CURRENT MICROBIOLOGY Vol. 43 (2001), pp. 260 –264 DOI: 10.1007/s002840010298
Current Microbiology An International Journal © Springer-Verlag New York Inc. 2001
Cloning, Characterization, and Functional Expression in Escherichia coli of argH Encoding Argininosuccinate Lyase in the Cyanobacterium Nostoc sp. Strain PCC 73102 Olga Troshina,1,2 Alfred Hansel,1,3 Peter Lindblad1 1
Department of Physiological Botany, EBC, Uppsala University, Villava¨gen 6, SE-752 36 Uppsala, Sweden Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region, 142 290, Russia 3 Molecular and Cellular Biophysics Research Unit, Medical Faculty of the Friedrich-Schiller University, Drackendorfer Strasse 1, D-07747 Jena, Germany 2
Received: 6 December 2000 / Accepted: 9 February 2001
Abstract. A gene argH, encoding argininosuccinate lyase (ASL), has been cloned from a cosmid library of the filamentous cyanobacterium Nostoc sp. strain PCC 73102. The argH open reading frame encodes a protein comprised of 461 amino acids with a calculated molecular mass of 51,349 Da. Protein sequence comparisons reveal significant similarities of the Nostoc PCC 73102 ASL to related proteins from other organisms. In an Escherichia coli ⌬argH strain, the Nostoc PCC 73102 ASL expressed from a recombinant plasmid could restore the ability to grow on medium without arginine. Moreover, cell extracts show a specific ASL activity of 16.2 nmoles of urea 䡠 min⫺1 䡠 (mg protein)⫺1. Partially purified, His-tagged ASL runs as a 53-kDa protein band in SDS-PAGE and about 215-kDa protein in nativePAGE, suggesting that the native protein is a tetramer.
Cyanobacteria constitute a large and diverse group of phototrophic prokaryotes capable of plant-type, oxygen-evolving photosynthesis [23]. They can use CO2 as carbon source, and e.g., ammonia and nitrate as nitrogen sources for growth. In addition, many cyanobacteria are able to fix atmospheric nitrogen. Under nitrogen repletion, cyanobacteria store nitrogen in a unique reserve compound, cyanophycin. It consists of L-aspartic acid and L-arginine in a 1:1 molar ratio [1, 22], although this composition might be dependent upon strain and growth conditions [16]. Arginine biosynthesis seems to be crucial for cyanobacterial strains fixing N2 under aerobic conditions, as the conditional arginine auxotroph Nostoc ellipsosporum NE1, which is unable to form cyanophycin, neither differentiates heterocysts nor fixes molecular nitrogen [13]. Arginine is synthesized in cyanobacteria, as in other photosynthetic bacteria, via the so-called “cyclic pathway of ornithine synthesis” [4, 8 –10, 19]. Some of the enzymes of the arginine biosynthesis pathway have been detected in cell-free extracts of the unicellular Synechococcus sp., as well as the filamentous strains Nostoc muscorum G [8], Correspondence to: Peter Lindblad; email:
[email protected]
Anabaena variabilis [8 –10], Anabaena cylindrica, Anabaena PCC 7120 [7], and Nostoc PCC 73102 [12, 15, 24]. However, only a few cyanobacterial genes encoding enzymes of the arginine biosynthetic pathway have been cloned and characterized: argC, and argD from Anabaena PCC 7120 encoding N-acetylglutamate semialdehyde dehydrogenase and N-acetylornithine aminotransferase respectively [5, 6], argL from Nostoc ellipsosporum NE1 encoding N-acetylglutamate semialdehyde dehydrogenase [13], and argF encoding ornithine carbamoyl transferase in Nostoc PCC 73102 [11]. The last step in the synthesis of arginine is catalyzed by the enzyme argininosuccinate lyase (ASL). We have demonstrated activity of this enzyme in cell-free extracts of Nostoc PCC 73102 [24]. In the present work we have cloned a gene encoding ASL in Nostoc PCC 73102, argH, and expressed it in an Escherichia coli ⌬argH strain, where it restored the ability to grow without the addition of arginine to the growth medium. Materials and Methods Bacterial strains and growth conditions. Nostoc sp. strain PCC 73102, a heterocyst-forming cyanobacterium originally isolated from
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O. Troshina et al.: argH Encoding Argininosuccinate Lyase in Nostoc coralloid roots of the cycad Macrozamia, and Synechocystis sp. strain PCC 6803 [20] were obtained from the Pasteur Culture Collection (PCC, Paris, France) and grown in BG110 medium (Nostoc PCC 73102) or BG110 medium supplemented with 10 mM nitrate (Synechocystis PCC 6803), as described [24]. E. coli strain XL1 Blue competent cells (Stratagene) were utilized for DNA manipulation. For gene expression and complementation studies, an E. coli C600 ⌬argH strain [2] was aerobically grown in liquid or on agar-solidified (1.5% agar) LB medium at 37°C [21], when needed on defined M9 medium supplemented with the required nutrients [2].
umn. Contaminating proteins were eluted by washing the column with 16 volumes of buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 20 mM imidazole). Bound ASL was eluted from the Ni-NTA column with elution buffer (4 ⫻ 0.5 ml of 50 mM NaH2PO4, pH 8.0, 300 mM NaCl, and 250 mM imidazole). Extracts were analyzed by SDS- and native-PAGE gels with a Pharmacia PhastSystem and 10 –15 % Phast gradient gels followed by staining with Coomassie blue R-350. The protein content was estimated by using Coomassie brilliant blue and bovine serum albumin as the standard [18].
DNA manipulations. Degenerated primers for PCR-based synthesis of an argH-specific probe were constructed after aligning ArgH protein sequences from several organisms using the EMBL data library and the GenBank database. The primers were synthesized on the basis of conserved regions as well as the corresponding DNA sequence of the ORF (slr1133) putatively encoding ArgH in Synechocystis PCC 6803 [17]. Genomic DNA was isolated from Nostoc PCC 73102 as described [11]. Cosmid and plasmid DNA were isolated from E. coli by using the Wizard Minipreps Plus kit (Promega). DNA fragments were purified from agarose gels with the Qiaquick Gel Extraction kit (Qiagen). Approximately 2000 cosmid-containing E. coli colonies representing a Nostoc PCC 73102 genomic library were plated and transferred onto Hybond-N⫹ nylon membranes (Amersham-Pharmacia Biotech) by standard procedures [21], and the membranes were further treated according to the manufacturer’s instructions. Probes for use in colony hybridization were labeled by using the Rediprime kit (Amersham-Pharmacia Biotech) and 32P-dCTP (Amersham-Pharmacia Biotech) and were hybridized at 55°C [21]. Probes for use in Southern blot hybridization were labeled with digoxigenin-11-dUTP (“DIG DNA Labelling and Detection Kit”; Roche Molecular), hybridized, and detected according to manufacturer’s instructions. Preparation of competent E. coli cells, transformation of E. coli, digestion of DNA with restriction endonucleases, and ligation with T4 ligase were performed by standard procedures [21].
Amino acid sequencing. For N-terminal amino acid sequencing, the proteins separated by SDS–PAGE were transferred electrophoretically onto a polyvinyldifluoride (PVDF) membrane and visualized by staining with Coomassie blue R-350. The polypeptide of about 53 kDa was excised from the membrane, and the N-terminal amino acids were analyzed in an ABI 476A protein sequenator (Applied Biosystem) according to the manufacturer’s instructions. Sequence call was made after manual inspection of the chromatograms.
Heterologous expression of argH and isolation of recombinant argininosuccinate lyase. For heterologous expression, the argH ORF was amplified via PCR (using Expand High Fidelity PCR System (Roche Diagnostics)) from genomic DNA isolated from Nostoc PCC 73102 as template with specific primers flanking the gene at 5⬘ and 3⬘ termini. The primers introduced restriction sites BamHI at the 5⬘-end and SalI at the 3⬘-end of the amplified DNA. The PCR product was digested with BamHI/SalI and cloned into the expression vector pQE30 carrying the 6 ⫻ His tag coding sequence 5⬘ to the multiple cloning site (Qiagen). Recombinant expression plasmids were transformed into the E. coli C600 ⌬argH strain [2] harboring pREP4 plasmid (Qiagen), the presence of which allows for the controlled expression of the argH gene. An overnight culture of E. coli cells, transformed with the expression vector, was inoculated into 100 ml of LB medium with antibiotics (50 mg/L kanamycin, 50 mg/L tetracyclin, 200 mg/L ampicillin), grown to the log phase (OD590 ⫽ 0.6), and then induced with 1 mM IPTG at 37°C for 4 h. The cells were harvested after induction by centrifugation (10,000 g, 5 min, 4°C), the pellet was washed with 20 mM K-phosphate buffer (pH 7.4), and the cells were stored frozen at ⫺20°C. Thawed cells were resuspended in lysis buffer (50 mM NaH2PO4, pH 8.0; 300 mM NaCl, 10 mM imidazole) and disrupted by sonication for 7 ⫻ 20 s with 2 min cooling (ice) intervals. Cell extract was centrifuged (10,000 g, 1 h, 4°C) and the supernatant used in the purification. The supernatant was incubated with 1 ml of 50% nickelnitrilotriacetic acid-loaded agarose (Ni-NTA) (Qiagen) for 1 h at 4°C with shaking, before the extract-resin mixture was loaded into a col-
Preparation of crude extracts of E. coli and ASL activity measurements. Extracts of E. coli C600 ⌬argH cells transformed with expression vector were obtained as described above, except that cells were resuspended in 20 mM potassium phosphate buffer (pH 7.4). In vitro ASL activity was determined by measuring the formation of urea produced from arginine in an excess of arginase as described previously [24].
Results and Discussion Cloning and sequencing of the Nostoc PCC 73102 argH. PCRs with degenerated primers and Synechocystis PCC 6803 DNA as a template resulted in amplification of one fragment with the expected size of 540 bp (data not shown). Sequencing of the fragment established its identity as part of the putative Synechocystis PCC 6803 argH gene. However, no amplification product was obtained when using genomic DNA isolated from Nostoc PCC 73102. The fragment obtained with Synechocystis PCC 6803 DNA was, therefore, used for screening a cosmid library of Nostoc PCC 73102. A single positive colony was identified and its cosmid purified, used for subcloning, and subsequent sequencing. The restriction map of the DNA region containing the cloned putative argH in Nostoc PCC 73102 is shown in Fig. 1a. Another ORF is located 419 bp upstream of argH. It has the same orientation as argH and putatively encodes cpmA, a gene involved in an output pathway of the cyanobacterial circadian system [14]. Sequence analysis of the Nostoc PCC 73102 argH. The identified ORF encoding Nostoc PCC 73102 ASL is 1386 bp long and translates into a polypeptide of 461 amino acids with a predicted molecular mass of 51,349 Da. The deduced sequence shows high similarity to ASLs from different organisms including bacteria, plants, and animals. It shares 78% identical and 86% similar amino acids with the presumptive ASL of Syn-
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Fig. 1. argH in Nostoc sp. strain PCC 73102. (a) Physical map of the cloned argH (1386bp) with some restriction enzyme sites indicated. Another ORF is located 419 bp upstream of argH. It has the same orientation as argH and putatively encodes cpmA, a gene involved in an output pathway of the cyanobacterial circadian system [14]. (b) Alignment of the deduced amino acid sequences of ArgH from Nostoc PCC 73102, E. coli, and duck ␦-crystallin. Identical/similar amino acids are boxed and shaded. Conserved regions (I, II, and III, respectively) and amino acids known to be involved in catalysis are indicated by arrows. The Nostoc PCC 73102 argH and deduced protein sequences are deposited in GenBank under the accession No. AF143209.
echocystis PCC 6803 and 40 –50% of identity (60 – 65 % of similarity) with ASLs from other organisms. The deduced Nostoc PCC 73102 ArgH also shows 20 –25 % identity, and approximately 40% similarity, with other enzymes of the fumarase superfamily (class II fumarase, aspartase, adenylosuccinate lyase, carboxy-cis,cis-muconate cycloisomerase, and ␦II-crystallin—the structural protein of bird eye lenses, homologous to ASL, and possessing ASL activity). All members of the fumarase superfamily share a short conserved sequence motif,
G-S-x-x-M-x-x-K-x-N, are biologically active as homotetramers, and catalyze the formation of fumarate via a -elimination reaction involving cleavage of a C␣–N or a C␣–O bond [3, 25, 26]. Three highly conserved regions of amino acid sequences (according to the numbering scheme of duck ␦II-crystallin: conserved region I, the residues 114 –121; conserved region II, the residues 159 –168; conserved region III, the residues 282–296; see Fig. 1b) have been proposed to be involved in forming the active site [3, 25]. These conserved regions,
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although dispersed in monomer, are brought together in the tetramer structure. Site-directed mutagenesis of duck ␦II-crystallin and studies of the kinetic and thermodynamic properties of mutant enzymes, as well as crystal structure analysis of an inactive duck ␦II-crystallin mutant with bound argininosuccinate, revealed that S29, D33, H91, S114, R115, N116, T161, H162, S283, S284, K289, N291, E296, Y323, L327, D330, and K331 are essential for binding and stabilizing the substrate and for catalytic activity [3, 25]. These amino acid residues are highly conserved across all ASL sequences studied, including Nostoc PCC 73102 ArgH, stressing their importance for structure/functional relationship within the ASL/␦-crystallin superfamily members. Complementation of an E. coli argH mutant and expression of the Nostoc PCC 73102 argH in E. coli. E. coli C600 ⌬argH is a mutant with an absolute requirement for exogenous arginine [2]. Expression of the Nostoc PCC 73102 argH in an E. coli C600 ⌬argH led to growth of the strain on minimal M9 medium containing no arginine. Despite the fact that the gene was put under control of the lac repressor, slow growth on solidified M9 medium without arginine was observed even in the absence of IPTG due to “leaky” expression of argH. However, addition of IPTG induced growth significantly, and the expression of the recombinant protein was not toxic for the host cells. Transformation of the expression vector alone, however, did not result in any colonies on minimal medium. E. coli C600 ⌬argH cells transformed with expression vector with/or without Nostoc argH were examined for ASL activity by using an in vitro assay. E. coli cells containing pQE30 without insert did not possess any detectable ASL activity. Cells transformed with a vector containing Nostoc argH and induced with IPTG had a specific activity of 16.2 nmoles of urea produced min⫺1 (mg protein)⫺1, whereas cells not induced with IPTG showed an ASL activity of 6.9 nmoles of urea produced min⫺1 (mg protein)⫺1. These results confirm that the cyanobacterial protein derived from the recombinant plasmid has ASL activity and that this Nostoc ASL is functional in E. coli despite the fact that its protein sequence reveals an identity of only 45% (64% similarity) to the E. coli argH product (Fig. 1b). The presence of six consecutive histidine residues (6 ⫻ His tag) at the N-terminus of the recombinant ASL expressed from the pQE30 plasmid allowed partial purification of the protein in one step by affinity chromatography with Ni-NTA-agarose. SDS-PAGE of the crude extract from induced cells and fractions eluted from the Ni-NTA agarose column showed one dominant protein band with a molecular mass of about
Fig. 2. SDS-PAGE (a) and native-PAGE (b) analysis of samples at different stages of purification of an overexpressed Nostoc PCC 73102 argininosuccinate lyase in E. coli. (a) SDS-PAGE. Lane 1, Noninduced culture; lane 2, culture induced with IPTG; lane 3, crude extract from cells induced with IPTG (protein content is about 6.5 g); lane 4, flow-through; lanes 5, 6, and 7, first (protein is about 2 g), second, and third (protein is about 4 g) 250 mM elution fractions. (b) Native-PAGE. Lane 1, crude extract (protein is about 6.5 g); lane 2, flow-through; lane 3, elution fraction (protein is about 0.24 g). Molecular mass standards, in kDa, are indicated on the right. Protein bands were visualized by Coomassie blue staining.
53 kDa (Fig. 2a). The identity of the 53-kDa polypeptide was confirmed by N-terminal sequence analysis (data not shown). On native-PAGE, both crude extract of induced cells and the fraction eluted from Ni-NTA agarose yielded a single strong band of about 215 kDa (Fig. 2b), putatively corresponding to a homotetrameric oligomer. In summary, we have identified, cloned, and sequenced argH encoding argininosuccinate lyase in the filamentous heterocyst-forming cyanobacterium Nostoc PCC 73102. The Nostoc PCC 73102 ArgH is highly homologous to the presumptive ArgH of the unicellular cyanobacterium Synechocystis PCC 6803 and ArgH from other organisms. It also shares all conserved residues/regions with other enzymes of the fumarase superfamily. The Nostoc PCC 73102 argH has been functionally expressed in an E. coli ⌬argH mutant, and a recombinant ASL has been partially purified.
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ACKNOWLEDGMENTS The authors thank Dr. A. Auchincloss (University of Geneva, Geneva, Switzerland) and P. Paulsrud (Uppsala University, Uppsala, Sweden) for providing the E. coli C600 ⌬argH strain and the Nostoc PCC 73102 cosmid library, respectively. This work was financially supported by research grants awarded by RFFI (99-04-49124), the Swedish Natural Science Research Council, and the Royal Swedish Academy of Sciences (co-operation between Sweden and the former Soviet Union, # 12609).
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