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Alpha-amylase gene of thermophilic Streptomyces sp. TO1: nucleotide sequence, transcriptional and amino acid sequence analysis
FEMS Microbiology Letters 160 (1998) 17^23
Alpha-amylase gene of thermophilic Streptomyces sp. TO1: nucleotide sequence, transcriptional and amino acid sequence analysis Lot¢ Mellouli a; *, Raoudha Ghorbel a , Marie Joelle Virolle b , Samir Bejar b
a
a Centre de Biotechnologie de Sfax, B.P. 358, 3018 Sfax, Tunisie Institut de Geèneètique et Microbiologie, URA CNRS 1354, Baêtiment 400, Universiteè Paris-Sud, F-91405 Orsay Cedex, France
Received 28 November 1997; accepted 20 December 1997
Abstract The nucleotide sequence of a 1860-bp region encoding a thermostable K-amylase of Streptomyces sp. TO1 was determined. Frame analysis revealed the presence of a 1359-bp long open reading frame (amy TO1) encoding a 453 amino acid protein with a deduced Mr of 49 kDa. Northern blot analysis revealed that amy TO1 gene was expressed as approximately 1.5-kbp monocistronic transcript in both SL1326/pLM1 and Streptomyces sp. TO1 strains. Primer extension experiments indicated that the transcriptional start site lies 30 bp upstream of the ATG start codon, and allowed the identification of 335 (TTGCTG) and 310 (TACGCG) eubacterial-like promoter sequences. Amy TO1 exhibits strong amino acid identities with those from other Streptomyces species with a maximum of 78% with S. thermoviolaceus K-amylase. Nevertheless, subtle amino acid changes such as the substitution of four conserved residues found at similar positions in other Streptomyces K-amylases by proline residues, and the substitution of three conserved hydrophilic amino acids by hydrophobic ones in Amy TO1 might account for the thermostable properties of Amy TO1. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Thermostability; Sequence comparison; Hydrophobicity; Proline residue
1. Introduction Streptomyces are saprophytic soil bacteria produc* Corresponding author. Tel.: +216 (4) 274 110; Fax: +216 (4) 275 970. Abbreviations : aa, amino acid(s) ; amy, gene encoding Kamylase; Amy, K-amylase; bp, basepair(s); kbp, kilo basepair(s) or 1000 basepairs; kDa, kilo dalton; Mr , relative molecular mass; nt, nucleotide(s); ORF, open reading frame; tsp, transcription start point; RBS, ribosome-binding site; SL 1326, Streptomyces lividans 1326
ing several kinds of secondary metabolites, including most of the known antibiotics. In order to obtain nutrients from organic debris in the soil, they secrete a variety of hydrolytic enzymes, such as protease, nucleases, lipases and other di¡erent enzymes degrading polysaccharides such as cellulose, chitin, starch and xylan. Genes encoding a variety of extracellular enzymes from Streptomyces species have been cloned and characterised. These include endoglycosidase H [1], agarase [2], xylanase [3], and amylases [4^9].
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 5 8 5 - 5
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L. Mellouli et al. / FEMS Microbiology Letters 160 (1998) 17^23
K-Amylase (EC 3.2.1.1) is widely used in starch processing industries. These secreted endoenzymes randomly cleave K(1,4) linkages in starch and usually produce a mixture consisting of maltose, malto-oligosaccharides and K-limit dextrins as ¢nal products. In starch industry, high hydrolysis temperature is desirable because starch granules cannot be attacked by K-amylase unless they have been ruptured by heat. We have previously reported the cloning and expression of a thermostable K-amylase isolated from a new thermophilic Streptomyces species strain TO1 [10]. The thermostable Amy TO1 works at an optimal T³ of 70³C and might be of industrial interest to produce glucose syrup from starch. In this paper, we are reporting the sequencing of the gene encoding the thermostable K-amylase of the TO1 strain, its transcriptional analysis as well as detailed amino acid (aa) comparisons of Amy TO1 with other Streptomyces K-amylases. This comparison pointed toward unexpected amino acid changes that might account for the thermostable properties of Amy TO1.
2. Materials and methods 2.1. Bacterial strains and plasmids Streptomyces sp. TO1 strain and Streptomyces lividans 1326 containing pLM1 [10] were used in this work. E. coli DH5K (F3 P80 dlacZvM15 v(lacZYAargF) U169 endA1 recA1 hsdR17 (rk 3 , mk ) deoR thi-1 susE44 V3 gyrA96 relA1) and TG1 (supE hsdv5 thi v (lac-proAB) FP (traD36 proAB lacIq lacZvM15) were used as host strains. pLM1 and pLM2 [10] recombinant plasmids carrying amy TO1 were used as a source of sub-cloning fragments for sequencing, and M13 derivatives [11] were used as sequencing vectors. 2.2. DNA isolation and manipulation Plasmid puri¢cation, DNA digestion with restriction enzymes and cloning were done according to Sambrook et al. [12] for E. coli and Hopwood et al. [13] for Streptomyces.
2.3. DNA sequencing and sequence analysis DNA fragments were cloned into M13 derivatives, and unidirectional nested deletions were created using exonuclease III as described in Pharmacia protocols. The nucleotide sequence of the 1860-bp fragment containing the amy TO1 gene was determined on both strands using the chain-termination method of Sanger et al. [14] with universal primers. Sequence analysis and comparisons were done using the PC gene Software package 6.85 (Intelligenetics Inc., University of Geneva, Switzerland). 2.4. RNA isolation and manipulation 2.4.1. Culture conditions for RNA isolation Spores were pre-germinated as described previously by Hopwood et al. [13] and grown in 150-ml Erlenmeyer £asks containing 20 ml of liquid minimal medium with glycerol (1%, w/v) as carbon source for SL 1326/pLM1 and starch (1%, w/v) for Streptomyces sp. TO1. Cultures were grown under constant agitation at 150 rpm at 30³C for SL 1326/pLM1 and 45³C for Streptomyces sp. TO1. RNA was prepared from cultures actively producing K-amylase. This activity excreted in the culture medium was assayed at 37³C by the method described by Virolle et al. [15] using a Beckman DU640 spectrophotometer. The highest K-amylase activities were obtained in 45-h and 32-h grown cultures of Streptomyces sp. TO1 and SL 1326/pLM1, respectively. 2.4.2. Northern blot analysis Total RNAs were isolated and re-suspended in a glyoxal and dimethyl sulfoxide denaturing solution, electrophoresed on a 1% agarose gel, and blotted on Hybond nylon membrane (Amersham) according to Ausubel et al. [16]. The membrane was baked at 80³C for 2 h, and hybridised for 24 h at 65³C in 78 ml of a hybridisation solution containing the 0.75-kbp StuI-StuI fragment internal to amy TO1 as a radiolabelled probe. After a stringent wash procedure (twice 20 min in 0.2USSC^0.1% SDS at room temperature and again twice at 65³C), the membrane was exposed to X-ray ¢lms for visualisation of the hybridisation pattern.
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Fig. 1. The nt sequence of the coding and regulatory regions of amy TO1 gene. The tsp determined by primer extension is indicated by open circles, and the associated arrow represents the direction of transcription. RBS site is indicated in bold letters. Putative 310 and 335 regions are underlined and overscored. The probable signal peptidase cleavage site is indicated by an upward arrow. Convergent arrows represent inverted repeats, and an asterisk denotes the stop codon. The proposed aa binding sequence of tendamistat is underlined. The aa residues of the four conserved regions found in all Amy are underlined and italicised. The nucleotide sequence of the K-amylase gene of Streptomyces TO1 is deposited with the EMBL Database, accession no. Y13332.
2.4.3. Primer extension analysis Determination of the transcriptional start point (tsp) was performed using the primer extension technique according to the Sambrook et al. [12] manual. A synthetic oligonucleotide (5P-AGGCCTGCGCGGTGGTCGGGG-3P) complementary to nucleotides (nt) 347 to 367 of the amy TO1 sequence was labelled at the 5P-terminus with [Q-32 P]ATP (3000 Ci/mmol) and the polynucleotide kinase, hybridised to speci¢c RNA and elongated by the reverse transcriptase (Promega) in the presence of deoxynucleotides. Speci¢c RNA was thus reverse transcribed into a labelled cDNA that was run on a denaturing polyacrylamide gel, close to a sequence ladder of the appropriate fragment made with the same oligonucleotide.
3. Results and discussion 3.1. Nucleotide sequence of the amy gene of the TO1 strain The 1860-bp DNA fragment containing the amy TO1 gene was sequenced (Fig. 1). Frame analysis revealed the presence of a unique open reading frame (ORF) starting with an ATG codon at nt position 295 and ending with a TGA stop codon at nt position 1654. The base composition of this ORF is 68% G+C, in good agreement with the average G+C content of Streptomyces DNA [17]. This high G+C content results in an extremely biased usage of synonymous codons with 97% of the codons possessing a G or a C at the third position. This ORF could
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L. Mellouli et al. / FEMS Microbiology Letters 160 (1998) 17^23 Fig. 2. A : Northern blot analysis of the amy TO1 gene. The radiolabelled 0.75 StuI^StuI DNA fragment internal to the amy TO1 gene was used as a probe. Lane 1: radiolabelled 1-kbp ladder (Gibco-BRL) ; lane 2: RNA from S. lividans 1326/pLM1 (10 Wg); lane 3: RNA from Streptomyces sp. TO1 (20 Wg). The arrow indicates the K-amylase transcripts. B: Primer extension analysis of amy TO1 gene. DNA sequences shown on the left indicate the position of the tsp of amy TO1 (boxed residues).
6
encode a 453-aa protein with an estimated Mr of 49 kDa. A comparison of the N-terminus of Amy TO1 with some other Streptomyces K-amylases suggested that the putative signal sequence of Amy TO1 was Met-Ala-Arg-Arg-Thr-Leu-Ala-Gly-Ala-Ala-Arg-SerArg-Ser-Ala-Leu-Val-Met-Thr-Pro-Thr-Thr-Ala-GlnAla. A hexanucleotide sequence 5P-AAGGAA-3P located 6 bp upstream of the putative start codon and showing a fairly good complementarity to the 3P end of the 16S rRNA of S. lividans [18] is likely to constitute the RBS of the amy TO1 transcript. Ten nucleotides downstream from the stop codon there is an inverted repeat of 6 nucleotides that may play a role in transcription termination [19]. 3.2. Northern blot and primer extension analysis Northern blot analysis has shown that amy TO1 gene was transcribed approximately as a 1.5-kbp monocistronic mRNA in both SL 1326/pLM1 and Streptomyces sp. TO1 strains (Fig. 2A). This size was in good agreement with that expected from the sequence data. Primer extension mapping (Fig. 2B) showed the presence of a unique tsp at the C at position 264^ 265. This tsp is located 30^31 bp upstream of the ATG start codon. This experiment allowed the determination of eubacterial like 310 (TACGCG) and 335 (TTGCTG) promoter sequences separated by 18 bp and located 5^6 nt upstream of the tsp. Comparison of the amy gene promoters among known Streptomyces species showed that corresponding 310 and 335 regions were, respectively, (T/C)A(C/ G)G(G/C)T and TTG(A/C)C(C/G) [8]. Therefore, the 310 (TACGCG) and 335 (TTGCTG) regions of amy TO1 gene are slightly di¡erent from proceeding consensus.
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Fig. 3. Di¡erent motifs of the optimal alignment of amino acid sequence of Amy TO1 with those of S. thermoviolaceus (AMYST), S. venezuelae (AMYSV), S. limosus (AMYSL), S. griseus (AMYSG), Streptomyces sp. WL16 (AMYSS) and S. hygroscopicus (AMYHT). Symbols: 8, substitution of Gly57 , Arg87 , Arg251 and Ala414 or Gly414 by Pro residues in Amy TO1; 9, substitution of Ile151 and Lys302 by Val and Arg, respectively, in Amy TO1; B, substitution of hydrophilic aa Tyr56 , Arg294 and Asp334 by hydrophobic residues Leu, Ala and Ala, respectively, in Amy TO1.
3.3. Comparison of amino acid sequence of Amy TO1 with other Streptomyces K-amylases This comparison revealed that Amy TO1 shows high aa identities with K-amylases from other Streptomyces (Table 1). This identity reaches 78% with the less thermostable K-amylase of S. thermoviolaceus CUB74 [20]. Amy TO1 contains in its amino-terminal part the `Phe-Glu-Trp triplet', which has been suggested as a likely candidate for interaction with the tendamistat inhibitor [4]. Amy TO1, like other Streptomyces K-amylases, contains the four conserved aa regions (R1 to R4) characteristic of starch hydrolytic enzymes. These regions are also present in the pullulanases, isoamylases, neo-pullulanases and Kamylase-pullulanases, and are thought to be involved in substrate and calcium binding and catalysis [21].
3.4. What features make Amy TO1 thermostable? Many thermostable proteins have potential biotechnological applications. In consequence, the topic of protein thermostability has been extensively reviewed. Most evidence indicates that thermostable proteins are homologous to their mesophilic counterparts. In fact, thermozymes contain the same cataTable 1 Percentage of similarity between Amy TO1 and other Streptomyces K-amylases Amy of
Similarity (%)
Streptomyces sp. TO1 S. thermoviolaceus S. venezuelae S. limosus S. griseus Streptomyces sp. WL16 S. hygroscopicus
100 78.60 70.40 68.20 67.80 55.00 52.30
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L. Mellouli et al. / FEMS Microbiology Letters 160 (1998) 17^23
lytic consensus and in many cases the same structural backbone as their mesophilic counterparts [22]. No important structural features unique to thermozymes can account for their stability at elevated temperatures and their thermal properties reside in subtle aa di¡erences. Comparison of Amy TO1 with other Streptomyces K-amylases revealed the following di¡erences (Fig. 3): 1.
2.
3.
At position 57 and 87 (N-terminal parts), position 251 (central parts) and position 414 (Cterminal parts), respectively residues Gly, Arg and Ala or Gly found in all Streptomyces Kamylases were substituted by Pro residues in Amy TO1. Some authors emphasised the role of proline that could impose rigidity to polypeptide chains when it replaces small amino acid residues such as glycine and alanine [22]. Rigidity is an essential factor in protein thermostability. A high rigidity would preserve the catalytic active structure of thermozymes and protects them from unfolding. It was supposed that proline constrained loop regions to prevent the sequential dissociation of numerous coulombic stabilising interactions between two adjacent core elements [23]. At position 151 and 302 of Amy TO1, we noticed the replacement of Ile and Lys by Val and Arg, respectively. Comparing 70 proteins from mesophilic and thermophilic organisms, Menendez and Argos [24] ranked the most frequent amino acid changes from cold to hot as follows: Lys to Arg, Ser to Ala, Ser to Thr, Gly to Ala and Ile to Val. At positions 56, 294 and 334 the hydrophilic aa Tyr, Arg and Asp found in all Streptomyces Kamylases were substituted respectively by the hydrophobic residues Leu, Ala and Ala in Amy TO1. Bealin-Kelly et al. [25] established that thermostable Bacillus caldovelox K-amylase contains more hydrophobic interaction-forming residues than their mesophilic counterparts. It has been proposed that hydrophobic interactions play a signi¢cant role in enzyme thermostability by providing the energy needed to fold proteins in aqueous solutions.
Acknowledgments We thank Prof. R. Ellouz (Director of the Biotechnology Centre of Sfax-Tunisia) for invaluable discussions throughout this study, and Dr. M. Guerineau (DR1, CNRS, Orsay-France) for helpful advice. This research was supported by the Tunisian Government and European Commission (CII*-CT 94-0121). References [1] Robbins, P.W., Wirth, D.F. and Hering, C. (1981) Expression of the endoglycosidase H in Escherichia coli. J. Biol. Chem. 256, 10640^10644. [2] Kendhall, K. and Cullem, J. (1984) Cloning and expression of an extracellular agarase from Streptomyces coelicolor A3(2) in Streptomyces lividans 66. Gene 29, 315^321. [3] Iwasaki, A., Kishida, H. and Okanishi, M. (1987) Molecular cloning of a xylanase gene from Streptomyces sp. No. 36a and its expression in Streptomycetes. J. Antibiot. 39, 985^988. [4] Long, M.C., Virolle, M.J., Chang, Y.S., Chang, S. and Bibb, J.M. (1987) Alpha-amylase gene of Streptomyces limosus: Nucleotide sequence, expression motifs, and amino acid sequence homology to mammalian and invertebrate alpha-amylases. J. Bacteriol. 196, 5745- 5754. [5] Virolle, M.J. and Bibb, M.J. (1988) Cloning, characterisation and regulation of an alpha-amylase gene from Streptomyces limosus. Mol. Microbiol. 2, 197^208. [6] Bahri, M.S. and Ward, M.J. (1990) Cloning and expression of an alpha-amylase gene from Streptomyces thermoviolaceus CUB74 in E. coli JM107 and S. lividans TK24. J. Microbiol. 136, 811^818. [7] Vigal, T., Gill, J.A., Daza, A., Garcia-Gonzalez, M.D. and Martin, J.F. (1991) Cloning, characterization and expression of an alpha-amylase gene from Streptomyces griseus IMRU3570. Mol. Gen. Genet. 225, 278^288. [8] Tsao, L.S., Lin, L.L., Chen, J.C., Chen, J.H. and Hsu, W.H. (1993) Cloning and characterization of an alpha-amylase gene from Streptomyces lividans. Biochim. Biophys. Acta 117, 255^ 262. [9] Chen, I., Marcos, T.A., Costa, S., Martin, F.J. and Padilla, G. (1995) Cloning and characterisation of an K-amylase gene from Streptomyces sp. WL 16. Biochem. Mol. Biol. Int. 1059^1067. [10] Mellouli, L., Ghorbel, R., Kammoun, A., Mezghani, A. and Bejar, S. (1996) Characterisation and molecular cloning of thermostable alpha-amylase from Streptomyces sp. TO1. Biotechnol. Lett. 18, 809^814. [11] Yanish-Pierron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103^119.
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L. Mellouli et al. / FEMS Microbiology Letters 160 (1998) 17^23 [12] 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. [13] Hopwood, D.A., Bibb, M.J., Chater, K.F., Kieser, T., Bruton, C.J., Kieser, H.M., Lydiate, D.J., Smith, C.P., Ward, J.M. and Schremph, H. (1985) Genetic manipulation of Streptomyces. A Laboratory Manual. John Innes Foundation, Norwich. [14] Sanger, F., Coulson, A.R., Barrel, B.G., Smith, A.J.H. and Roe, B.A. (1980) Cloning in single stranded bacteriophage as an aid to rapid DNA sequencing. J. Mol. 143, 161^178. [15] Virolle, M.J., Morris, V.J. and Bibb, M.J. (1990) A simple and reliable turbidimetric and kinetic assay for alpha-amylase that is readily applied to culture supernatants and cell extracts. J. Ind. Microbiol. 5, 295^302. [16] Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.O., Seidman, J.S., Smith, J.A. and Struhl, K. (1987) In: Current Protocols in Molecular Biology (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.O., Seidman, J.S., Smith, J.A. and Struhl, K., Eds.) Section 2.4.1. Green Publishing Associates, New York. [17] Gladek, A. and Zakrzewska, J. (1984) Genome size of Streptomyces. FEMS Microbiol. Lett. 24, 73^76. [18] Bibb, M.J. and Cohen, S.N. (1982) Gene expression in Streptomyces: Construction and application of promoter-probe
[19]
[20]
[21]
[22]
[23]
[24]
[25]
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plasmids vectors in Streptomyces lividans. Mol. Gen. Genet. 87, 265^277. Rosenberg, M. and Court, D. (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu. Rev. Genet. 13, 319^359. Bahri, M.S. and Ward, M.J. (1993) Sequence of the Streptomyces thermoviolaceus CUB74 alpha-amylase encoding gene and its transcription analysis in Streptomyces lividans. Gene 127, 133^137. Janse, B.J., Steyn, A.J. and Pretorius, I.S. (1993) Regional sequence homologies in starch-degrading enzymes. Curr. Genet. 24, 400^407. Ostendorp, R., Liebl, W., Schurig, H. and Jaenick, R. (1993) The L-lactate dehydrogenase gene of the hyperthermophilic bacterium Thermotoga maritima cloned by complementation in E. coli. Eur. J. Biochem. 216, 709^715. Claire, V., Burdette, D.S. and Zeikus, J.G. (1996) In: Biotechnology Annual (El-Gewely, M.R., Ed.) pp. 1^83, Elsevier Science B.V., Amsterdam, The Netherlands. Menendez, A.P. and Argos, P. (1989) Engineering protein thermal stability. Sequence statistics point to residue substitutions in alpha-helices. J. Mol. Biol. 206, 397^406. Bealin-Kelly, F., Kelly, C.T. and Forgarty, W.M. (1991) Studies on the thermostability of the K-amylase of Bacillus caldovelox. Appl. Microbiol. Biotechnol. 36, 332^336.
FEMSLE 8011 17-2-98
Alpha-amylase gene of thermophilic Streptomyces sp. TO1: nucleotide sequence, transcriptional and amino acid sequence analysis Lot¢ Mellouli a; *, Raoudha Ghorbel a , Marie Joelle Virolle b , Samir Bejar b
a
a Centre de Biotechnologie de Sfax, B.P. 358, 3018 Sfax, Tunisie Institut de Geèneètique et Microbiologie, URA CNRS 1354, Baêtiment 400, Universiteè Paris-Sud, F-91405 Orsay Cedex, France
Received 28 November 1997; accepted 20 December 1997
Abstract The nucleotide sequence of a 1860-bp region encoding a thermostable K-amylase of Streptomyces sp. TO1 was determined. Frame analysis revealed the presence of a 1359-bp long open reading frame (amy TO1) encoding a 453 amino acid protein with a deduced Mr of 49 kDa. Northern blot analysis revealed that amy TO1 gene was expressed as approximately 1.5-kbp monocistronic transcript in both SL1326/pLM1 and Streptomyces sp. TO1 strains. Primer extension experiments indicated that the transcriptional start site lies 30 bp upstream of the ATG start codon, and allowed the identification of 335 (TTGCTG) and 310 (TACGCG) eubacterial-like promoter sequences. Amy TO1 exhibits strong amino acid identities with those from other Streptomyces species with a maximum of 78% with S. thermoviolaceus K-amylase. Nevertheless, subtle amino acid changes such as the substitution of four conserved residues found at similar positions in other Streptomyces K-amylases by proline residues, and the substitution of three conserved hydrophilic amino acids by hydrophobic ones in Amy TO1 might account for the thermostable properties of Amy TO1. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Thermostability; Sequence comparison; Hydrophobicity; Proline residue
1. Introduction Streptomyces are saprophytic soil bacteria produc* Corresponding author. Tel.: +216 (4) 274 110; Fax: +216 (4) 275 970. Abbreviations : aa, amino acid(s) ; amy, gene encoding Kamylase; Amy, K-amylase; bp, basepair(s); kbp, kilo basepair(s) or 1000 basepairs; kDa, kilo dalton; Mr , relative molecular mass; nt, nucleotide(s); ORF, open reading frame; tsp, transcription start point; RBS, ribosome-binding site; SL 1326, Streptomyces lividans 1326
ing several kinds of secondary metabolites, including most of the known antibiotics. In order to obtain nutrients from organic debris in the soil, they secrete a variety of hydrolytic enzymes, such as protease, nucleases, lipases and other di¡erent enzymes degrading polysaccharides such as cellulose, chitin, starch and xylan. Genes encoding a variety of extracellular enzymes from Streptomyces species have been cloned and characterised. These include endoglycosidase H [1], agarase [2], xylanase [3], and amylases [4^9].
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 5 8 5 - 5
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L. Mellouli et al. / FEMS Microbiology Letters 160 (1998) 17^23
K-Amylase (EC 3.2.1.1) is widely used in starch processing industries. These secreted endoenzymes randomly cleave K(1,4) linkages in starch and usually produce a mixture consisting of maltose, malto-oligosaccharides and K-limit dextrins as ¢nal products. In starch industry, high hydrolysis temperature is desirable because starch granules cannot be attacked by K-amylase unless they have been ruptured by heat. We have previously reported the cloning and expression of a thermostable K-amylase isolated from a new thermophilic Streptomyces species strain TO1 [10]. The thermostable Amy TO1 works at an optimal T³ of 70³C and might be of industrial interest to produce glucose syrup from starch. In this paper, we are reporting the sequencing of the gene encoding the thermostable K-amylase of the TO1 strain, its transcriptional analysis as well as detailed amino acid (aa) comparisons of Amy TO1 with other Streptomyces K-amylases. This comparison pointed toward unexpected amino acid changes that might account for the thermostable properties of Amy TO1.
2. Materials and methods 2.1. Bacterial strains and plasmids Streptomyces sp. TO1 strain and Streptomyces lividans 1326 containing pLM1 [10] were used in this work. E. coli DH5K (F3 P80 dlacZvM15 v(lacZYAargF) U169 endA1 recA1 hsdR17 (rk 3 , mk ) deoR thi-1 susE44 V3 gyrA96 relA1) and TG1 (supE hsdv5 thi v (lac-proAB) FP (traD36 proAB lacIq lacZvM15) were used as host strains. pLM1 and pLM2 [10] recombinant plasmids carrying amy TO1 were used as a source of sub-cloning fragments for sequencing, and M13 derivatives [11] were used as sequencing vectors. 2.2. DNA isolation and manipulation Plasmid puri¢cation, DNA digestion with restriction enzymes and cloning were done according to Sambrook et al. [12] for E. coli and Hopwood et al. [13] for Streptomyces.
2.3. DNA sequencing and sequence analysis DNA fragments were cloned into M13 derivatives, and unidirectional nested deletions were created using exonuclease III as described in Pharmacia protocols. The nucleotide sequence of the 1860-bp fragment containing the amy TO1 gene was determined on both strands using the chain-termination method of Sanger et al. [14] with universal primers. Sequence analysis and comparisons were done using the PC gene Software package 6.85 (Intelligenetics Inc., University of Geneva, Switzerland). 2.4. RNA isolation and manipulation 2.4.1. Culture conditions for RNA isolation Spores were pre-germinated as described previously by Hopwood et al. [13] and grown in 150-ml Erlenmeyer £asks containing 20 ml of liquid minimal medium with glycerol (1%, w/v) as carbon source for SL 1326/pLM1 and starch (1%, w/v) for Streptomyces sp. TO1. Cultures were grown under constant agitation at 150 rpm at 30³C for SL 1326/pLM1 and 45³C for Streptomyces sp. TO1. RNA was prepared from cultures actively producing K-amylase. This activity excreted in the culture medium was assayed at 37³C by the method described by Virolle et al. [15] using a Beckman DU640 spectrophotometer. The highest K-amylase activities were obtained in 45-h and 32-h grown cultures of Streptomyces sp. TO1 and SL 1326/pLM1, respectively. 2.4.2. Northern blot analysis Total RNAs were isolated and re-suspended in a glyoxal and dimethyl sulfoxide denaturing solution, electrophoresed on a 1% agarose gel, and blotted on Hybond nylon membrane (Amersham) according to Ausubel et al. [16]. The membrane was baked at 80³C for 2 h, and hybridised for 24 h at 65³C in 78 ml of a hybridisation solution containing the 0.75-kbp StuI-StuI fragment internal to amy TO1 as a radiolabelled probe. After a stringent wash procedure (twice 20 min in 0.2USSC^0.1% SDS at room temperature and again twice at 65³C), the membrane was exposed to X-ray ¢lms for visualisation of the hybridisation pattern.
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Fig. 1. The nt sequence of the coding and regulatory regions of amy TO1 gene. The tsp determined by primer extension is indicated by open circles, and the associated arrow represents the direction of transcription. RBS site is indicated in bold letters. Putative 310 and 335 regions are underlined and overscored. The probable signal peptidase cleavage site is indicated by an upward arrow. Convergent arrows represent inverted repeats, and an asterisk denotes the stop codon. The proposed aa binding sequence of tendamistat is underlined. The aa residues of the four conserved regions found in all Amy are underlined and italicised. The nucleotide sequence of the K-amylase gene of Streptomyces TO1 is deposited with the EMBL Database, accession no. Y13332.
2.4.3. Primer extension analysis Determination of the transcriptional start point (tsp) was performed using the primer extension technique according to the Sambrook et al. [12] manual. A synthetic oligonucleotide (5P-AGGCCTGCGCGGTGGTCGGGG-3P) complementary to nucleotides (nt) 347 to 367 of the amy TO1 sequence was labelled at the 5P-terminus with [Q-32 P]ATP (3000 Ci/mmol) and the polynucleotide kinase, hybridised to speci¢c RNA and elongated by the reverse transcriptase (Promega) in the presence of deoxynucleotides. Speci¢c RNA was thus reverse transcribed into a labelled cDNA that was run on a denaturing polyacrylamide gel, close to a sequence ladder of the appropriate fragment made with the same oligonucleotide.
3. Results and discussion 3.1. Nucleotide sequence of the amy gene of the TO1 strain The 1860-bp DNA fragment containing the amy TO1 gene was sequenced (Fig. 1). Frame analysis revealed the presence of a unique open reading frame (ORF) starting with an ATG codon at nt position 295 and ending with a TGA stop codon at nt position 1654. The base composition of this ORF is 68% G+C, in good agreement with the average G+C content of Streptomyces DNA [17]. This high G+C content results in an extremely biased usage of synonymous codons with 97% of the codons possessing a G or a C at the third position. This ORF could
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L. Mellouli et al. / FEMS Microbiology Letters 160 (1998) 17^23 Fig. 2. A : Northern blot analysis of the amy TO1 gene. The radiolabelled 0.75 StuI^StuI DNA fragment internal to the amy TO1 gene was used as a probe. Lane 1: radiolabelled 1-kbp ladder (Gibco-BRL) ; lane 2: RNA from S. lividans 1326/pLM1 (10 Wg); lane 3: RNA from Streptomyces sp. TO1 (20 Wg). The arrow indicates the K-amylase transcripts. B: Primer extension analysis of amy TO1 gene. DNA sequences shown on the left indicate the position of the tsp of amy TO1 (boxed residues).
6
encode a 453-aa protein with an estimated Mr of 49 kDa. A comparison of the N-terminus of Amy TO1 with some other Streptomyces K-amylases suggested that the putative signal sequence of Amy TO1 was Met-Ala-Arg-Arg-Thr-Leu-Ala-Gly-Ala-Ala-Arg-SerArg-Ser-Ala-Leu-Val-Met-Thr-Pro-Thr-Thr-Ala-GlnAla. A hexanucleotide sequence 5P-AAGGAA-3P located 6 bp upstream of the putative start codon and showing a fairly good complementarity to the 3P end of the 16S rRNA of S. lividans [18] is likely to constitute the RBS of the amy TO1 transcript. Ten nucleotides downstream from the stop codon there is an inverted repeat of 6 nucleotides that may play a role in transcription termination [19]. 3.2. Northern blot and primer extension analysis Northern blot analysis has shown that amy TO1 gene was transcribed approximately as a 1.5-kbp monocistronic mRNA in both SL 1326/pLM1 and Streptomyces sp. TO1 strains (Fig. 2A). This size was in good agreement with that expected from the sequence data. Primer extension mapping (Fig. 2B) showed the presence of a unique tsp at the C at position 264^ 265. This tsp is located 30^31 bp upstream of the ATG start codon. This experiment allowed the determination of eubacterial like 310 (TACGCG) and 335 (TTGCTG) promoter sequences separated by 18 bp and located 5^6 nt upstream of the tsp. Comparison of the amy gene promoters among known Streptomyces species showed that corresponding 310 and 335 regions were, respectively, (T/C)A(C/ G)G(G/C)T and TTG(A/C)C(C/G) [8]. Therefore, the 310 (TACGCG) and 335 (TTGCTG) regions of amy TO1 gene are slightly di¡erent from proceeding consensus.
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L. Mellouli et al. / FEMS Microbiology Letters 160 (1998) 17^23
21
Fig. 3. Di¡erent motifs of the optimal alignment of amino acid sequence of Amy TO1 with those of S. thermoviolaceus (AMYST), S. venezuelae (AMYSV), S. limosus (AMYSL), S. griseus (AMYSG), Streptomyces sp. WL16 (AMYSS) and S. hygroscopicus (AMYHT). Symbols: 8, substitution of Gly57 , Arg87 , Arg251 and Ala414 or Gly414 by Pro residues in Amy TO1; 9, substitution of Ile151 and Lys302 by Val and Arg, respectively, in Amy TO1; B, substitution of hydrophilic aa Tyr56 , Arg294 and Asp334 by hydrophobic residues Leu, Ala and Ala, respectively, in Amy TO1.
3.3. Comparison of amino acid sequence of Amy TO1 with other Streptomyces K-amylases This comparison revealed that Amy TO1 shows high aa identities with K-amylases from other Streptomyces (Table 1). This identity reaches 78% with the less thermostable K-amylase of S. thermoviolaceus CUB74 [20]. Amy TO1 contains in its amino-terminal part the `Phe-Glu-Trp triplet', which has been suggested as a likely candidate for interaction with the tendamistat inhibitor [4]. Amy TO1, like other Streptomyces K-amylases, contains the four conserved aa regions (R1 to R4) characteristic of starch hydrolytic enzymes. These regions are also present in the pullulanases, isoamylases, neo-pullulanases and Kamylase-pullulanases, and are thought to be involved in substrate and calcium binding and catalysis [21].
3.4. What features make Amy TO1 thermostable? Many thermostable proteins have potential biotechnological applications. In consequence, the topic of protein thermostability has been extensively reviewed. Most evidence indicates that thermostable proteins are homologous to their mesophilic counterparts. In fact, thermozymes contain the same cataTable 1 Percentage of similarity between Amy TO1 and other Streptomyces K-amylases Amy of
Similarity (%)
Streptomyces sp. TO1 S. thermoviolaceus S. venezuelae S. limosus S. griseus Streptomyces sp. WL16 S. hygroscopicus
100 78.60 70.40 68.20 67.80 55.00 52.30
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L. Mellouli et al. / FEMS Microbiology Letters 160 (1998) 17^23
lytic consensus and in many cases the same structural backbone as their mesophilic counterparts [22]. No important structural features unique to thermozymes can account for their stability at elevated temperatures and their thermal properties reside in subtle aa di¡erences. Comparison of Amy TO1 with other Streptomyces K-amylases revealed the following di¡erences (Fig. 3): 1.
2.
3.
At position 57 and 87 (N-terminal parts), position 251 (central parts) and position 414 (Cterminal parts), respectively residues Gly, Arg and Ala or Gly found in all Streptomyces Kamylases were substituted by Pro residues in Amy TO1. Some authors emphasised the role of proline that could impose rigidity to polypeptide chains when it replaces small amino acid residues such as glycine and alanine [22]. Rigidity is an essential factor in protein thermostability. A high rigidity would preserve the catalytic active structure of thermozymes and protects them from unfolding. It was supposed that proline constrained loop regions to prevent the sequential dissociation of numerous coulombic stabilising interactions between two adjacent core elements [23]. At position 151 and 302 of Amy TO1, we noticed the replacement of Ile and Lys by Val and Arg, respectively. Comparing 70 proteins from mesophilic and thermophilic organisms, Menendez and Argos [24] ranked the most frequent amino acid changes from cold to hot as follows: Lys to Arg, Ser to Ala, Ser to Thr, Gly to Ala and Ile to Val. At positions 56, 294 and 334 the hydrophilic aa Tyr, Arg and Asp found in all Streptomyces Kamylases were substituted respectively by the hydrophobic residues Leu, Ala and Ala in Amy TO1. Bealin-Kelly et al. [25] established that thermostable Bacillus caldovelox K-amylase contains more hydrophobic interaction-forming residues than their mesophilic counterparts. It has been proposed that hydrophobic interactions play a signi¢cant role in enzyme thermostability by providing the energy needed to fold proteins in aqueous solutions.
Acknowledgments We thank Prof. R. Ellouz (Director of the Biotechnology Centre of Sfax-Tunisia) for invaluable discussions throughout this study, and Dr. M. Guerineau (DR1, CNRS, Orsay-France) for helpful advice. This research was supported by the Tunisian Government and European Commission (CII*-CT 94-0121). References [1] Robbins, P.W., Wirth, D.F. and Hering, C. (1981) Expression of the endoglycosidase H in Escherichia coli. J. Biol. Chem. 256, 10640^10644. [2] Kendhall, K. and Cullem, J. (1984) Cloning and expression of an extracellular agarase from Streptomyces coelicolor A3(2) in Streptomyces lividans 66. Gene 29, 315^321. [3] Iwasaki, A., Kishida, H. and Okanishi, M. (1987) Molecular cloning of a xylanase gene from Streptomyces sp. No. 36a and its expression in Streptomycetes. J. Antibiot. 39, 985^988. [4] Long, M.C., Virolle, M.J., Chang, Y.S., Chang, S. and Bibb, J.M. (1987) Alpha-amylase gene of Streptomyces limosus: Nucleotide sequence, expression motifs, and amino acid sequence homology to mammalian and invertebrate alpha-amylases. J. Bacteriol. 196, 5745- 5754. [5] Virolle, M.J. and Bibb, M.J. (1988) Cloning, characterisation and regulation of an alpha-amylase gene from Streptomyces limosus. Mol. Microbiol. 2, 197^208. [6] Bahri, M.S. and Ward, M.J. (1990) Cloning and expression of an alpha-amylase gene from Streptomyces thermoviolaceus CUB74 in E. coli JM107 and S. lividans TK24. J. Microbiol. 136, 811^818. [7] Vigal, T., Gill, J.A., Daza, A., Garcia-Gonzalez, M.D. and Martin, J.F. (1991) Cloning, characterization and expression of an alpha-amylase gene from Streptomyces griseus IMRU3570. Mol. Gen. Genet. 225, 278^288. [8] Tsao, L.S., Lin, L.L., Chen, J.C., Chen, J.H. and Hsu, W.H. (1993) Cloning and characterization of an alpha-amylase gene from Streptomyces lividans. Biochim. Biophys. Acta 117, 255^ 262. [9] Chen, I., Marcos, T.A., Costa, S., Martin, F.J. and Padilla, G. (1995) Cloning and characterisation of an K-amylase gene from Streptomyces sp. WL 16. Biochem. Mol. Biol. Int. 1059^1067. [10] Mellouli, L., Ghorbel, R., Kammoun, A., Mezghani, A. and Bejar, S. (1996) Characterisation and molecular cloning of thermostable alpha-amylase from Streptomyces sp. TO1. Biotechnol. Lett. 18, 809^814. [11] Yanish-Pierron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103^119.
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L. Mellouli et al. / FEMS Microbiology Letters 160 (1998) 17^23 [12] 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. [13] Hopwood, D.A., Bibb, M.J., Chater, K.F., Kieser, T., Bruton, C.J., Kieser, H.M., Lydiate, D.J., Smith, C.P., Ward, J.M. and Schremph, H. (1985) Genetic manipulation of Streptomyces. A Laboratory Manual. John Innes Foundation, Norwich. [14] Sanger, F., Coulson, A.R., Barrel, B.G., Smith, A.J.H. and Roe, B.A. (1980) Cloning in single stranded bacteriophage as an aid to rapid DNA sequencing. J. Mol. 143, 161^178. [15] Virolle, M.J., Morris, V.J. and Bibb, M.J. (1990) A simple and reliable turbidimetric and kinetic assay for alpha-amylase that is readily applied to culture supernatants and cell extracts. J. Ind. Microbiol. 5, 295^302. [16] Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.O., Seidman, J.S., Smith, J.A. and Struhl, K. (1987) In: Current Protocols in Molecular Biology (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.O., Seidman, J.S., Smith, J.A. and Struhl, K., Eds.) Section 2.4.1. Green Publishing Associates, New York. [17] Gladek, A. and Zakrzewska, J. (1984) Genome size of Streptomyces. FEMS Microbiol. Lett. 24, 73^76. [18] Bibb, M.J. and Cohen, S.N. (1982) Gene expression in Streptomyces: Construction and application of promoter-probe
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plasmids vectors in Streptomyces lividans. Mol. Gen. Genet. 87, 265^277. Rosenberg, M. and Court, D. (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu. Rev. Genet. 13, 319^359. Bahri, M.S. and Ward, M.J. (1993) Sequence of the Streptomyces thermoviolaceus CUB74 alpha-amylase encoding gene and its transcription analysis in Streptomyces lividans. Gene 127, 133^137. Janse, B.J., Steyn, A.J. and Pretorius, I.S. (1993) Regional sequence homologies in starch-degrading enzymes. Curr. Genet. 24, 400^407. Ostendorp, R., Liebl, W., Schurig, H. and Jaenick, R. (1993) The L-lactate dehydrogenase gene of the hyperthermophilic bacterium Thermotoga maritima cloned by complementation in E. coli. Eur. J. Biochem. 216, 709^715. Claire, V., Burdette, D.S. and Zeikus, J.G. (1996) In: Biotechnology Annual (El-Gewely, M.R., Ed.) pp. 1^83, Elsevier Science B.V., Amsterdam, The Netherlands. Menendez, A.P. and Argos, P. (1989) Engineering protein thermal stability. Sequence statistics point to residue substitutions in alpha-helices. J. Mol. Biol. 206, 397^406. Bealin-Kelly, F., Kelly, C.T. and Forgarty, W.M. (1991) Studies on the thermostability of the K-amylase of Bacillus caldovelox. Appl. Microbiol. Biotechnol. 36, 332^336.
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