- Email: [email protected]
Molecular cloning and functional characterization of a novel decarboxylase from uncultured microorganisms
Biochemical and Biophysical Research Communications 357 (2007) 421–426 www.elsevier.com/locate/ybbrc
Molecular cloning and functional characterization of a novel decarboxylase from uncultured microorganisms Chengjian Jiang, Bo Wu
*
Guangxi Key Laboratory of Subtropical Bioresources Conservation and Utilization, The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, Guangxi University, 100 Daxue Road, Nanning, Guangxi 530004, People’s Republic of China Received 22 March 2007 Available online 3 April 2007
Abstract The metagenomic library approach has been used successfully to isolate novel biocatalyst genes from uncultured microorganisms. We report the cloning of a novel decarboxylase gene by sequence-based screening of a plasmid metagenomic library constructed with DNA from alkaline polluted soils. The gene was named undec1A and had an open reading frame of 1077 base pairs. It encoded a 359 amino acid polypeptide with a molecular mass of 38 kDa. The predicted protein had 58% similarity to a decarboxylase from Chlorobium phaeobacteroides BS1. The putative decarboxylase gene was subcloned into pETBlue-2 vector and overexpressed in Escherichia coli Tuner (DE3) pLac. The recombinant protein was purified to homogeneity. Functional characterization with liquid chromatography-mass spectrometry confirmed that the recombinant Undec1A protein catalyzed the decarboxylation of L-cysteine to form cysteamine. 2007 Elsevier Inc. All rights reserved. Keywords: Metagenomic library; Uncultured microorganisms; Sequence-based screening; Decarboxylase; Cysteamine
Soil microorganisms are the source of many important biocatalysts [1]. Recent studies have shown that one gram of soil may contain several thousand species of different microorganisms [2]. However, 99% of the bacteria in soil cannot be cultured with conventional methods, which exclude a large fraction of the soil microbial population from being harnessed [3]. Recently, a new strategy that involves the cloning of the total microbial genome, or ‘‘metagenome’’, directly isolated from natural environments into cultivable bacterium such as Escherichia coli is developed [4]. This approach does not require the in vitro culturing of microorganisms from environmental samples and can avoid the enrichment of dominant strains under specific selective conditions [5]. Various vectors, such as plasmids, fosmids, cosmids with small inserts and bacterial artificial chromosomes (BACs) with large inserts, have been used successfully to construct the metagenomic
*
Corresponding author. Fax: +86 (0771) 3237873. E-mail address: [email protected] (B. Wu).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.03.159
libraries [6]. Previous studies using this strategy have identified a large pool of genes of enzymes, such as lipase [7], esterase [8], hydrolase [9], amylase [10], alcohol/aldehyde dehydrogenase [11], and complex gene clusters encoding the enzymes in antibiotic production [12–14]. The most important advantage of the metagenomic libraries is that they contain genomes from the whole population of microorganisms in a specific sample rather than cultivated or enriched subpopulations and thus provide a more comprehensive collection of global microbiological information [15]. Decarboxylation is a common reaction in living organisms. Decarboxylase occurs in various catabolic and anabolic processes and is carried out by a variety of enzymes. Studying decarboxylase is of general biochemical interest. During the biosynthesis of coenzyme A, decarboxylation of (R)-4 0 -phospho-N-pantothenoylcysteine to form 4 0 -phosphopantotheine is one key step [16]. Decarboxylase is also an interesting target for the development of new antibacterial agents [17]. Several genes encoding different decarboxylases [18–20], such as AtHAL3a, Dfp, MrsD,
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C. Jiang, B. Wu / Biochemical and Biophysical Research Communications 357 (2007) 421–426
and EpiD proteins from different organisms, have been cloned and partially characterized by polymerase chain reaction (PCR) and comparative genomics methods. However, cloning of decarboxylases from uncultured microorganisms has not been reported. In this study, a plasmid metagenomic library was constructed from alkaline polluted soil samples. Through sequence-based screening of the DNA library, a gene encoding a novel decarboxylase (named Undec1A) was isolated. The deduced amino acid sequence had moderate similarity to a family of cysteine decarboxylase proteins. Biochemical analysis of the overexpressed recombinant protein revealed that Undec1A catalyzed decarboxylation of L-cysteine to form cysteamine. To our knowledge, this is the first report of a decarboxylase isolated from soil metagenome. Materials and methods Plasmid vectors, bacterium strains, and growth conditions. The soil metagenomic library was constructed with E. coli DH5a as the host and pGEM-3Zf (+) as the cloning vector. The protein expression vector was pETBlue-2, and E. coli Novablue (Novagen) strain was used as the screening host for pETBlue-2 bearing inserts. Bacterial strain for expression of recombinant protein was Tuner (DE3) pLac (Novagen). All the E. coli bacterial cultures were grown at 37 C on Luria-Bertani (LB) agar plates or in LB medium. Where appropriate, media were amended with 100 lg ampicillin/ml, 50 lg carbenicillin/ml, 15 lg tetracycline/ml, or 34 lg chloramphenicol/ml. DNA manipulation and protein analysis. All DNA manipulations, including cloning and subcloning, transformation of E. coli cells, and PCR were performed according to standard techniques [21] or following the manufacturer’s instructions unless indicated otherwise. Protein preparation and analysis including protein extraction from E. coli, protein quantification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) were performed as described in the standard protocols [22]. DNA extraction from soil samples and construction of metagenomic library. Soil samples were collected from the ground surface of a stream located in Guangxi Province, South China. The ground soil has been enriched with alkaline pollutants for over 20 years. Water drainage from the soil sample has a pH of approximately 9.5. The metagenomic DNA was extracted from the alkaline polluted soils following the direct lysis method of Zhou [23] and Park [24]. Briefly, the DNA pellet from 5 g of soil sample was washed twice with 75% ethanol, air dried and dissolved in 1· TE buffer (10 mM Tris–HCl pH 8.0 and 1 mM Na2EDTA). To remove the contaminants, the crude metagenomic DNA extract was further purified on a 0.6% low-melting agarose gel, which was run at 60 V for 3 h. DNA fragments (ca. 23 kb) were recollected from the gel. The purified soil DNA was partially digested with EcoRI followed by size fractionation with lowmelting agarose gel as described [21]. Fractions containing DNA fragments of 1.0–15 kb were ligated into EcoRI-cleaved and dephosphorylated pGEM-3Zf (+) vector, and the ligation products were used to transfect E. coli DH5a. After overnight growth on LB agar plates containing ampicillin at 100 lg/ml, white colonies harboring plasmids bearing inserts were collected and used to construct the metagenomic library. This library was stored at 80 C until screening. DNA sequence analysis, database search and gene structure characterization. DNA sequence analysis was performed with the BigDye Terminator Cycle sequencing kit on an ABI Prism 3700 DNA analyzer (Applied Biosystems, USA). Protein translation was carried out with the web-based translation tool at the Expasy homepage (http://www.expasy.org/tools/ dna.html). Sequence predictions were retrieved from the protein and nucleotide databases at the National Center for Biotechnology Informa-
tion (NCBI) Entrez page (http://www.ncbi.nlm.nih.gov/Entrez/). Sequence similarity searches were performed with the BLAST 2.0 program. Amino acid sequence alignment of Undec1A with homologous proteins was done with the Align X program, a component of the Vector NT1 suite (Informax, North Bethesda, MD, USA) using the blosum62mt2 scoring matrix. Based on the comparison of the deduced amino acids, a putative gene (undec1A) encoding a novel decarboxylase was identified and further characterized. A phylogenetic tree was constructed using the neighbor-joining method with Molecular Evolutionary Genetics Analysis 3.1 software (MEGA, Version 3.1) [25]. Boot-strapping value was used to estimate the reliability of phylogenetic reconstructions (1000 replicates). Overexpression and purification of the recombinant decarboxylase protein. The Undec1A nucleotide sequence was amplified from plasmid (pGEXC0426) isolated from a decarboxylase producing clone. PCR was carried out in a total volume of 50 ll containing 2.5 mM MgCl2, 10 mM Tris–HCl (pH 8.4), 50 mM KCl, 0.2 mM each dNTP, 0.4 lM each primer, 1.0 U Vent DNA polymerase (NEB, USA), and 10 ng plasmid template. Restriction enzyme sites (underlined) for SalI and MluI were designed in the forward primer/reverse primer (5 0 -ATAGTCGACATGATCACCCC TCTTACGCTGGCAAC-3 0 /5 0 -CGACGCGTGTGAACCAGGGTAAGT ATCTTCCG-3 0 ). The PCR cycling consisted of a denaturation step (96 C for 2 min), 30 cycles of 94 C for 40 s, 65 C for 30 s and 72 C for 2 min, and a final extension step at 72 C for 10 min. After amplification, the PCR product mixture was digested with SalI and MluI and ligated directly into pETBlue-2 (Novagen) expression vector cleaved with the same enzymes. The resulting recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and plated on LB selection plates. After overnight incubation at 37 C, positive white colonies were picked for isolation of recombinant expression plasmid, which was subsequently introduced into E. coli Tuner (DE3) pLac (Novagen) to express the target protein. The transformed bacterial cells were cultured in LB medium containing carbenicillin 50 lg/ml and chloramphenicol 100 lg/ml at 37 C, and protein expression was induced by the addition of 1 mM isopropyl-b-Dthiogalactopyranoside when the optical density at 660 nm reached 0.6. After incubation for additional 6 h, the cells were harvested, washed twice with phosphate-buffered saline (pH 7.6), and lysed by sonication in 10 ml of 20 mM Tris–HCl, pH 8.0. The lysate was centrifuged twice at 30,000g for 20 min at 4 C and 1 ml of the supernatant was diluted with 5 ml of column buffer (20 mM Tris–HCl, pH 8.0, 10 mM imidazole, 300 mM NaCl) and applied onto an equilibrated nickel nitrilotriacetic acid (Ni– NTA) column containing 1 ml of Ni–NTA–agarose (Novagen). After a wash with 5 ml of column buffer containing 10 mM imidazole, the recombinant protein was eluted with the same buffer but containing 250 mM imidazole and the yellow peak fraction (about 600 ll) was collected. 1,4-Dithiothreitol (DTT) (Promega) was added to a final concentration of 5 mM into the protein immediately after its elution from the column. The isolated protein was further purified by gel filtration. A 25 ll aliquot of the Ni–NTA collection was loaded onto a Superdex 200 PC 3.2/ 30 column equilibrated with the running buffer (20 mM Tris–HCl, pH 8.0, 200 mM NaCl) and eluted at a flow rate of 40 ll/min. The Superdex 200 PC 3.2/30 column and the standard proteins used for calibration were obtained from Amersham Pharmacia Biotech, USA. The protein concentration was determined by a Bio-Rad protein assay kit with bovine serum albumin as the standard. The protein purified with Ni–NTA column and gel filtration was used for enzyme activity assays. Identification of the decarboxylation product. Liquid chromatography–mass spectrometry (LC–MS) was used to identify the decarboxylation product of the Undec1A recombinant protein. The LCQ mass spectrometer (Thermo Finnigan, San Jose, CA) was equipped with an electrospray ionization source and coupled with a HP 1100 HPLC system (Hewlett Packard, USA). The enzymatic reaction mixture contained 5 mM DTT, 1 mg/ml L-cysteine, 20 mM Tris–HCl, pH 7.0, and ca. 40 lg purified recombinant enzyme protein. The reaction was conducted in a total volume of 5.0 ml at 35 C for 30 min. Upon termination of the reaction, the residual protein was removed by centrifugation through a membrane (Vivaspin 500, Vivascience). The filtrate was separated on a C8 reverse phase column (150 · 4.6 mm,
C. Jiang, B. Wu / Biochemical and Biophysical Research Communications 357 (2007) 421–426 5 lm) (Thermo Finnigan, San Jose, CA) with acetic acid (0.02 mol/l) and acetonitrile (94:6, v/v) as the mobile phase at the flow rate of 0.2 ml/min. Mass spectrum data were acquired in positive mode with one full scan followed by two data-dependent scans of the most intense and the second most intense ions.
Results and discussion Construction of the metagenomic library The metagenomic DNA was directly extracted from alkaline polluted soils. The constructed library should contain a genome pool of all the microorganisms in the soil, including those of uncultivable bacteria. Approximately 15 lg of DNA was obtained from 1 g of soil sample. Majority of the DNA molecules had a size about 23 kb, which was shown as a strong band on the agarose gel (data
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not shown). However, the crude DNA extraction solution appeared yellowish dark brown due to possible contamination of water-soluble organic substances in the soil, which could hamper further enzymatic manipulation. To obtain more pure DNA, the crude extract was further separated on a low-melting agarose gel. The DNA collected from the agarose gel was partially digested with EcoRI and ligated with the EcoRI-cleaved pGEM-3Zf (+) vector. The final plasmid metagenomic library constructed from 1 lg purified soil DNA contained approximately 30,000 clones. In addition, restriction analysis of randomly chosen recombinant plasmids revealed a high level of diversity of the foreign DNA fragments in pGEM-3Zf (+). When EcoRI digested plasmids from 16 randomly picked recombinant positive plasmids were analyzed on an agarose gel, sizes of the inserts ranged from 1 kb to 15 kb, with an average of ca. 3.5 kb.
Fig. 1. Sequence alignment of Undec1A protein with other decarboxylases in the HFCD family. Decarboxylases are identified by their GenBank accession number. The highly conserved regions of insertion His motif, PXMNXXMW motif, substrate recognition clamp, dimerization motif, AAVAD motif and KXKK motif are boxed in shadow.
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Bioinformatics analysis and the cloning of a decarboxylase gene The environmentally derived plasmid library was grown in microplates. The bacteria were pelleted and lysed, and some randomly chosen recombinant plasmids were sequenced and analyzed. Totally, the DNA sequence analysis and database search revealed that 2 out of the 25 ORFs or predicted genes had over 80% similarities to known genes, and the other 23 recombinant plasmids had no good match with database sequences (detailed data not shown). This result confirmed that the metagenomic library contained DNA molecules from uncharacterized genomes and also demonstrated that the metagenome of naturally occurring microbacteria contained an immense pool of genes, most of which are not represented by pure and enrichment cultures established under certain selective conditions [26]. We identified an interesting clone, named pGEXC0426, and had its insert further characterized. The insert DNA had a length of 1146 base pairs (bp), and it had no good match with known genes at DNA level in the database. However, it was most similar to a cysteine decarboxylase (GenBank Accession No. AAQ66840) with 56% identity and 70% similarity at amino acid level. Based on the database comparison, we considered the cloned gene on pGEXC0426 novel and named it undec1A. The gene had a long open reading frame of 1077 bp. The deduced peptide had a predicted molecular mass of 38 kDa and an isoelectric point of 6.04. The undec1A nucleotide sequence has been deposited in the GenBank under Accession No. EF015465. When the deduced amino acid sequence of Undec1A was searched against the NCBI and Expasy databases, it was found that Undec1A had some similarity with cysteine decarboxylases from
Porphyromonas gingivalis W83 and Cytophaga hutchinsonii and several cysteine decarboxylases belonging to the homooligomeric flavin-containing cysteine decarboxylase (HFCD) protein family. These cysteine decarboxylases in HFCD protein family include DNA/pantothenate metabolism flavoprotein (GenBank Accession No. ZP_00532913) from Chlorobium phaeobacteroides BS1 (58% identitical and 74% similar), cysteine decarboxylase (GenBank Accession No. AAQ66840) from P. gingivalis W83 (56% identitical and 70% similar), PPC decarboxylase (GenBank Accession No. ZP_00307777.1) from C. hutchinsonii (50% identitical and 66% similar), and cysteine decarboxylase (GenBank Accession No. YP_037930.1) from Bacillus thuringiensis serovar konkukian strain 97-27 (46% identitical and 64% similar). Multiple alignments of the deduced amino acids of Undec1A with the most homologous decarboxylase proteins (above 60% similarity) in the HFCD family (NCBI database) were presented in Fig. 1. The putative protein had the EpiD conserved domain [19] for the catalytic activity of decarboxylases. Furthermore, amino acid sequence comparison revealed that the deduced Undec1A peptide shared the flavin-binding motif, the conserved active-site residues, and decarboxylation participating cysteine residues with other decarboxylase members in the HFCD protein family. Therefore, Undec1A should be classified as a new member of the HFCD family. Fig. 2 presents the phylogenetic tree, showing relationship with other decarboxylases, based on amino acids. The phylogenetic tree also revealed that Undec1A was most closely related to the decarboxylase of P. gingivalis W83 and it clustered together with the HFCD proteins from several bacterium species of C. hutchinsonii and Bacillus thuringiensis.
Fig. 2. Phylogenetic relationship of Undec1A protein with other decarboxylases in the HFCD family. The phylogenetic tree was constructed by the neighbor-joining method with MEGA, version 3.1, software. The numbers associated with the branches refer to bootstrap values (confidence limits) representing the substitution frequencies per amino acid residue. Decarboxylases are identified by their GenBank accession number.
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Overexpression and purification of recombinant Undec1A In order to investigate the biochemical properties of Undec1A, the gene was subcloned in frame with a six-histidine tag sequence into expression vector pETBlue-2 and expressed in E. coli Tuner (DE3) pLacI. The initial analysis with crude cell lysate showed that the bacteria containing recombinant plasmid pETBlue-2-undec1A produced a substantial amount of the expected recombinant protein, while this protein was not detectable in the culture of the bacteria containing the parent vector pETBlue-2. The recombinant Undec1A protein was initially purified with Ni–NTA magnetic agarose chromatography. Degraded and/or nonspecifically bound polypeptides in the elutes from Ni–NTA agarose column were removed by subsequent gel filtration chromatography (GFC). As shown in Fig. 3, the recombinant Undec1A protein was purified to homogeneity, as demonstrated by a single band (ca. 38 kDa) on SDS–
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PAGE gel. The decarboxylase was purified about 281.5fold to homogeneity with an overall enzyme yield of 20.8%. Functional characterization of recombinant Undec1A Through reaction product identification by LC–MS, our initial studies confirmed that the purified recombinant Undec1A protein had cysteine decarboxylase activity. As shown in Fig. 4, the enzymatic product showed a molecular ion at m/z 78.0, which is the same as the protonated cysteamine, suggesting cleavage of the –COOH group from Lcysteine. HighChem Mass Frontier software 3.0 (Thermo Finnigan, San Jose, CA) was used to verify that the molecular ion observed correlated with the proposed structure. The mass spectrum of the reaction product is identical to that of cysteamine. These results confirmed that Undec1A protein catalyzed the formation of cysteamine from L-cysteine. Our study of Undec1A may provide new insight into the relationship between the sequence, structure, and activity of HFCD protein family. For example, several bacterial conserved regions in the HFCD family are the N-terminal G-G/S-I-A-X-Y-K motif, PXMNXXMW motif, the substrate recognition clamp, and the histidine insertion motif, dimerization motif, AAVAD motif, and KXKK motif. However, the G-G/S-I-A-X-Y-K motif of the deduced Undec1A peptide is missing (Fig. 1). The absence of the N-terminal motif may affect its enzymatic activity. It would be interesting to elucidate the catalyzing mechanism through studies of the 3D structures of Undec1A protein with X-ray diffraction methods. Conclusion
Fig. 3. The SDS–PAGE of Undec1A. Proteins were separated by 12% SDS–PAGE and then stained with Coomassie brilliant blue G-250. Lane 1, molecular weight standards. Lane 2, purified recombinant Undec1A protein. The Undec1A protein is indicated by the black arrow.
It is well accepted that the field of metagenomic gene discovery offers enormous potential for fundamental microbiology study and biotechnology development. We constructed a plasmid library from alkaline polluted soils and cloned a novel decarboxylase gene (undec1A) by sequence-based screening strategy. Sequence analysis demonstrated that the identified gene product was
Fig. 4. Mass spectrum of the reaction product of Undec1A with L-cysteine as the substrate. The electrospray ionization conditions were: sheath gas flow rate, 80 arbitrary units; auxiliary gas, 210 arbitrary units; spray voltage, 4.5 kV; capillary temperature, 350 C; capillary voltage, 4.9 V; and tube lens offset 25 V.
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related with the HFCD family of cysteine decarboxylases. Characterization with LC–MS confirmed that the recombinant Undec1A protein catalyzed decarboxylation of L-cysteine to form cysteamine. The finding of a new HFCD family decarboxylase from alkaline polluted soils points out the advantage of metagenomic library in cloning novel genes through sequence-based screening using the E. coli host. Similar approach can also be employed for other broad applications in the field of biotechnology.
[11]
[12]
[13]
Acknowledgment This research was supported as a part of a program through Grant (No. 2001AA214141) from the Chinese National Programs for High Technology Research and Development.
[14] [15]
References
[16]
[1] D. Cowan, Q. Meyer, W. Stafford, S. Muyanga, R. Cameron, P. Wittwer, Metagenomic gene discovery: past, present and future, Trends Biotechnol. 23 (2005) 321–329. [2] V. Torsvik, L. Ovreas, Microbial diversity and function in soil: from genes to ecosystems, Curr. Opin. Microbiol. 5 (2002) 240–245. [3] R.I. Amann, W. Ludwig, K.H. Schleifer, Phylogenetic identification and in situ detection of individual microbial cells without cultivation, Microbiol. Rev. 59 (1995) 43–169. [4] J. Handelsman, Metagenomics: application of genomics to uncultured microorganisms, Microbiol. Mol. Biol. Rev. 68 (2004) 669–685. [5] K. Zengler, G. Toledo, M. Rappe, J. Elkins, E.J. Mathur, J.M. Short, M. Keller, Cultivating the uncultured, Proc. Natl. Acad. Sci. USA 99 (2002) 15681–15686. [6] C.S. Riesenfeld, P.D. Schloss, J. Handelsman, Metagenomics: genomic analysis of microbial communities, Annu. Rev. Genet. 38 (2004) 525–552. [7] M.H. Lee, C.H. Lee, T.K. Oh, J.K. Song, J.H. Yoon, Isolation and characterization of a novel lipase from a metagenomic library of tidal flat sediments: evidence for a new family of bacterial lipases, Appl. Environ. Microbiol. 72 (2006) 7406–7409. [8] C. Elend, C. Schmeisser, C. Leggewie, P. Babiak, J.D. Carballeira, H.L. Steele, J.L. Reymond, K.E. Jaeger, W.R. Streit, Isolation and biochemical characterization of two novel metagenome-derived esterases, Appl. Environ. Microbiol. 72 (2006) 3637–3645. [9] M. Ferrer, O.V. Golyshina, T.N. Chernikova, A.N. Khachane, D. Reyes-Duarte, V.A. Santos, C. Strompl, K. Elborough, G. Jarvis, A. Neef, M.M. Yakimov, K.N. Timmis, P.N. Golyshin, Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora, Environ. Microbiol. 7 (2005) 1996–2010. [10] J. Yun, S. Kang, S. Park, H. Yoon, M.J. Kim, S. Heu, S. Ryu, Characterization of a novel amylolytic enzyme encoded by a gene
[17]
[18]
[19]
[20] [21]
[22] [23] [24]
[25]
[26]
from a soil-derived metagenomic library, Appl. Environ. Microbiol. 70 (2004) 7229–7235. M. Wexler, P.L. Bond, D.J. Richardson, A.W. Johnston, A wide host-range metagenomic library from a waste water treatment plant yields a novel alcohol/aldehyde dehydrogenase, Environ. Microbiol. 7 (2005) 1917–1926. D.E. Gillespie, S.F. Brady, A.D. Bettermann, N.P. Cianciotto, M.R. Liles, M.R. Rondon, J. Clardy, R.M. Goodman, J. Handelsman, Isolation of antibiotics turbomycin A and B from a metagenomic library of soil microbial DNA, Appl. Environ. Microbiol. 68 (2002) 4301–4306. A. Martinez, S.J. Kolvek, J. Hopke, C.L. Tiong Yip, M.S. Osburne, Environmental DNA fragment conferring early and increased sporulation and antibiotic production in Streptomyces species, Appl. Environ. Microbiol. 71 (2005) 1638–1641. R. Daniel, The soil metagenome—a rich resource for the discovery of novel natural products, Curr. Opin. Biotechnol. 15 (2004) 199–204. G.W. Tyson, J. Chapman, P. Hugenholtz, E.E. Allen, R.J. Ram, P.M. Richardson, V.V. Solovyev, E.M. Rubin, D.S. Rokhsar, J.F. Banfield, Community structure and metabolism through reconstruction of microbial genomes from the environment, Nature 428 (2004) 37–43. T.P. Begley, C. Kinsland, E. Strauss, The biosynthesis of coenzyme A in bacteria, Vitam. Horm. 61 (2001) 157–171. S.Y. Gerdes, M.D. Scholle, M. D’Souza, A. Bernal, M.V. Baev, M. Farrell, O.V. Kurnasov, M.D. Daugherty, F. Mseeh, B.M. Polanuyer, J.W. Campbell, S. Anantha, K.Y. Shatalin, S.A. Chowdhury, M.Y. Fonstein, A.L. Osterman, From genetic footprinting to antimicrobial drug targets: examples in cofactor biosynthetic pathways, J. Bacteriol. 184 (2002) 4555–4572. P. Hernandez-Acosta, D.G. Schmid, G. Jung, F.A. Culianez-Macia, T. Kupke, Molecular characterization of the Arabidopsis thaliana flavoprotein AtHAL3a reveals the general reaction mechanism of 4 0 phosphopantothenoylcysteine decarboxylases, J. Biol. Chem. 277 (2002) 20490–20498. T. Kupke, Molecular characterization of the 4 0 -phosphopantothenoylcysteine decarboxylase domain of bacterial Dfp flavoproteins, J. Biol. Chem. 276 (2001) 27597–27604. T. Kupke, W. Schwarz, 4 0 -Phosphopantetheine biosynthesis in Archaea, J. Biol. Chem. 281 (2006) 5435–5444. J. Sambrook, D.W. Russell, Molecular Cloning: A Laboratory Manual, third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. J. Zhou, M.A. Bruns, J.M. Tiedje, DNA recovery from soils of diverse composition, Appl. Environ. Microbiol. 62 (1996) 316–322. J.W. Park, D.E. Crowley, Normalization of soil DNA extraction for accurate quantification of target genes by real-time PCR and DGGE, Biotechniques 38 (2005) 579–586. S. Kumar, K. Tamura, M. Nei, MEGA3: integrated software for molecular evolutionary genetics Analysis and sequence alignment, Brief. Bioinformat. 5 (2004) 150–163. P.D. Schloss, J. Handelsman, Biotechnological prospects from metagenomics, Curr. Opin. Biotechnol. 14 (2003) 303–310.
Molecular cloning and functional characterization of a novel decarboxylase from uncultured microorganisms Chengjian Jiang, Bo Wu
*
Guangxi Key Laboratory of Subtropical Bioresources Conservation and Utilization, The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, Guangxi University, 100 Daxue Road, Nanning, Guangxi 530004, People’s Republic of China Received 22 March 2007 Available online 3 April 2007
Abstract The metagenomic library approach has been used successfully to isolate novel biocatalyst genes from uncultured microorganisms. We report the cloning of a novel decarboxylase gene by sequence-based screening of a plasmid metagenomic library constructed with DNA from alkaline polluted soils. The gene was named undec1A and had an open reading frame of 1077 base pairs. It encoded a 359 amino acid polypeptide with a molecular mass of 38 kDa. The predicted protein had 58% similarity to a decarboxylase from Chlorobium phaeobacteroides BS1. The putative decarboxylase gene was subcloned into pETBlue-2 vector and overexpressed in Escherichia coli Tuner (DE3) pLac. The recombinant protein was purified to homogeneity. Functional characterization with liquid chromatography-mass spectrometry confirmed that the recombinant Undec1A protein catalyzed the decarboxylation of L-cysteine to form cysteamine. 2007 Elsevier Inc. All rights reserved. Keywords: Metagenomic library; Uncultured microorganisms; Sequence-based screening; Decarboxylase; Cysteamine
Soil microorganisms are the source of many important biocatalysts [1]. Recent studies have shown that one gram of soil may contain several thousand species of different microorganisms [2]. However, 99% of the bacteria in soil cannot be cultured with conventional methods, which exclude a large fraction of the soil microbial population from being harnessed [3]. Recently, a new strategy that involves the cloning of the total microbial genome, or ‘‘metagenome’’, directly isolated from natural environments into cultivable bacterium such as Escherichia coli is developed [4]. This approach does not require the in vitro culturing of microorganisms from environmental samples and can avoid the enrichment of dominant strains under specific selective conditions [5]. Various vectors, such as plasmids, fosmids, cosmids with small inserts and bacterial artificial chromosomes (BACs) with large inserts, have been used successfully to construct the metagenomic
*
Corresponding author. Fax: +86 (0771) 3237873. E-mail address: [email protected] (B. Wu).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.03.159
libraries [6]. Previous studies using this strategy have identified a large pool of genes of enzymes, such as lipase [7], esterase [8], hydrolase [9], amylase [10], alcohol/aldehyde dehydrogenase [11], and complex gene clusters encoding the enzymes in antibiotic production [12–14]. The most important advantage of the metagenomic libraries is that they contain genomes from the whole population of microorganisms in a specific sample rather than cultivated or enriched subpopulations and thus provide a more comprehensive collection of global microbiological information [15]. Decarboxylation is a common reaction in living organisms. Decarboxylase occurs in various catabolic and anabolic processes and is carried out by a variety of enzymes. Studying decarboxylase is of general biochemical interest. During the biosynthesis of coenzyme A, decarboxylation of (R)-4 0 -phospho-N-pantothenoylcysteine to form 4 0 -phosphopantotheine is one key step [16]. Decarboxylase is also an interesting target for the development of new antibacterial agents [17]. Several genes encoding different decarboxylases [18–20], such as AtHAL3a, Dfp, MrsD,
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and EpiD proteins from different organisms, have been cloned and partially characterized by polymerase chain reaction (PCR) and comparative genomics methods. However, cloning of decarboxylases from uncultured microorganisms has not been reported. In this study, a plasmid metagenomic library was constructed from alkaline polluted soil samples. Through sequence-based screening of the DNA library, a gene encoding a novel decarboxylase (named Undec1A) was isolated. The deduced amino acid sequence had moderate similarity to a family of cysteine decarboxylase proteins. Biochemical analysis of the overexpressed recombinant protein revealed that Undec1A catalyzed decarboxylation of L-cysteine to form cysteamine. To our knowledge, this is the first report of a decarboxylase isolated from soil metagenome. Materials and methods Plasmid vectors, bacterium strains, and growth conditions. The soil metagenomic library was constructed with E. coli DH5a as the host and pGEM-3Zf (+) as the cloning vector. The protein expression vector was pETBlue-2, and E. coli Novablue (Novagen) strain was used as the screening host for pETBlue-2 bearing inserts. Bacterial strain for expression of recombinant protein was Tuner (DE3) pLac (Novagen). All the E. coli bacterial cultures were grown at 37 C on Luria-Bertani (LB) agar plates or in LB medium. Where appropriate, media were amended with 100 lg ampicillin/ml, 50 lg carbenicillin/ml, 15 lg tetracycline/ml, or 34 lg chloramphenicol/ml. DNA manipulation and protein analysis. All DNA manipulations, including cloning and subcloning, transformation of E. coli cells, and PCR were performed according to standard techniques [21] or following the manufacturer’s instructions unless indicated otherwise. Protein preparation and analysis including protein extraction from E. coli, protein quantification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) were performed as described in the standard protocols [22]. DNA extraction from soil samples and construction of metagenomic library. Soil samples were collected from the ground surface of a stream located in Guangxi Province, South China. The ground soil has been enriched with alkaline pollutants for over 20 years. Water drainage from the soil sample has a pH of approximately 9.5. The metagenomic DNA was extracted from the alkaline polluted soils following the direct lysis method of Zhou [23] and Park [24]. Briefly, the DNA pellet from 5 g of soil sample was washed twice with 75% ethanol, air dried and dissolved in 1· TE buffer (10 mM Tris–HCl pH 8.0 and 1 mM Na2EDTA). To remove the contaminants, the crude metagenomic DNA extract was further purified on a 0.6% low-melting agarose gel, which was run at 60 V for 3 h. DNA fragments (ca. 23 kb) were recollected from the gel. The purified soil DNA was partially digested with EcoRI followed by size fractionation with lowmelting agarose gel as described [21]. Fractions containing DNA fragments of 1.0–15 kb were ligated into EcoRI-cleaved and dephosphorylated pGEM-3Zf (+) vector, and the ligation products were used to transfect E. coli DH5a. After overnight growth on LB agar plates containing ampicillin at 100 lg/ml, white colonies harboring plasmids bearing inserts were collected and used to construct the metagenomic library. This library was stored at 80 C until screening. DNA sequence analysis, database search and gene structure characterization. DNA sequence analysis was performed with the BigDye Terminator Cycle sequencing kit on an ABI Prism 3700 DNA analyzer (Applied Biosystems, USA). Protein translation was carried out with the web-based translation tool at the Expasy homepage (http://www.expasy.org/tools/ dna.html). Sequence predictions were retrieved from the protein and nucleotide databases at the National Center for Biotechnology Informa-
tion (NCBI) Entrez page (http://www.ncbi.nlm.nih.gov/Entrez/). Sequence similarity searches were performed with the BLAST 2.0 program. Amino acid sequence alignment of Undec1A with homologous proteins was done with the Align X program, a component of the Vector NT1 suite (Informax, North Bethesda, MD, USA) using the blosum62mt2 scoring matrix. Based on the comparison of the deduced amino acids, a putative gene (undec1A) encoding a novel decarboxylase was identified and further characterized. A phylogenetic tree was constructed using the neighbor-joining method with Molecular Evolutionary Genetics Analysis 3.1 software (MEGA, Version 3.1) [25]. Boot-strapping value was used to estimate the reliability of phylogenetic reconstructions (1000 replicates). Overexpression and purification of the recombinant decarboxylase protein. The Undec1A nucleotide sequence was amplified from plasmid (pGEXC0426) isolated from a decarboxylase producing clone. PCR was carried out in a total volume of 50 ll containing 2.5 mM MgCl2, 10 mM Tris–HCl (pH 8.4), 50 mM KCl, 0.2 mM each dNTP, 0.4 lM each primer, 1.0 U Vent DNA polymerase (NEB, USA), and 10 ng plasmid template. Restriction enzyme sites (underlined) for SalI and MluI were designed in the forward primer/reverse primer (5 0 -ATAGTCGACATGATCACCCC TCTTACGCTGGCAAC-3 0 /5 0 -CGACGCGTGTGAACCAGGGTAAGT ATCTTCCG-3 0 ). The PCR cycling consisted of a denaturation step (96 C for 2 min), 30 cycles of 94 C for 40 s, 65 C for 30 s and 72 C for 2 min, and a final extension step at 72 C for 10 min. After amplification, the PCR product mixture was digested with SalI and MluI and ligated directly into pETBlue-2 (Novagen) expression vector cleaved with the same enzymes. The resulting recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and plated on LB selection plates. After overnight incubation at 37 C, positive white colonies were picked for isolation of recombinant expression plasmid, which was subsequently introduced into E. coli Tuner (DE3) pLac (Novagen) to express the target protein. The transformed bacterial cells were cultured in LB medium containing carbenicillin 50 lg/ml and chloramphenicol 100 lg/ml at 37 C, and protein expression was induced by the addition of 1 mM isopropyl-b-Dthiogalactopyranoside when the optical density at 660 nm reached 0.6. After incubation for additional 6 h, the cells were harvested, washed twice with phosphate-buffered saline (pH 7.6), and lysed by sonication in 10 ml of 20 mM Tris–HCl, pH 8.0. The lysate was centrifuged twice at 30,000g for 20 min at 4 C and 1 ml of the supernatant was diluted with 5 ml of column buffer (20 mM Tris–HCl, pH 8.0, 10 mM imidazole, 300 mM NaCl) and applied onto an equilibrated nickel nitrilotriacetic acid (Ni– NTA) column containing 1 ml of Ni–NTA–agarose (Novagen). After a wash with 5 ml of column buffer containing 10 mM imidazole, the recombinant protein was eluted with the same buffer but containing 250 mM imidazole and the yellow peak fraction (about 600 ll) was collected. 1,4-Dithiothreitol (DTT) (Promega) was added to a final concentration of 5 mM into the protein immediately after its elution from the column. The isolated protein was further purified by gel filtration. A 25 ll aliquot of the Ni–NTA collection was loaded onto a Superdex 200 PC 3.2/ 30 column equilibrated with the running buffer (20 mM Tris–HCl, pH 8.0, 200 mM NaCl) and eluted at a flow rate of 40 ll/min. The Superdex 200 PC 3.2/30 column and the standard proteins used for calibration were obtained from Amersham Pharmacia Biotech, USA. The protein concentration was determined by a Bio-Rad protein assay kit with bovine serum albumin as the standard. The protein purified with Ni–NTA column and gel filtration was used for enzyme activity assays. Identification of the decarboxylation product. Liquid chromatography–mass spectrometry (LC–MS) was used to identify the decarboxylation product of the Undec1A recombinant protein. The LCQ mass spectrometer (Thermo Finnigan, San Jose, CA) was equipped with an electrospray ionization source and coupled with a HP 1100 HPLC system (Hewlett Packard, USA). The enzymatic reaction mixture contained 5 mM DTT, 1 mg/ml L-cysteine, 20 mM Tris–HCl, pH 7.0, and ca. 40 lg purified recombinant enzyme protein. The reaction was conducted in a total volume of 5.0 ml at 35 C for 30 min. Upon termination of the reaction, the residual protein was removed by centrifugation through a membrane (Vivaspin 500, Vivascience). The filtrate was separated on a C8 reverse phase column (150 · 4.6 mm,
C. Jiang, B. Wu / Biochemical and Biophysical Research Communications 357 (2007) 421–426 5 lm) (Thermo Finnigan, San Jose, CA) with acetic acid (0.02 mol/l) and acetonitrile (94:6, v/v) as the mobile phase at the flow rate of 0.2 ml/min. Mass spectrum data were acquired in positive mode with one full scan followed by two data-dependent scans of the most intense and the second most intense ions.
Results and discussion Construction of the metagenomic library The metagenomic DNA was directly extracted from alkaline polluted soils. The constructed library should contain a genome pool of all the microorganisms in the soil, including those of uncultivable bacteria. Approximately 15 lg of DNA was obtained from 1 g of soil sample. Majority of the DNA molecules had a size about 23 kb, which was shown as a strong band on the agarose gel (data
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not shown). However, the crude DNA extraction solution appeared yellowish dark brown due to possible contamination of water-soluble organic substances in the soil, which could hamper further enzymatic manipulation. To obtain more pure DNA, the crude extract was further separated on a low-melting agarose gel. The DNA collected from the agarose gel was partially digested with EcoRI and ligated with the EcoRI-cleaved pGEM-3Zf (+) vector. The final plasmid metagenomic library constructed from 1 lg purified soil DNA contained approximately 30,000 clones. In addition, restriction analysis of randomly chosen recombinant plasmids revealed a high level of diversity of the foreign DNA fragments in pGEM-3Zf (+). When EcoRI digested plasmids from 16 randomly picked recombinant positive plasmids were analyzed on an agarose gel, sizes of the inserts ranged from 1 kb to 15 kb, with an average of ca. 3.5 kb.
Fig. 1. Sequence alignment of Undec1A protein with other decarboxylases in the HFCD family. Decarboxylases are identified by their GenBank accession number. The highly conserved regions of insertion His motif, PXMNXXMW motif, substrate recognition clamp, dimerization motif, AAVAD motif and KXKK motif are boxed in shadow.
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Bioinformatics analysis and the cloning of a decarboxylase gene The environmentally derived plasmid library was grown in microplates. The bacteria were pelleted and lysed, and some randomly chosen recombinant plasmids were sequenced and analyzed. Totally, the DNA sequence analysis and database search revealed that 2 out of the 25 ORFs or predicted genes had over 80% similarities to known genes, and the other 23 recombinant plasmids had no good match with database sequences (detailed data not shown). This result confirmed that the metagenomic library contained DNA molecules from uncharacterized genomes and also demonstrated that the metagenome of naturally occurring microbacteria contained an immense pool of genes, most of which are not represented by pure and enrichment cultures established under certain selective conditions [26]. We identified an interesting clone, named pGEXC0426, and had its insert further characterized. The insert DNA had a length of 1146 base pairs (bp), and it had no good match with known genes at DNA level in the database. However, it was most similar to a cysteine decarboxylase (GenBank Accession No. AAQ66840) with 56% identity and 70% similarity at amino acid level. Based on the database comparison, we considered the cloned gene on pGEXC0426 novel and named it undec1A. The gene had a long open reading frame of 1077 bp. The deduced peptide had a predicted molecular mass of 38 kDa and an isoelectric point of 6.04. The undec1A nucleotide sequence has been deposited in the GenBank under Accession No. EF015465. When the deduced amino acid sequence of Undec1A was searched against the NCBI and Expasy databases, it was found that Undec1A had some similarity with cysteine decarboxylases from
Porphyromonas gingivalis W83 and Cytophaga hutchinsonii and several cysteine decarboxylases belonging to the homooligomeric flavin-containing cysteine decarboxylase (HFCD) protein family. These cysteine decarboxylases in HFCD protein family include DNA/pantothenate metabolism flavoprotein (GenBank Accession No. ZP_00532913) from Chlorobium phaeobacteroides BS1 (58% identitical and 74% similar), cysteine decarboxylase (GenBank Accession No. AAQ66840) from P. gingivalis W83 (56% identitical and 70% similar), PPC decarboxylase (GenBank Accession No. ZP_00307777.1) from C. hutchinsonii (50% identitical and 66% similar), and cysteine decarboxylase (GenBank Accession No. YP_037930.1) from Bacillus thuringiensis serovar konkukian strain 97-27 (46% identitical and 64% similar). Multiple alignments of the deduced amino acids of Undec1A with the most homologous decarboxylase proteins (above 60% similarity) in the HFCD family (NCBI database) were presented in Fig. 1. The putative protein had the EpiD conserved domain [19] for the catalytic activity of decarboxylases. Furthermore, amino acid sequence comparison revealed that the deduced Undec1A peptide shared the flavin-binding motif, the conserved active-site residues, and decarboxylation participating cysteine residues with other decarboxylase members in the HFCD protein family. Therefore, Undec1A should be classified as a new member of the HFCD family. Fig. 2 presents the phylogenetic tree, showing relationship with other decarboxylases, based on amino acids. The phylogenetic tree also revealed that Undec1A was most closely related to the decarboxylase of P. gingivalis W83 and it clustered together with the HFCD proteins from several bacterium species of C. hutchinsonii and Bacillus thuringiensis.
Fig. 2. Phylogenetic relationship of Undec1A protein with other decarboxylases in the HFCD family. The phylogenetic tree was constructed by the neighbor-joining method with MEGA, version 3.1, software. The numbers associated with the branches refer to bootstrap values (confidence limits) representing the substitution frequencies per amino acid residue. Decarboxylases are identified by their GenBank accession number.
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Overexpression and purification of recombinant Undec1A In order to investigate the biochemical properties of Undec1A, the gene was subcloned in frame with a six-histidine tag sequence into expression vector pETBlue-2 and expressed in E. coli Tuner (DE3) pLacI. The initial analysis with crude cell lysate showed that the bacteria containing recombinant plasmid pETBlue-2-undec1A produced a substantial amount of the expected recombinant protein, while this protein was not detectable in the culture of the bacteria containing the parent vector pETBlue-2. The recombinant Undec1A protein was initially purified with Ni–NTA magnetic agarose chromatography. Degraded and/or nonspecifically bound polypeptides in the elutes from Ni–NTA agarose column were removed by subsequent gel filtration chromatography (GFC). As shown in Fig. 3, the recombinant Undec1A protein was purified to homogeneity, as demonstrated by a single band (ca. 38 kDa) on SDS–
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PAGE gel. The decarboxylase was purified about 281.5fold to homogeneity with an overall enzyme yield of 20.8%. Functional characterization of recombinant Undec1A Through reaction product identification by LC–MS, our initial studies confirmed that the purified recombinant Undec1A protein had cysteine decarboxylase activity. As shown in Fig. 4, the enzymatic product showed a molecular ion at m/z 78.0, which is the same as the protonated cysteamine, suggesting cleavage of the –COOH group from Lcysteine. HighChem Mass Frontier software 3.0 (Thermo Finnigan, San Jose, CA) was used to verify that the molecular ion observed correlated with the proposed structure. The mass spectrum of the reaction product is identical to that of cysteamine. These results confirmed that Undec1A protein catalyzed the formation of cysteamine from L-cysteine. Our study of Undec1A may provide new insight into the relationship between the sequence, structure, and activity of HFCD protein family. For example, several bacterial conserved regions in the HFCD family are the N-terminal G-G/S-I-A-X-Y-K motif, PXMNXXMW motif, the substrate recognition clamp, and the histidine insertion motif, dimerization motif, AAVAD motif, and KXKK motif. However, the G-G/S-I-A-X-Y-K motif of the deduced Undec1A peptide is missing (Fig. 1). The absence of the N-terminal motif may affect its enzymatic activity. It would be interesting to elucidate the catalyzing mechanism through studies of the 3D structures of Undec1A protein with X-ray diffraction methods. Conclusion
Fig. 3. The SDS–PAGE of Undec1A. Proteins were separated by 12% SDS–PAGE and then stained with Coomassie brilliant blue G-250. Lane 1, molecular weight standards. Lane 2, purified recombinant Undec1A protein. The Undec1A protein is indicated by the black arrow.
It is well accepted that the field of metagenomic gene discovery offers enormous potential for fundamental microbiology study and biotechnology development. We constructed a plasmid library from alkaline polluted soils and cloned a novel decarboxylase gene (undec1A) by sequence-based screening strategy. Sequence analysis demonstrated that the identified gene product was
Fig. 4. Mass spectrum of the reaction product of Undec1A with L-cysteine as the substrate. The electrospray ionization conditions were: sheath gas flow rate, 80 arbitrary units; auxiliary gas, 210 arbitrary units; spray voltage, 4.5 kV; capillary temperature, 350 C; capillary voltage, 4.9 V; and tube lens offset 25 V.
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related with the HFCD family of cysteine decarboxylases. Characterization with LC–MS confirmed that the recombinant Undec1A protein catalyzed decarboxylation of L-cysteine to form cysteamine. The finding of a new HFCD family decarboxylase from alkaline polluted soils points out the advantage of metagenomic library in cloning novel genes through sequence-based screening using the E. coli host. Similar approach can also be employed for other broad applications in the field of biotechnology.
[11]
[12]
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Acknowledgment This research was supported as a part of a program through Grant (No. 2001AA214141) from the Chinese National Programs for High Technology Research and Development.
[14] [15]
References
[16]
[1] D. Cowan, Q. Meyer, W. Stafford, S. Muyanga, R. Cameron, P. Wittwer, Metagenomic gene discovery: past, present and future, Trends Biotechnol. 23 (2005) 321–329. [2] V. Torsvik, L. Ovreas, Microbial diversity and function in soil: from genes to ecosystems, Curr. Opin. Microbiol. 5 (2002) 240–245. [3] R.I. Amann, W. Ludwig, K.H. Schleifer, Phylogenetic identification and in situ detection of individual microbial cells without cultivation, Microbiol. Rev. 59 (1995) 43–169. [4] J. Handelsman, Metagenomics: application of genomics to uncultured microorganisms, Microbiol. Mol. Biol. Rev. 68 (2004) 669–685. [5] K. Zengler, G. Toledo, M. Rappe, J. Elkins, E.J. Mathur, J.M. Short, M. Keller, Cultivating the uncultured, Proc. Natl. Acad. Sci. USA 99 (2002) 15681–15686. [6] C.S. Riesenfeld, P.D. Schloss, J. Handelsman, Metagenomics: genomic analysis of microbial communities, Annu. Rev. Genet. 38 (2004) 525–552. [7] M.H. Lee, C.H. Lee, T.K. Oh, J.K. Song, J.H. Yoon, Isolation and characterization of a novel lipase from a metagenomic library of tidal flat sediments: evidence for a new family of bacterial lipases, Appl. Environ. Microbiol. 72 (2006) 7406–7409. [8] C. Elend, C. Schmeisser, C. Leggewie, P. Babiak, J.D. Carballeira, H.L. Steele, J.L. Reymond, K.E. Jaeger, W.R. Streit, Isolation and biochemical characterization of two novel metagenome-derived esterases, Appl. Environ. Microbiol. 72 (2006) 3637–3645. [9] M. Ferrer, O.V. Golyshina, T.N. Chernikova, A.N. Khachane, D. Reyes-Duarte, V.A. Santos, C. Strompl, K. Elborough, G. Jarvis, A. Neef, M.M. Yakimov, K.N. Timmis, P.N. Golyshin, Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora, Environ. Microbiol. 7 (2005) 1996–2010. [10] J. Yun, S. Kang, S. Park, H. Yoon, M.J. Kim, S. Heu, S. Ryu, Characterization of a novel amylolytic enzyme encoded by a gene
[17]
[18]
[19]
[20] [21]
[22] [23] [24]
[25]
[26]
from a soil-derived metagenomic library, Appl. Environ. Microbiol. 70 (2004) 7229–7235. M. Wexler, P.L. Bond, D.J. Richardson, A.W. Johnston, A wide host-range metagenomic library from a waste water treatment plant yields a novel alcohol/aldehyde dehydrogenase, Environ. Microbiol. 7 (2005) 1917–1926. D.E. Gillespie, S.F. Brady, A.D. Bettermann, N.P. Cianciotto, M.R. Liles, M.R. Rondon, J. Clardy, R.M. Goodman, J. Handelsman, Isolation of antibiotics turbomycin A and B from a metagenomic library of soil microbial DNA, Appl. Environ. Microbiol. 68 (2002) 4301–4306. A. Martinez, S.J. Kolvek, J. Hopke, C.L. Tiong Yip, M.S. Osburne, Environmental DNA fragment conferring early and increased sporulation and antibiotic production in Streptomyces species, Appl. Environ. Microbiol. 71 (2005) 1638–1641. R. Daniel, The soil metagenome—a rich resource for the discovery of novel natural products, Curr. Opin. Biotechnol. 15 (2004) 199–204. G.W. Tyson, J. Chapman, P. Hugenholtz, E.E. Allen, R.J. Ram, P.M. Richardson, V.V. Solovyev, E.M. Rubin, D.S. Rokhsar, J.F. Banfield, Community structure and metabolism through reconstruction of microbial genomes from the environment, Nature 428 (2004) 37–43. T.P. Begley, C. Kinsland, E. Strauss, The biosynthesis of coenzyme A in bacteria, Vitam. Horm. 61 (2001) 157–171. S.Y. Gerdes, M.D. Scholle, M. D’Souza, A. Bernal, M.V. Baev, M. Farrell, O.V. Kurnasov, M.D. Daugherty, F. Mseeh, B.M. Polanuyer, J.W. Campbell, S. Anantha, K.Y. Shatalin, S.A. Chowdhury, M.Y. Fonstein, A.L. Osterman, From genetic footprinting to antimicrobial drug targets: examples in cofactor biosynthetic pathways, J. Bacteriol. 184 (2002) 4555–4572. P. Hernandez-Acosta, D.G. Schmid, G. Jung, F.A. Culianez-Macia, T. Kupke, Molecular characterization of the Arabidopsis thaliana flavoprotein AtHAL3a reveals the general reaction mechanism of 4 0 phosphopantothenoylcysteine decarboxylases, J. Biol. Chem. 277 (2002) 20490–20498. T. Kupke, Molecular characterization of the 4 0 -phosphopantothenoylcysteine decarboxylase domain of bacterial Dfp flavoproteins, J. Biol. Chem. 276 (2001) 27597–27604. T. Kupke, W. Schwarz, 4 0 -Phosphopantetheine biosynthesis in Archaea, J. Biol. Chem. 281 (2006) 5435–5444. J. Sambrook, D.W. Russell, Molecular Cloning: A Laboratory Manual, third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. J. Zhou, M.A. Bruns, J.M. Tiedje, DNA recovery from soils of diverse composition, Appl. Environ. Microbiol. 62 (1996) 316–322. J.W. Park, D.E. Crowley, Normalization of soil DNA extraction for accurate quantification of target genes by real-time PCR and DGGE, Biotechniques 38 (2005) 579–586. S. Kumar, K. Tamura, M. Nei, MEGA3: integrated software for molecular evolutionary genetics Analysis and sequence alignment, Brief. Bioinformat. 5 (2004) 150–163. P.D. Schloss, J. Handelsman, Biotechnological prospects from metagenomics, Curr. Opin. Biotechnol. 14 (2003) 303–310.