- Email: [email protected]
Complete Nucleotide Sequence and Overexpression of cat1 Gene Cluster, and Roles of the Putative Transcriptional Activator CatR1 in Acinetobacter lwoffii K24 Capable of Aniline Degradation
Biochemical and Biophysical Research Communications 288, 645– 649 (2001) doi:10.1006/bbrc.2001.5818, available online at http://www.idealibrary.com on
Complete Nucleotide Sequence and Overexpression of cat 1 Gene Cluster, and Roles of the Putative Transcriptional Activator CatR1 in Acinetobacter lwoffii K24 Capable of Aniline Degradation Seung Il Kim,* ,1 Yong-Cheol Yoo,* and Hyung-Yeel Kahng† *Biomolecule Research Team, Korea Basic Science Institute, Daejon 305-333, Korea; and †Research Institute for Basic Sciences, Cheju National University, Jeju 690-756, Korea
Received September 17, 2001
The aniline-assimilating bacterium Acinetobacter lwoffii K24 has two cat gene clusters (cat 1 and cat 2). In this study, we completely sequenced 10-kb DNA fragment of cat 1 genes of A. lwoffii K24, which had been cloned in plasmid pCD1-1. Sequence analysis revealed that the order of genes in the cat 1 operon-containing gene cluster was ORF porin, catR 1, catB 1C 1A 1D, ORF1, and ORF2. Two ORFs located immediately downstream catD were most similar with two ORFs in cat gene cluster of Acinetobacter calcoaceticus ADP1 but the gene structure of catR 1B 1C 1A 1 was closest to that found in Frateurua sp. ANA-18 or Pseudomonas putida PRS2000. CatA 1 gene product was significantly overexpressed and detected in SDS–PAGE when four cat 1 genes (catB 1C 1A 1D) were placed under the control of a lac promoter in pUC118 while overexpressions of other cat genes were accomplished under the control of a lac promoter in pET vector system. All gene products were verified by N-terminal amino acid sequencing. Gel retardation assay revealed that the putative regulatory gene activator CatR 1 for the catB 1C 1A 1 operon could bind the promoter region of catB 2 as well as catB 1, suggesting that transcription of catB 1 or catB 2 might be controlled by the putative gene activator CatR 1. However, the promoter regions of catA 1 and catA 2 were found to have no affinity with catR 1. © 2001 Academic Press
Key Words: Acinetobacter lwoffii K24; aniline degradation; cat1 gene cluster; CatR1; overexpression; gel retardation assay.
Previous studies demonstrated that aniline is degraded to catechol, which can be metabolized by ortho cleavage (-ketoadipate) pathway or meta cleavage To whom correspondence should be addressed. Fax: ⫹82-42-8653451. E-mail: [email protected]. 1
pathway by dioxygenases (1–3). It has been reported that the -ketoadipate pathway is accomplished by a chromosomally encoded biodegradation gene cluster while the genes of meta cleavage pathway are on the catabolic plasmid (4). The -ketoadipate pathway genes are diversely distributed in different microorganisms, along with their various organizations, transcriptions and regulation patterns (4, 5). Notably, two copies of degrading genes have been found in several aniline degrading bacteria containing -ketoadipate pathways (6, 7). Acinetobacter lwoffii K24 was also found to have two copies of catRABC (cat 1 and cat 2 genes) on the chromosome (6). Though two cat gene clusters, cat1 and cat2 demonstrate different order of gene arrangements (catB 1C 1A 1 and catB 2A 2C 2) and different transcriptional patterns, they were simultaneously expressed in response to aniline (8). Two catRs (catR 1 and catR 2) in A. lwoffii K24 were assumed to be transcriptional activators in that each cat gene cluster has its own catR and catR binding site between catR and catB. However, roles of two cat gene clusters in the cell are not clear yet. In our effort to elucidate the role of the gene clusters in A. lwoffii K24, we analyzed the complete DNA sequence containing 10-kb containing cat 1 gene clusters and report in this study. Furthermore, possible roles of two CatRs also will be described based on expression pattern of cat genes and gel retardation assay using CatR 1. MATERIALS AND METHODS DNA sequencing and analysis. The nucleotide sequences of 10-kb containing cat 1 genes were determined by the dideoxy-chain termination methods using an ABI Prism DyeDeoxy Terminator cycles sequencing kit (Perkin–Elmer). Sequencing reactions were prepared according to the supplier’s instructions and analyzed by electrophoresis using the Perkin Elmer Model 377 DNA sequencer. Mac DNASIS of Hitachi Software was used for DNA analysis. The nucleotide sequence has been submitted to the GenBank database under the Accession No. U77658.
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0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Vol. 288, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
PCR Primers for Overexpression and Amplification of Promoter Regions Gene CatR 1 CatB 1 CatC 1 CatD 1 CatB 1 promoter CatA 1 promoter CatB 2 promoter CatA 2 promoter
Primer
Sequence
Oligo#120 Oligo#121 Oligo#148 Oligo#149 Oligo#150 Oligo#151 Oligo#152 Oligo#153 Oligo#137.1 Oligo#138.1 Oligo#154.1 Oligo#113.1 Oligo#139.1 Oligo#140.1 Oligo#116.1 Oligo#11
5⬘-CTC TAT CAC ATA TGG ATC TGC GCC AGT TTC G-3⬘ 5⬘-AGC TGG ATC CCG TCT ATC AAT TGT GAG T-3⬘ 5⬘-CTC TAT CAC ATA TGT CCA GTG TAA CGA TT-3⬘ 5⬘-AGC TGG ATC CTT ACG CCT TCG TGA CCT T-3⬘ 5⬘-CTC TAT CAC ATA TGC TTT TCC ATG TAC GC-3⬘ 5⬘-AGC TGG ATC CTC ACG ACT CGT CGT CGC G-3⬘ 5⬘-ACT ATG GCT AGC ATG TAT GAA GGC GAA CGG-3⬘ 5⬘-AGC TGG ATC CGA TTT CTC ACT CGA TCA G-3⬘ 5⬘- 4001GAC GAA GTA GCG AAA CTG 4018-3⬘ 5⬘- 4222CTG GAC ATG TCG CAG GT 4206-3⬘ 5⬘- 5331CTG ATC AAG GTC ACG AAG 5348-3⬘ 5⬘- 5785ATG CTC ATG ATG CCA GTC 5768-3⬘ 5⬘- 202GCT TTC CTC GAT CTG CTG 219-3⬘ 5⬘- 437GCT ATC ATT CCT TGC CCT 420-3⬘ 5⬘- 1429TTG CTG CTG ACC GAA GAA 1446-3⬘ 5⬘- 1757CTT CGA TCG TAT AGA ACA GAT CCC TGA CG 1728-3⬘
Overexpression of cat genes in E. coli. The oligonucleotide primers for overexpression of cat genes were listed on the Table 1. Forward primers have NdeI or NheI recognition site and reverse primers have BamH1 recognition site, respectively. PCR reactions were run for 25 cycle using the following condition; denaturation at 95°C for 1 min, annealing at 65°C for 1 min, and extension at 72°C for 1 min. PCR products were run on 1% agarose gel and purified using QIAquick Spin column (Qiagen). The PCR products were digested with NdeI (or NheI) and BamH1. The DNA fragments were ligated into plasmid pET-3 (Stratagene) treated with the same enzymes and ligated DNA mixtures were used to transform E. coli DH5␣. New vectors, pSK251 (containing catR 1), pSK264 (containing catB 1), pSK265 (containing catC 1) and SK266 (containing catD) were obtained (Table 2), and they were transformed into E. coli BL21 for overexpression. After the transformants were cultured in 100 ml LB broth containing Ap (50 g/ml) until A 600 nm is 0.4, 0.1 mM isopropyl-D-thiogalactopyranoside (IPTG) was added for protein induction. After induction of 4 – 6 h, cells were harvested and lysed for protein analysis as described in previous method (6). The protein was analyzed on 10% SDS–PAGE. Plasmid isolation, plasmid construction, and transformation were performed according to the protocol of Maniatis (9). Identification of protein products of cat genes. To verify the expression of cat gene products, crude extracts of E. coli transformants were loaded on the SDS–PAGE and electro-transferred
onto PVDF membrane by the ABI protocol. Protein bands were excised from the PVDF membrane and installed into the blot cartridge of a Model 491A protein sequencer (Perkin–Elmer, Foster City, CA) for sequencing analysis. The obtained N-terminal sequence was used for protein identification by BLAST search of NCBI. Partial purification of CatR 1. After harvesting 100 ml of E. coli BL21 (pSK251), the cells were suspended in 5 ml of 50 mM Tris–HCl (pH 8.0) and broken by two cycles through a French pressure cell (SLM-AMINCO, Urbana, IL) at 15,000 psi. The crude extract was centrifuged at 15,000g for 50 min. Supernatant fractions were loaded onto a HR5/5 MonoQ column of FPLC (Pharmacia, Uppsala, Sweden). Protein was eluted with a linear gradient of NaCl from 0.1 to 1.0 M at the flow rate of 1.0 ml/min for 10 min. Each fraction (1.0 ml) was collected and used for SDS–PAGE to identify CatR 1. CatR 1 fraction was used for gel retardation assay. PCR and radioactive labeling of DNA. DNA fragments containing catB 1, catA 1, catB 2 or catA 2 promoter regions were obtained by PCR amplification with eight primers listed on the Table 1. PCR reactions were performed for 25 cycles of 1 min at 95°C, 1 min at 50°C and 1 min at 72°C. PCR products were purified on the 1% agarose gel. End labeling of PCR products was performed by Mega labeling kit (Takara, Japan). [␥- 32P]ATP (370 MBq/ml) of NEN (U.S.A.) was used for labeling.
TABLE 2
Plasmids Used in This Study Plasmid
Characteristics
Source or reference
pUC118 pET-3 pCD1-1 pCD12 pCD13 pCD14 pSK251 pSK264 pSK265 pSK266
Cloning vector, Ap r T7 RNA polymerase-based expression vector, Ap r pUC118 ⫹ Sau3 A fragment of cat 1 genes pUC118 ⫹ SacI–KpnI fragment of pCD1-1, catB 1C 1A 1 pUC118 ⫹ SacI–HindIII fragment of pCD1-1, catB 1C 1 pUC118 ⫹ HindIII–XbaI fragment of pCD1-1, catD pET-3 ⫹ catR 1(oligo#120 & oligo#121) pET-3 ⫹ catB 1(oligo#148 & oligo#149) pET-3 ⫹ catC 1(oligo#150 & oligo#151) pET-3 ⫹ catD (oligo#152 & oligo#153)
Takara Stratagene 1 1 1 This study This study This study This study This study
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The Summary of DNA Sequencing of cat 1 Gene Cluster in Plasmid pCD 1-1 Gene name
Position
Amino acid residues
MW (Da)
Putative porine CatR 1 CatB 1 CatC 1 CatA 1 CatD ORF1 ORF2
1,699–2,733 3,119–4,030(reverse) 4,215–5,354 5,384–5,674 5,778–6,713 6,896–7,693 7,711–8,964 8,961–(10,157)
344 a.a. 303 a.a. 379 a.a. 96 a.a. 311 a.a. 265 a.a. 417 a.a. (?)
36,520 34,264 40,788 11,047 33,376 28,880 45,688 (?)
Note. Total DNA 10,157 bp.
Gel retardation assay. Gel retardation assays were analyzed by the modified methods of Kaufman et al. (10). Labeled DNA fragments (catB 1, catA 1, catB 2 and catA 2 promoter regions) were mixed with partially purified CatR 1 in a total volume of 20 l and incubated at 22–25°C for 30 min. 20 l of binding reaction mixture consisted of 10 l of 2⫻ DNA–protein binding buffer (40 mM Tris–HCl, pH 7.9, 100 mM NaCl, 20% glycerol and 0.2 mM DTT), 1.5 l of competitor DNA [1 mg/ml poly(dI-dC)], 1.0 l of BSA (1 mg/ml), labeled DNA (0.1 pmol; about 1 ⫻ 10 5 to 1 ⫻ 10 6 cpm) and 0.1 g of CatR 1. Reaction mixtures were electrophoresed in a 4% polyacrylamide gel for 2 h at 100 V, using TBE as a running buffer. DNA migration was monitored by PhosphoImager of Molecular Dynamics.
than a 30% homology with putative porins of Alcaligenes eutrophous (I39570) and Pseudomonas cepacia (JC6314). The function of the putative porin for aniline metabolism warrants further intensive study. The other ORFs (ORF1 and ORF2) have a similar gene structure with ORFs on the cat genes of A. calcoaceticus ADP1 (11). The ORF1 has about 48% sequence homology with ORF1 (P07776) of A. calcoaceticus ADP1. The ORF2 has more than 399 a.a. (not including the stop codon in the 10-kb DNA fragment) and about 50% sequence homology with ORF2 (P07777) in 300 a.a. of N-terminal region. Mutagenesis of two ORFs in A. calcoaceticus ADP1 was reported not to make any effect on growth in benzoate, catechol, p-hydroxybenzoate and muconate except for accumulation of catechol (11). Though the function of two ORFs of A. calcoaceticus ADP1 was not clear, it is interesting that A. lwoffii K24 has similar genes in cat gene region. However, cat gene structure (catR 1B 1C 1A 1D) of A. lwoffii K24 is similar with that found in Frateuria sp. ANA-18 or P. putida PRS2000. Comparative analysis of cat genes with others suggests that cat 1 genes of A. lwoffii K24 might have been evolved in the different environment from that A. calcoaceticus ADP1 and P. putida PRS2000 had experienced. Overexpression of cat Genes in E. coli BL21
RESULTS AND DISCUSSION DNA Sequence Analysis of cat 1 Gene Region The DNA sequence of the 10,157-bp cat 1 gene region was analyzed and summarized in Table 3. In addition to five cat genes, three ORFs were found around the cat genes (Fig. 1). CatD that consists of 265 a.a. shared 97% identity with catD of Frateuria sp. ANA-18, 45% with pcaD of Pseudomonas aeruginosa PAO1 and 43% with pcaD of Acinetobacter calcoaceticus ADP1. ORF (ORFporin) located immediately upstream catR 1 has an estimated molecular weight of 36,520 Da (344 a.a.). Sequence analysis showed that the ORFporin has more
When the four plasmids (pCD1-1, pCD12, pCD13 and pCD14) were under IPTG induction for overexpression of cat genes, we could detect only catA 1 gene product on SDS–PAGE (Fig. 2). Therefore, four expression vectors (pSK251, pSK264, pSK265, and pSK266) were constructed for the expression of other cat gene products (CatR 1, CatD, CatC 1, and CatB 1). From E. coli BL21 containing these vectors, overexpressed protein bands were detected and analyzed by N-terminal amino acid sequencer (Fig. 3). We could confirm the expression of CatR 1, CatD, CatC 1 and CatB 1 with N-terminal amino acid sequences.
FIG. 1. Organization of 10-kb cat 1 gene portion in A. lwoffii K24. Eight ORFs are shown, and arrows below the ORF designations indicate the direction of transcription. Plasmid pCD12, pCD13, and pCD14 were subcloned from pCD1-1. Restriction endonuclease: Sp, SphI; Sa, SacI; Sm, SmaI; E, EcoRI; N, NheI; H, HindIII; K, KpnI. 647
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FIG. 2. Induction of protein product of cat 1 gene under lac promoter of pUC118. Lane 1, pCD14; lane 2, pCD13; lane 3, pCD12; lane 4, pCD1-1; lane 5, E. coli DH5␣ as control.
Characterization of catR 1 Binding to cat Genes of A. lwoffii K24 In the previous study, CatR 1 of A. lwoffii K24 was shown to have a significant homology to other LysR family regulators including CatRs (12). To confirm the function of CatR 1 of A. lwoffii K24 as a transcriptional activator, gel retardation assay was performed using partially purified CatR 1. When crude proteins containing CatR 1 were applied to MonoQ column (0.5 by 5 cm; Pharmacia), partially purified catR 1 was eluted at 0.85 M NaCl. CatR of P. putida was eluted at about 0.9 M on an S-Sepharose column (13). Four promoter regions of
FIG. 3. Expression of CatR 1, CatD, CatC 1, and CatB 1 by using the T7 RNA polymerase promoter of pET vector. Arrowheads indicate the overexpressed proteins. Lane 1, pSK251 (CatR 1); lane 2, pSK266 (CatD); lane 3, pSK265 (CatC 1); lane 4, pSK264 (CatB 1); lane 5, pCD12 (CatA 1). N-terminal sequences of proteins: CatR 1, 1 XDLRQ 5; CatD, 1MASMYE 6; CatC 1, 1MLFHVR 6; CatB 1, 1MSSVT 5. Because NheI site was used for construction of expression vector (pSK266), vector sequence (MAS) was attached to N-terminal sequence of CatD.
FIG. 4. Gel retardation assay of CatR 1 binding to the catB 1, catB 2, catA 1, and catA 2 promoter region. CatB 1 promoter region, 222-bp DNA fragment (lanes 1 and 2); catA 1 promoter region, 455-bp DNA fragment (lanes 3 and 4); catA 2 promoter region, 329-bp DNA fragment (lanes 5 and 6); catB 2 promoter region, 236-bp DNA fragment (lanes 7 and 8). Gel retardation assay was performed with 0.1 g of partially purified CatR 1 and 32P-labeled DNA fragments (lanes 1, 3, 5, and 7). C1, faster migrating complex; C2, slower migrating complex.
cat 1 and cat 2 genes (catB 1, catA 1, catB 2 and catA 2) were amplified by PCR reaction, 32P-labeled and used in gel retardation assay in order to know whether these DNA fragments interact with CatR 1. CatR 1 could bind on the catB 1 promoter region (Fig. 4). As reported in gel retardation assay of P. putida, two complexes (C1 and C2) were detected on the gel (13). C1 complex was only shown when gel retardation assay was performed without cis,cis-muconate as inducer and was disappeared with increasing cis,cis-muconate. However, contrary to CatR of P. putida (14), CatR 1 had no binding activity to catA 1 promoter region (Fig. 4). The results of gel retardation as well as expression pattern of catA 1 gene products in E. coli DH5␣ suggest that catA 1 may be under independent transcriptional regulation without CatR 1. Because A. lwoffii K24 has other cat genes (cat 2 genes), we performed gel retardation assay to know whether CatR 1 can interact with catB 2 gene promoter region. Interestingly CatR 1 was capable of binding catB 2 promoter region (Fig. 4) but did not interact with catA 2 promoter region. These results imply that CatR 1 of A. lwoffii K24 can cross-interact with two catB genes in the cell. The ability of CatR 2 to interact with cat genes also should be studied. CatR 2 of A. lwoffii K24 has been cloned by PCR using the sequences of catR 2 of Frateuria sp. ANA-18 (1) because the two catRs have a very high sequence homology (98%). Target mutagenesis and complementation test are being conducted to understand the function of two CatRs in A. lwoffii K24. Cross-interaction of LysR transcriptional activator was well studied in P. putida PRS2000, which has CatR and ClcR as a transcriptional activator of cat and clc genes for catabolism of catechol, respectively (15). In this strain, CatR and ClcR could interact with other pathway genes. Additionally, CatR could complement a ClcR ⫺ mutant. In conclusion, this study revealed that complete DNA sequence of the 10-kb cat 1 operon-
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containing gene cluster in A. lwoffii K24 was organized into ORF porin, catR 1, catB 1C 1A 1D, ORF1, and ORF2. Gel retardation assay revealed that CatR 1, the putative regulator for the cat B 1C 1A 1 operon might function as a transcriptional activator for expression of cat 1 genes as well as cat 2 genes. REFERENCES
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9.
10.
1. Murakami, S., Takashima, A., Takemoto, J., Takenaka, S., Shinke, R., and Aoki, K. (1999) Cloning and sequence analysis of two catechol-degrading gene clusters from the aniline-assimilating bacterium Frateuria species ANA-18. Gene 226, 189 –198. 2. Fukumori, F., and Saint, C. P. (1997) Nucleotide sequences and regulation analysis of genes involved in conversion of aniline to catechol in Pseudomonas putida UCC22 (pTDN1). J. Bacteriol. 179, 399 – 408. 3. Fujii, T., Takeo, M., and Maeda, Y. (1997) Plasmid-encoded genes specifying aniline oxidation from Acinetobacter sp. Strain YAA. Microbiology 143, 93–99. 4. Harwood, C. S., and Parales, R. E. (1996) The -ketoadipate pathway and the biology of self-identity. Annu. Rev. Microbiol. 50, 553–590. 5. Kim, S. I., Ha, K. S., and Leem, S. H. (1999) Differential organization and transcription of cat 2 gene cluster in Acinetobacter lwoffii K24. J. Biosci. Bioeng. 88, 250 –257. 6. Kim, S. I., Leem, S. H., Choi, J. S., Chung, Y. H., Kim, S., Park, Y. M., Park, Y. K., Lee, Y. N., and Ha, K. S. (1997) Cloning and characterization of two catA genes in Acinetobacter lwoffii K24. J. Bacteriol. 179, 5226 –5231. 7. Aoki, K., Konohana, T., Shinke, R., and Nishira, H. (1984) Two
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catechol 1,2-dioxygenases from aniline-assimilating bacterium, Frateuria species ANA-18. Agric. Biol. Chem. 48, 2097–2104. Kim, S. I., Kim, S.-J., Nam, M. H., Kim, S., Ha, K.-S., Oh, K.-H., Yoo, J.-S., and Park, Y.-M. (2001) Proteome analysis of anilineinduced proteins in Acinetobacter lwoffii K24. Curr. Microbiol., in press. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Kaufman, P. B., Wu, W., Kim, D., and Cseke, L. J. (1995) Handbook of Molecular and Cellular Methods in Biology and Medicine, CRC Press, Boca Raton, FL. Neidle, E. L., Hartnett, C., and Ornston, L. N. (1989) Characterization of Acinetobacter calcoaceticus catM, a repressor gene homologous in sequence to transcriptional activator genes. J. Bacteriol. 171, 5410 –5421. Kim, S. I., Leem, S. H., Choi, J. S., and Ha, K. S. (1998) Organization and transcriptional characterization of the cat 1 gene cluster in Acinetobacter lwoffii K24. Biochem. Biophys. Res. Commun. 243, 289 –294. Parsek, M. R., Shinabarger, D. L., Rothmel, R. K., and Charkrabarty, A. M. (1992) Roles of CatR and cis,cis-muconate in activation of the catBC operon, which is involved in benzoate degradation in Pseudomonas putida. J. Bacteriol. 174, 7798 – 7806. Houghton, J. E., Brown, T. M., Appel, A. J., Hughes, E. J., and Ornston, L. N. (1995) Discontinuities in the evolution of Pseudomonas putida cat genes. J. Bacteriol. 177, 401– 412. Parsek, M. R., McFall, S. M., Shinabarger, D. L., and Charkrabarty, A. M. (1994) Interaction of two LysR-type regulatory proteins CatR and ClcR with heterologous promoters: Functional and evolutionary implications. Proc. Natl. Acad. Sci. USA 91, 12393–12397.
Complete Nucleotide Sequence and Overexpression of cat 1 Gene Cluster, and Roles of the Putative Transcriptional Activator CatR1 in Acinetobacter lwoffii K24 Capable of Aniline Degradation Seung Il Kim,* ,1 Yong-Cheol Yoo,* and Hyung-Yeel Kahng† *Biomolecule Research Team, Korea Basic Science Institute, Daejon 305-333, Korea; and †Research Institute for Basic Sciences, Cheju National University, Jeju 690-756, Korea
Received September 17, 2001
The aniline-assimilating bacterium Acinetobacter lwoffii K24 has two cat gene clusters (cat 1 and cat 2). In this study, we completely sequenced 10-kb DNA fragment of cat 1 genes of A. lwoffii K24, which had been cloned in plasmid pCD1-1. Sequence analysis revealed that the order of genes in the cat 1 operon-containing gene cluster was ORF porin, catR 1, catB 1C 1A 1D, ORF1, and ORF2. Two ORFs located immediately downstream catD were most similar with two ORFs in cat gene cluster of Acinetobacter calcoaceticus ADP1 but the gene structure of catR 1B 1C 1A 1 was closest to that found in Frateurua sp. ANA-18 or Pseudomonas putida PRS2000. CatA 1 gene product was significantly overexpressed and detected in SDS–PAGE when four cat 1 genes (catB 1C 1A 1D) were placed under the control of a lac promoter in pUC118 while overexpressions of other cat genes were accomplished under the control of a lac promoter in pET vector system. All gene products were verified by N-terminal amino acid sequencing. Gel retardation assay revealed that the putative regulatory gene activator CatR 1 for the catB 1C 1A 1 operon could bind the promoter region of catB 2 as well as catB 1, suggesting that transcription of catB 1 or catB 2 might be controlled by the putative gene activator CatR 1. However, the promoter regions of catA 1 and catA 2 were found to have no affinity with catR 1. © 2001 Academic Press
Key Words: Acinetobacter lwoffii K24; aniline degradation; cat1 gene cluster; CatR1; overexpression; gel retardation assay.
Previous studies demonstrated that aniline is degraded to catechol, which can be metabolized by ortho cleavage (-ketoadipate) pathway or meta cleavage To whom correspondence should be addressed. Fax: ⫹82-42-8653451. E-mail: [email protected]. 1
pathway by dioxygenases (1–3). It has been reported that the -ketoadipate pathway is accomplished by a chromosomally encoded biodegradation gene cluster while the genes of meta cleavage pathway are on the catabolic plasmid (4). The -ketoadipate pathway genes are diversely distributed in different microorganisms, along with their various organizations, transcriptions and regulation patterns (4, 5). Notably, two copies of degrading genes have been found in several aniline degrading bacteria containing -ketoadipate pathways (6, 7). Acinetobacter lwoffii K24 was also found to have two copies of catRABC (cat 1 and cat 2 genes) on the chromosome (6). Though two cat gene clusters, cat1 and cat2 demonstrate different order of gene arrangements (catB 1C 1A 1 and catB 2A 2C 2) and different transcriptional patterns, they were simultaneously expressed in response to aniline (8). Two catRs (catR 1 and catR 2) in A. lwoffii K24 were assumed to be transcriptional activators in that each cat gene cluster has its own catR and catR binding site between catR and catB. However, roles of two cat gene clusters in the cell are not clear yet. In our effort to elucidate the role of the gene clusters in A. lwoffii K24, we analyzed the complete DNA sequence containing 10-kb containing cat 1 gene clusters and report in this study. Furthermore, possible roles of two CatRs also will be described based on expression pattern of cat genes and gel retardation assay using CatR 1. MATERIALS AND METHODS DNA sequencing and analysis. The nucleotide sequences of 10-kb containing cat 1 genes were determined by the dideoxy-chain termination methods using an ABI Prism DyeDeoxy Terminator cycles sequencing kit (Perkin–Elmer). Sequencing reactions were prepared according to the supplier’s instructions and analyzed by electrophoresis using the Perkin Elmer Model 377 DNA sequencer. Mac DNASIS of Hitachi Software was used for DNA analysis. The nucleotide sequence has been submitted to the GenBank database under the Accession No. U77658.
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0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Vol. 288, No. 3, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
PCR Primers for Overexpression and Amplification of Promoter Regions Gene CatR 1 CatB 1 CatC 1 CatD 1 CatB 1 promoter CatA 1 promoter CatB 2 promoter CatA 2 promoter
Primer
Sequence
Oligo#120 Oligo#121 Oligo#148 Oligo#149 Oligo#150 Oligo#151 Oligo#152 Oligo#153 Oligo#137.1 Oligo#138.1 Oligo#154.1 Oligo#113.1 Oligo#139.1 Oligo#140.1 Oligo#116.1 Oligo#11
5⬘-CTC TAT CAC ATA TGG ATC TGC GCC AGT TTC G-3⬘ 5⬘-AGC TGG ATC CCG TCT ATC AAT TGT GAG T-3⬘ 5⬘-CTC TAT CAC ATA TGT CCA GTG TAA CGA TT-3⬘ 5⬘-AGC TGG ATC CTT ACG CCT TCG TGA CCT T-3⬘ 5⬘-CTC TAT CAC ATA TGC TTT TCC ATG TAC GC-3⬘ 5⬘-AGC TGG ATC CTC ACG ACT CGT CGT CGC G-3⬘ 5⬘-ACT ATG GCT AGC ATG TAT GAA GGC GAA CGG-3⬘ 5⬘-AGC TGG ATC CGA TTT CTC ACT CGA TCA G-3⬘ 5⬘- 4001GAC GAA GTA GCG AAA CTG 4018-3⬘ 5⬘- 4222CTG GAC ATG TCG CAG GT 4206-3⬘ 5⬘- 5331CTG ATC AAG GTC ACG AAG 5348-3⬘ 5⬘- 5785ATG CTC ATG ATG CCA GTC 5768-3⬘ 5⬘- 202GCT TTC CTC GAT CTG CTG 219-3⬘ 5⬘- 437GCT ATC ATT CCT TGC CCT 420-3⬘ 5⬘- 1429TTG CTG CTG ACC GAA GAA 1446-3⬘ 5⬘- 1757CTT CGA TCG TAT AGA ACA GAT CCC TGA CG 1728-3⬘
Overexpression of cat genes in E. coli. The oligonucleotide primers for overexpression of cat genes were listed on the Table 1. Forward primers have NdeI or NheI recognition site and reverse primers have BamH1 recognition site, respectively. PCR reactions were run for 25 cycle using the following condition; denaturation at 95°C for 1 min, annealing at 65°C for 1 min, and extension at 72°C for 1 min. PCR products were run on 1% agarose gel and purified using QIAquick Spin column (Qiagen). The PCR products were digested with NdeI (or NheI) and BamH1. The DNA fragments were ligated into plasmid pET-3 (Stratagene) treated with the same enzymes and ligated DNA mixtures were used to transform E. coli DH5␣. New vectors, pSK251 (containing catR 1), pSK264 (containing catB 1), pSK265 (containing catC 1) and SK266 (containing catD) were obtained (Table 2), and they were transformed into E. coli BL21 for overexpression. After the transformants were cultured in 100 ml LB broth containing Ap (50 g/ml) until A 600 nm is 0.4, 0.1 mM isopropyl-D-thiogalactopyranoside (IPTG) was added for protein induction. After induction of 4 – 6 h, cells were harvested and lysed for protein analysis as described in previous method (6). The protein was analyzed on 10% SDS–PAGE. Plasmid isolation, plasmid construction, and transformation were performed according to the protocol of Maniatis (9). Identification of protein products of cat genes. To verify the expression of cat gene products, crude extracts of E. coli transformants were loaded on the SDS–PAGE and electro-transferred
onto PVDF membrane by the ABI protocol. Protein bands were excised from the PVDF membrane and installed into the blot cartridge of a Model 491A protein sequencer (Perkin–Elmer, Foster City, CA) for sequencing analysis. The obtained N-terminal sequence was used for protein identification by BLAST search of NCBI. Partial purification of CatR 1. After harvesting 100 ml of E. coli BL21 (pSK251), the cells were suspended in 5 ml of 50 mM Tris–HCl (pH 8.0) and broken by two cycles through a French pressure cell (SLM-AMINCO, Urbana, IL) at 15,000 psi. The crude extract was centrifuged at 15,000g for 50 min. Supernatant fractions were loaded onto a HR5/5 MonoQ column of FPLC (Pharmacia, Uppsala, Sweden). Protein was eluted with a linear gradient of NaCl from 0.1 to 1.0 M at the flow rate of 1.0 ml/min for 10 min. Each fraction (1.0 ml) was collected and used for SDS–PAGE to identify CatR 1. CatR 1 fraction was used for gel retardation assay. PCR and radioactive labeling of DNA. DNA fragments containing catB 1, catA 1, catB 2 or catA 2 promoter regions were obtained by PCR amplification with eight primers listed on the Table 1. PCR reactions were performed for 25 cycles of 1 min at 95°C, 1 min at 50°C and 1 min at 72°C. PCR products were purified on the 1% agarose gel. End labeling of PCR products was performed by Mega labeling kit (Takara, Japan). [␥- 32P]ATP (370 MBq/ml) of NEN (U.S.A.) was used for labeling.
TABLE 2
Plasmids Used in This Study Plasmid
Characteristics
Source or reference
pUC118 pET-3 pCD1-1 pCD12 pCD13 pCD14 pSK251 pSK264 pSK265 pSK266
Cloning vector, Ap r T7 RNA polymerase-based expression vector, Ap r pUC118 ⫹ Sau3 A fragment of cat 1 genes pUC118 ⫹ SacI–KpnI fragment of pCD1-1, catB 1C 1A 1 pUC118 ⫹ SacI–HindIII fragment of pCD1-1, catB 1C 1 pUC118 ⫹ HindIII–XbaI fragment of pCD1-1, catD pET-3 ⫹ catR 1(oligo#120 & oligo#121) pET-3 ⫹ catB 1(oligo#148 & oligo#149) pET-3 ⫹ catC 1(oligo#150 & oligo#151) pET-3 ⫹ catD (oligo#152 & oligo#153)
Takara Stratagene 1 1 1 This study This study This study This study This study
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The Summary of DNA Sequencing of cat 1 Gene Cluster in Plasmid pCD 1-1 Gene name
Position
Amino acid residues
MW (Da)
Putative porine CatR 1 CatB 1 CatC 1 CatA 1 CatD ORF1 ORF2
1,699–2,733 3,119–4,030(reverse) 4,215–5,354 5,384–5,674 5,778–6,713 6,896–7,693 7,711–8,964 8,961–(10,157)
344 a.a. 303 a.a. 379 a.a. 96 a.a. 311 a.a. 265 a.a. 417 a.a. (?)
36,520 34,264 40,788 11,047 33,376 28,880 45,688 (?)
Note. Total DNA 10,157 bp.
Gel retardation assay. Gel retardation assays were analyzed by the modified methods of Kaufman et al. (10). Labeled DNA fragments (catB 1, catA 1, catB 2 and catA 2 promoter regions) were mixed with partially purified CatR 1 in a total volume of 20 l and incubated at 22–25°C for 30 min. 20 l of binding reaction mixture consisted of 10 l of 2⫻ DNA–protein binding buffer (40 mM Tris–HCl, pH 7.9, 100 mM NaCl, 20% glycerol and 0.2 mM DTT), 1.5 l of competitor DNA [1 mg/ml poly(dI-dC)], 1.0 l of BSA (1 mg/ml), labeled DNA (0.1 pmol; about 1 ⫻ 10 5 to 1 ⫻ 10 6 cpm) and 0.1 g of CatR 1. Reaction mixtures were electrophoresed in a 4% polyacrylamide gel for 2 h at 100 V, using TBE as a running buffer. DNA migration was monitored by PhosphoImager of Molecular Dynamics.
than a 30% homology with putative porins of Alcaligenes eutrophous (I39570) and Pseudomonas cepacia (JC6314). The function of the putative porin for aniline metabolism warrants further intensive study. The other ORFs (ORF1 and ORF2) have a similar gene structure with ORFs on the cat genes of A. calcoaceticus ADP1 (11). The ORF1 has about 48% sequence homology with ORF1 (P07776) of A. calcoaceticus ADP1. The ORF2 has more than 399 a.a. (not including the stop codon in the 10-kb DNA fragment) and about 50% sequence homology with ORF2 (P07777) in 300 a.a. of N-terminal region. Mutagenesis of two ORFs in A. calcoaceticus ADP1 was reported not to make any effect on growth in benzoate, catechol, p-hydroxybenzoate and muconate except for accumulation of catechol (11). Though the function of two ORFs of A. calcoaceticus ADP1 was not clear, it is interesting that A. lwoffii K24 has similar genes in cat gene region. However, cat gene structure (catR 1B 1C 1A 1D) of A. lwoffii K24 is similar with that found in Frateuria sp. ANA-18 or P. putida PRS2000. Comparative analysis of cat genes with others suggests that cat 1 genes of A. lwoffii K24 might have been evolved in the different environment from that A. calcoaceticus ADP1 and P. putida PRS2000 had experienced. Overexpression of cat Genes in E. coli BL21
RESULTS AND DISCUSSION DNA Sequence Analysis of cat 1 Gene Region The DNA sequence of the 10,157-bp cat 1 gene region was analyzed and summarized in Table 3. In addition to five cat genes, three ORFs were found around the cat genes (Fig. 1). CatD that consists of 265 a.a. shared 97% identity with catD of Frateuria sp. ANA-18, 45% with pcaD of Pseudomonas aeruginosa PAO1 and 43% with pcaD of Acinetobacter calcoaceticus ADP1. ORF (ORFporin) located immediately upstream catR 1 has an estimated molecular weight of 36,520 Da (344 a.a.). Sequence analysis showed that the ORFporin has more
When the four plasmids (pCD1-1, pCD12, pCD13 and pCD14) were under IPTG induction for overexpression of cat genes, we could detect only catA 1 gene product on SDS–PAGE (Fig. 2). Therefore, four expression vectors (pSK251, pSK264, pSK265, and pSK266) were constructed for the expression of other cat gene products (CatR 1, CatD, CatC 1, and CatB 1). From E. coli BL21 containing these vectors, overexpressed protein bands were detected and analyzed by N-terminal amino acid sequencer (Fig. 3). We could confirm the expression of CatR 1, CatD, CatC 1 and CatB 1 with N-terminal amino acid sequences.
FIG. 1. Organization of 10-kb cat 1 gene portion in A. lwoffii K24. Eight ORFs are shown, and arrows below the ORF designations indicate the direction of transcription. Plasmid pCD12, pCD13, and pCD14 were subcloned from pCD1-1. Restriction endonuclease: Sp, SphI; Sa, SacI; Sm, SmaI; E, EcoRI; N, NheI; H, HindIII; K, KpnI. 647
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FIG. 2. Induction of protein product of cat 1 gene under lac promoter of pUC118. Lane 1, pCD14; lane 2, pCD13; lane 3, pCD12; lane 4, pCD1-1; lane 5, E. coli DH5␣ as control.
Characterization of catR 1 Binding to cat Genes of A. lwoffii K24 In the previous study, CatR 1 of A. lwoffii K24 was shown to have a significant homology to other LysR family regulators including CatRs (12). To confirm the function of CatR 1 of A. lwoffii K24 as a transcriptional activator, gel retardation assay was performed using partially purified CatR 1. When crude proteins containing CatR 1 were applied to MonoQ column (0.5 by 5 cm; Pharmacia), partially purified catR 1 was eluted at 0.85 M NaCl. CatR of P. putida was eluted at about 0.9 M on an S-Sepharose column (13). Four promoter regions of
FIG. 3. Expression of CatR 1, CatD, CatC 1, and CatB 1 by using the T7 RNA polymerase promoter of pET vector. Arrowheads indicate the overexpressed proteins. Lane 1, pSK251 (CatR 1); lane 2, pSK266 (CatD); lane 3, pSK265 (CatC 1); lane 4, pSK264 (CatB 1); lane 5, pCD12 (CatA 1). N-terminal sequences of proteins: CatR 1, 1 XDLRQ 5; CatD, 1MASMYE 6; CatC 1, 1MLFHVR 6; CatB 1, 1MSSVT 5. Because NheI site was used for construction of expression vector (pSK266), vector sequence (MAS) was attached to N-terminal sequence of CatD.
FIG. 4. Gel retardation assay of CatR 1 binding to the catB 1, catB 2, catA 1, and catA 2 promoter region. CatB 1 promoter region, 222-bp DNA fragment (lanes 1 and 2); catA 1 promoter region, 455-bp DNA fragment (lanes 3 and 4); catA 2 promoter region, 329-bp DNA fragment (lanes 5 and 6); catB 2 promoter region, 236-bp DNA fragment (lanes 7 and 8). Gel retardation assay was performed with 0.1 g of partially purified CatR 1 and 32P-labeled DNA fragments (lanes 1, 3, 5, and 7). C1, faster migrating complex; C2, slower migrating complex.
cat 1 and cat 2 genes (catB 1, catA 1, catB 2 and catA 2) were amplified by PCR reaction, 32P-labeled and used in gel retardation assay in order to know whether these DNA fragments interact with CatR 1. CatR 1 could bind on the catB 1 promoter region (Fig. 4). As reported in gel retardation assay of P. putida, two complexes (C1 and C2) were detected on the gel (13). C1 complex was only shown when gel retardation assay was performed without cis,cis-muconate as inducer and was disappeared with increasing cis,cis-muconate. However, contrary to CatR of P. putida (14), CatR 1 had no binding activity to catA 1 promoter region (Fig. 4). The results of gel retardation as well as expression pattern of catA 1 gene products in E. coli DH5␣ suggest that catA 1 may be under independent transcriptional regulation without CatR 1. Because A. lwoffii K24 has other cat genes (cat 2 genes), we performed gel retardation assay to know whether CatR 1 can interact with catB 2 gene promoter region. Interestingly CatR 1 was capable of binding catB 2 promoter region (Fig. 4) but did not interact with catA 2 promoter region. These results imply that CatR 1 of A. lwoffii K24 can cross-interact with two catB genes in the cell. The ability of CatR 2 to interact with cat genes also should be studied. CatR 2 of A. lwoffii K24 has been cloned by PCR using the sequences of catR 2 of Frateuria sp. ANA-18 (1) because the two catRs have a very high sequence homology (98%). Target mutagenesis and complementation test are being conducted to understand the function of two CatRs in A. lwoffii K24. Cross-interaction of LysR transcriptional activator was well studied in P. putida PRS2000, which has CatR and ClcR as a transcriptional activator of cat and clc genes for catabolism of catechol, respectively (15). In this strain, CatR and ClcR could interact with other pathway genes. Additionally, CatR could complement a ClcR ⫺ mutant. In conclusion, this study revealed that complete DNA sequence of the 10-kb cat 1 operon-
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containing gene cluster in A. lwoffii K24 was organized into ORF porin, catR 1, catB 1C 1A 1D, ORF1, and ORF2. Gel retardation assay revealed that CatR 1, the putative regulator for the cat B 1C 1A 1 operon might function as a transcriptional activator for expression of cat 1 genes as well as cat 2 genes. REFERENCES
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