucleotide sequence of the Tn903 kanamycin resistance determinant in pUC4K. Nucleic Acids Res. 16:358.[Free Full Text]
44
44. Tobes, R., and L. J. Ramos. 2002. AraC-XylS database: a family of positive transcriptional regulators in bacteria. Nucleic Acids Res. 30:318-321.[Abstract/Free Full Text]
45
45. Watanabe, I., Y. Satoh, and K. Enomoto. 1987. Screening, isolation and taxonomical properties of microorganisms having acrylonitrile-hydrating activity. Agric. Biol. Chem. 51:3193-3199.
46
46. Yamada, H., and M. Kobayashi. 1996. Nitrile hydratase and its application to industrial production of acrylamide. Biosci. Biotechnol. Biochem. 60:1391-1400.[Medline]
47
47. Yamada, H., K. Ryuno, T. Nagasawa, K. Enomoto, and I. Watanabe. 1986. Optimum culture conditions for production by Pseudomonas chlororaphis B23 of nitrile hydratase. Agric. Biol. Chem. 50:2859-2865.
48
48. Yamada, H., S. Shimizu, and M. Kobayashi. 2001. Hydratases involved in nitrile conversion: screening, characterization and application. Chem. Rec. 1:152-161.[CrossRef][Medline]
49
49. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33:103-119.[CrossRef][Medline]

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Journal of Bacteriology, June 2008, p. 4210-4217, Vol. 190, No. 12
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