Gene, 127 (1993) 133-137 Q 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l 1 i9/93/$06.00

GENE 07055

133

Sequence of the Strep tomyces thermoviolaceus CUB74 a-amylase-encoding gene and its transcription analysis in Streptomyces Zividans

~ucleotide sequence; amino acid comp~~son; promoter mapping; expression; thermophilic; inhibition by tendamistat)

Sami M. Bahri and John M. Ward

Biochemistry and Molecular Biology Department, University College London. Gower St., London WCIE 6BT, UK. Tel. (44-71) 3877050, ext. 2242; Fax

(44-71) 3807193

Received by C.R. Hutchinson: 22 June 1992; Revised/Accepted: 14 December/l9 December 1992; Received at publishers: 26 January 1993

SUMMARY

The a-amylase (Amy)-encoding gene (amy) of Streptomyces thermoviolaceus CUB74, previously cloned in Escherichia coli and S. liuidans and localised on a 1.7-kb BamHI-SphI genomic DNA fragment, has been sequenced. A single open reading frame of 1380 bp, which could encode an Amy protein of 460 amino acids (aa), was identified. The deduced aa sequence of the thermophilic Amy is similar (up to 69.5%) to the mesophilic Amy of S. griseus, S. limosus, S. uenezuelae

and S. hygroscopicus. A 40% sequence similarity was found between the extracellular forms of the S. thermoviolaceus and the pig pancreatic Amy. In addition, the activity of the S. thermoviolaceus Amy is strongly inhibited by tendamistat, a potent inhibitor of mammalian Amy. The nucleotide sequence at the 5’ end of umy was able to initiate trans~~ption in S. lividans and contains a promoter whose sequence is identical to the promoters of the S. lim~sus~ S. uenezuel~e and S. griseus amy.

INTRODUCTION

a-Amylases (Amy) are endoenzymes that randomly cleave 1,4-m-linkages in starch to produce oligosaccha- rides of different chain lengths (Guilbot and Mercier, 1985). They are found in both prokaryotes and eukary- otes, and genes encoding these enzymes have been iso- lated and characterized from many genera.

In the streptomycetes, reports of cloning and sequen- cing of amy genes have been limited to the mesophilic enzymes. These include the amy genes of S. hygroscopicus

Correspondence to: Dr. SM. Bahri at his present address: Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, 0511 Singapore. Tel. (65) 7723790; Fax (65) 7791117 e-mail:mcbsb@NUSVM

Abbreviations: aa, amino acid(s); Amy, a-amylase(s); amy, gene(s) encod- ing Amy; bp, base pair(s); kb, kilobase or 1000 bp; nt, nucieotid~s); ORF, open reading frame; P, promoter; PPA, pig pancreatic Amy; RBS, ribosome-binding site(s); S., Streptomycq tsp, transcription start point; [ 1, denotes plasmid-carrier state.

(McKillop et al., 1986; Hoshiko et al., 1987), S. limosus (Long et al., 1987), S. uenezuelae (Virolle et al., 1988), and S. griseus IMRV3570 (Vigal et al., 1990; Garcia-Gonzalez et al., 1991). The deduced aa sequences of these enzymes share a high degree of similarity (Virolle et al., 1988). In addition, streptomycete Amy were found to have con- siderable homology to mammalian and invertebrate Amy, but not to those of plants and fungi, or others of bacterial origin (Long et al., 1987). These relationships suggest that these genes may have derived from a common ancestral gene.

We previously reported the cloning and expression of a thermostable amy isolated from S. thermouiolaceus CUB74 (Bahri and Ward, 1990a). The thermophilic Amy is stable to heat in the presence of calcium. In addition, Amy production in S. thermoviolaceus was found to be induced by maltotriose but not maltose (Bahri and Ward, 1990b). The aim of the present study was to determine the sequence of amy including its flanking 5’ and 3’ non- coding regions and to analyse the transcription of this gene in S. fividans.

134

EXPERIMENTAL AND DISCUSSION

Our previous work had localised the coding sequence of amy to a 1.7-kb BarnHI-SphI DNA fragment (Bahri and Ward, 1990a). At present, we have sequenced this DNA fragment and identi~ed an ORF for amy which starts with an ATG at nt 215 and ends with a TGA stop codon at nt 1594 (Fig. 1). This ORF could encode a pro- tein of 460 aa (49.2 kDa), which is in reasonable agreement with the size of the Amy protein estimated by SDS-polyacrylamide gel analysis (Bahri and Ward, 1990a). Computer analysis (Heijne, 1986) of the predicted protein sequence revealed a putative signal sequence of

21 aa at its N terminus (Fig. 1). Cleavage of this signal sequence was predicted to occur after the LLA triplet.

Located 8 bp upstream from the putative start codon is the hexanucleotide sequence 5’-GAAAGA (Fig. 1) which shows perfect complementarity to the 3’ end of the 16s rRNA of S. lividans (Bibb and Cohen, 1982). Thus, this sequence is likely to be the RBS (Storm0 et al., 1982) of the amy transcript. The 69% G + C content of the Amy coding sequence is very close to the average G+ C content of Streptomyces DNA (Enquist and Bradley, 1971).

(b} The aa sequence homology The predicted aa sequence of the S. ther~ovioiaceus

thermostable Amy was optimally aligned with those of

~r3cmxGA-m

B.blXIT v- -oaJ-e

-BBS

~~GCAAGTlYTTCCA?XXCCTM-CCGTCCGACGCG MZ0ZlC~ACGGAGAMGAACCCXXACATGXCAGCAGAA cxclvltGGGCcx: 240

#JuTI MASRTLSGA'

<A%CGXGCCCAC~CG@XCCAA~~GA~~360

LALAAAATAVLAAPATVAHRSPPGTKDVTAVL- t

D Y V S V4'

TePCCCCGCCCA~AC~TACCAG OCTACM 480

AKKCTSTLGPAGYGYVQVSPPAEHIQGSQWWTSYQPVSYKag

~~~A~~~~AC~~~~~M~~A~~~~~600

IAGRLGDRAAFRSHVNTCHAAGVICVVV~ BSAGSGT 129

G wmGGccCCGGAs ‘zIxx%CcGCmTcACCG &.ZTACC!AGGG 720

GTGOSSYTKYDYPGLYSAPDFDDCTAEI TDYQDRWNVQHC16g

~~~~~AGTACGTGCCACAGACGA?COCCGGCTACA~~~~~~~ GCABAC *4o

ELVGLADLDTGEEYVRQTIAGYMNDLLSLGVDGFRIDAAT 209

O CCGAACCXCTACXZGMGCAGGAGGXA lcrACGGCcmGAACCrCC%ARGCCtGGCGh

Jf_IPAEDLANIKS RLSNPNAYWKQ

c?mCACcGG ;4";

IVIIGAGEPPKPGEYTG

p_Awmcwm CCCAGGAGCB A0XXXAGAAC.MZGtXGMBACTGXXXT ACC'lGAQZACXACGAcGoc ',ygo

TGDVQEPRYAYDLKRVFTQEHLAYLKNYGEDWGYLSSTTA

-mAACw CGCIXAACl?.CAAGA4(XXCGCCACCl.A~?CXT

GVrv,o

kzxxmxaa ;"2"9

TERNGSTLNYKNDATYTLANVFnLAWPYGAPD

~~~~~~~C~~~A~~~~ IT 1320

INSGYEWSDPDAGPPDGGGVl3ACWQNGWKCQHKWPPII,ASU 369

~~~~ATCGCCFIUXCCGCKXCA~AA~ ~ 1440

VAPRNATRGEPVTDWWDDGADAIAFGRGSKGFVAINHESA 409

mmm_CCT ACXCGACGlGCAG?XJCAACACCACGGXZt~ACGUFXACC~ 1560

TVQRTYQTSLPAGTYCDVQSNTTVTVDSAGRFTAALGPDT44g

v-p

ALA LHNGRTSC.' Am-===CCCAe CfxamxCGAwp roO

pv spar: 1712

Fig. 1. The nt sequence of S. thermovioluceus CUB74 amy. DNA fragments were cloned into Ml3 and pUC derivatives (Yanisch-Perron et al., 1985) and sequenced (Sanger et ai., 1977; 1980) using the 17-m universai and reverse primers (from Pharmacia). dITP was also used where necessary to

resolve band compressions. The tsp is indicated by open circles, and the associated arrow represents the direction of transc~ption, Putative -10 and -35 regions are overscored, and 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 italicized. GenBank accession No. is M34957.

135

the S. griseus, S. limosus, S. venezuelae, and S. hygroscopi- cus mesophilic Amy (Table I). S. thermoviolaceus Amy shows a greater degree of identity to the S. griseus and S. limosus Amy (69.5% similarity), compared with a sim- ilarity of 68.5% to the S. uenezuelae and 43.7% to the S. hygroscopicus Amy (Table I). The S. limosus, S. vene- zuelae, and S. griseus Amy have a long C-terminal por- tion which is not found in either the S. thermoviolaceus or S. hygroscopicus Amy. Svensson et al. (1989) have spec- ulated that this region may be a domain which binds to raw starch granules. The S. thermoviolaceus Amy is the smallest of the streptomycete family of amylases.

Alignment of the aa of the extracellular forms of S. thermoviolaceus Amy and the mammalian PPA shows that these enzymes are 40% similar (Table I), which is close to the degree of similarity between streptomycete and mammalian amylases (36%) reported by Long et al. (1987). The three-dimensional structure of PPA has been determined (Buisson et al., 1987), and the aa residues involved in calcium binding were shown to be N”‘, D159, D16’, and H 201 These aa residues also occur in S. thermo- .

violaceus Amy (NE’, H13’, D’45, and H’79; Fig. 1). The same aa are also conserved in the other streptomycete Amy, except in S. hygroscopicus where the first aa residue is replaced by T “’ It would be interesting to test whether .

or not Ca2+ ions can influence the stability of these meso- philic Amy as has been the case for the thermophilic Amy (Bahri and Ward, 1990a).

The close similarity between streptomycete and mam- malian Amy is further confirmed by the finding that both groups of enzymes are inhibited by tendamistat while all other eubacterial Amy are not (Long et al., 1987). S. thermoviolaceus Amy with an initial activity of 1.4 units/ml was partially inhibited in the presence of 1.75

TABLE I

The aa sequence similarity between several known Amy

Amy” Length

(aa)

Similarityb

(“/)

STT 460 100

STG 566 69.5

STL 566 69.5

STV 568 68.5

STH 478 43.7

PPA 496 40

“The Amy aa sequences were optimally aligned using the MicroGenie

sequence analysis computer program (Beckman-RIIC Ltd., High

Wycombe, Buckinghamshire, UK). STT, S. thermouio&eus; STG, S.

griseus; STL, S. limosus; STV, S. uenezuelae; STH, S. hygroscopicus; PPA, pig pancreatic Amy.

bCalculated as: N (id;tical) x 100, where N (identical)=number of

identical aa in each comparison, and N = total number of aa in the

shorter aa sequence.

and 3.5 pmol of tendamistat (Table II). Complete inhibi- tion was observed in the presence of 17.5 pmol of inhibi- tor. The profile of inhibition of S. thermoviolaceus Amy is similar to those of S. limosus (Long et al., 1987) and S. venezuelae (Virolle et al., 1988) Amy. The aa sequence FEW, which has been suggested as a possible candidate for interaction with the inhibitor tendamistat (Hofmann et al., 1985), is also present in S. thermoviolaceus Amy (Fig. 1).

(c) Transcription of amy in Streptomyces lividans

S. lividans is used extensively as a host for cloning and expressing genes from Streptomyces species, and being a streptomycete it should allow expression from the natural promoter of the S. thermoviolaceus amy. Therefore, a 1.7- kb BamHI-SphI genomic DNA fragment carrying amy was cloned between the BglII and SphI sites of S. lividans plasmid pIJ702 to produce pQR311. Total mRNA was prepared from S. lividansCpQR31 l] and analysed on a Northern blot (Fig. 2A).

The 1.7-kb genomic fragment carrying amy was radio- labeled and used as a probe. The probe hybridized to three mRNA transcripts of different sizes (1.5,2, and 3 kb) in the lane containing mRNA isolated from S. liuidansCpQR31 l] (Fig. 2A, lane 3) whereas no mRNA bands were detected in the control lane containing mRNA isolated from S. lividans[pIJ702] (Fig. 2A, lane 2). Given the size of the genomic insert (1.7 kb) in pQR3 11, it was concluded that the 1 S-kb mRNA species is the result of transcription from the amy promoter situ- ated on the genomic insert itself, whereas the other two mRNA species (2 and 3 kb) may have resulted from read-

TABLE II

Tendamistat assay

Inhibitor” Amy activity” Residual activity

(pmol) (units/ml) (X)

0.0 1.4 (100) 0.35 1.3 93

1.75 1.1 78

3.5 1.0 71

17.5 0.0 0

35.0 0.0 0

70.0 0.0 0

‘Methods: The activity of S. thermooiohceus Amy was determined using

the turbidity method as described by Long et al. (1987). In the inhibitor

assay, 100~nl samples of an Amy solution prepared from the supernatant

of a S. thermouiolaceus CUB74 liquid culture were incubated for 2 min

in the presence of 0 (control), 0.35, 1.75, 3.5, 17.5, 35, or 70pmol of

tendamistat inhibitor, respectively. The Amy samples were then mixed

with 900 ul of potato starch solution (in 50 mM Naeacetate pH

5.8/S mM CaCl,), and the rate of decrease of Asoon,,, was measured at

50°C. Tendamistat (Calbiochem) is a potent inhibitor of mammalian

Amy.

136

2.4_

1.4_

4

Fig. 2. Analysis of transcripts. (Panel A) Northern blotting. Total RNA was isolated from Streptomyces by the method of Covey and Smith (see Hopwood et al., 1985). RNA samples (40 ug per lane) were run in a 1% agarose gel, analysed by Northern blot and probed with [a- 32P]dCTP nick-translated I .%kb SUSHI-SpkI genomic DNA fragment carrying amy (Sambrook et al., 1989). Lanes: 1, RNA size markers (from Bethesda Research Laboratories); 2, RNA from S. liuidans[pIJ702] (control); 3, RNA from 8. liuidans[pQR311]. The three transcripts (1.5, 2, and 3 kb, respectively) detected with the amy DNA probe are indi- cated by arrows (lane 5). (Panel B) High resolution Sl mapping. This was done as described by Berk and Sharp (1977). A 258-bp BumHI- Nor1 DNA fragment labeled at the Not1 end (Sambrook et al., 1989) was used to map the tsp of amy in 5. liuidans[pQRfll] (lane 5). The size marker (lanes l-4) was obtained by cloning the 258-bp BarnHI- Not1 DNA fragment between the BumHI and SpkI sites of pUC18 and sequencing with the 17-nt primer. The size marker contains 27 extra nt comprising the 17-nt primer and IO-nt linker DNA generated from cloning. Lanes 1,2,3, and 4 are A, T, C, and G sequences, respectively. Lane 6 is RNA from S. ~i~jdu~s[pIJ702] (control). All samples were analysed on an 8% polyacrylamide-7 M urea sequencing gel. The three Sl-protected DNA fragments are indicated by arrows (lane 5).

through from other sequences located on pIJ702. This hypothesis was confirmed by high resolution Sl-mapping experiments.

In order to map the tsp of amy in S. lioidans[pQR311], a 258-bp BamHI-Nut1 DNA fragment (Fig. 1) was end- labeled at the Not1 site. In S. Z~~j~u~s~pQR31 I], three Sl- protected DNA fragments were detected (Fig. 2B, lane 5). The lowest band corresponds to nt GGC (nt 124-126). This tsp is located 91 bp upstream from the predicted

translation start codon (Fig. 1). Potential - 10 (TACGGT) and -35 (TTGACC) regions (separated by 17 bp) are located 2-4 bp upstream from the tsp (Fig. 1). Furthermore, the sequences of the - 10 and - 35 regions in the amy promoter are identical to those found in the promoter regions of the S. limosus, S. venezuelae, and S. griseus amy. Although the amy promoter shows sequence similarity to typical E. co&type promoters (Hawley and Ma&lure, 1983), the amy promoter is not functional in E. coli (Bahri and Ward, 1990a) . Similarly, the ermEP1 and ermEP2 promoters, which also resemble typical eubacterial promoters, do not function in E. cob (Bibb et al., 1986).

Another Sl-protected band was detected (Fig. ZB, lane 5) which may correspond to a second tsp 25-26 bp upstream from GGC. However, it is not known whether this tsp is used in the natural host, S. thermoviolaceus. The largest Sl-protected DNA fragment corresponds to the size of the full-length probe. This is probably due to transcriptional readthrough from the upstream pIJ702 sequences (also deduced from Northern blotting data). It is unlikely that this band is due to reannealing of the probe since no Sl-protected bands were detected in the control, S. ~i~~~~ns~pIJ702] (Fig. 2B, lane 6).

If the amy transcript is approximately 1.5 kb long and the tsp is at nt 126, then the transcription termination site probably lies between 20 and 50 bp downstream from the translational stop codon. There is a perfect inverted repeat of 11 bp in this region (spanning nt 1622-1651) which may be a transcription terminator. Inverted repeats are also found upstream from the potential amy promoter (Fig. 1). These repeats may play a role in terminating transcripts initiated from genes situated upstream from amy.

(d) Conclusions (I) Sequencing of the S. thermoviolaceus CUB74 amy

revealed an ORF of 1380 bp which could encode an Amy protein of 460 aa. The S. therm~vio~ace~s Amy has 69.5% similarity to the S. griseus and S. l~mosus Amy, 68.5% similarity to the S. venezuelae Amy, and 43.7% similarity to the S. hygroscopicus Amy. Furthermore, the S. thermo- violaceus Amy has a 40% similarity to the PPA.

(2) The tsp of the S. thermoviolaceus amy was identified by S 1 mapping in S. ~iv~~ans. The - 10 and - 35 regions of the S. therm~violace~s amy promoter were found to be identical to those of the S. limosus, S. venezuelae, and S. griseus amy promoters. Furthermore, the putative RBS has perfect complementarity to the 3’ end of the 16s rRNA of S. lividuns. The translation stop codon is fol- lowed by a perfect inverted repeat of 11 bp (nt 1622- 165 1) which may act as a transcription terminator.

(3) S. thermoviolaceus Amy activity was found to be

137

inhibited by tendamistat, a potent inhibitor of mamma-

lian Amy.

ACKNOWLEDGEMENTS

This work was supported by a grant from the Hariri foundation and the SERC Biotechnology Directorate to S.M.B. and J.M.W., respectively. We thank Drs. Sara Zaman and Thomas Dick for their comments on the manuscript.

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