269 Gene, 47 (1986) 269-211

Elsevier

GEN 01776

Cyclodextrin-glycosyltransferase from Klebsielh pneumoniue M5al: cloning, nucleotide sequence and expression

(Recombinant DNA; precursor protein; secretion; homologies to a-amylases)

Florian Binder”, Otto Huberb and August B&k”*

n Lehrstuhl fi3r Mikrobiologie der Universittit Miinchen. Maria- Ward-Strasse la, D-8000 Miinchen 19, Tel. 089/l 77084, and b Consortium ftir Elektrochemische Industrie GrnbH, Zielstattstrasse 20. D-8000 Miinchen 70 (F.R.G.) Tel. 089178741

(Received June 12th, 1986)

(Accepted September lst, 1986)

SUMMARY

The structural gene encoding cyclodextrin-glycosyltransferase of Klebsiella pneumoniae strain M5al was cloned; it is expressed both in Escheridzia coli and in K. pneumoniae and the gene product is secreted into the extracellular space. Determination of the nucleotide sequence revealed an open reading frame coding for a single polypeptide of 655 amino acid (aa) residues. The enzyme is synthesized as a precursor with an N-terminal signal peptide of 30 aa residues, which is proteolytically processed between two alanine residues during export. The primary structure of CGT bears homology with the sequences of amylases from both prokaryotic and eukaryotic

origins.

INTRODUCTION

Some microorganisms able to hydrolyze starch synthesize and export CGT (EC 2.4.1.19). This enzyme catalyzes the degradation of starch into CD (for review see Szejtli, 1982).

* To whom correspondence and reprint requests should be

addressed.

Abbreviations: aa, amino acid(s); bp, base pair(s); CAP,

catabolite activator protein; CD, cyclodextrin(s); CGT, cyclo-

dextrin-glycosyltransferase; cgr, cyclodextrin-glycosyltransferase

gene; Cm, chloramphenicol; Ig, immunoglobulin; kb, kilobase

or 1000 bp; LB, Luria broth; nt, nucleotide(s); ORF, open

reading frame: PAGE, polyacrylamide-gel electrophoresis; pFp,

parafluorophenylalanine; RBS, ribosome-binding site; SDS,

sodium dodecyl sulfate; [ 1, designates plasmid-carrier state.

Cyclodextrins (CD) are cyclic, non-reducing oligo- saccharides composed of six, seven or eight glucose units linked by a-1,Cbonds. According to the num- ber of their glucose units they are termed a-, j?-, and y-CD. They are able to form inclusion complexes with a number of organic and inorganic molecules, thereby changing the physical and chemical proper- ties of the included compounds (for review see Szejtli, 1982). This ability to form inclusion com- plexes is of potential industrial importance (Horikoshi and Teruhiko, 1982).

The CGT produced by K.pneumoniae M5al (Bender, 1977) is one of the few proteins which are exported by Gram-negative bacteria in high amounts. The formation of the enzyme is subject to strict regulation, namely by induction and by catabolite repression (Bender, 1977). In order to investigate the molecular basis of expression and of

0378-l 119/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)

270

~~quenc~~ of N-terminal aa r&dues was per- formed by the method described hy ~~~~- Liebold and Kimura (1984),

MATERLUS AND METRODS

(P) Reagents

271

K. pneumoniae strains: (i) K. pneumoniae KAY2026,

which is naturally devoid of CGT activity, and (ii) K. pneumoniae RClO, a mutant of K. pneumoniae

M5a1, whose cgt structural gene was deleted by UV mutagenesis (our unpublished results). After trans- formation with the recombinant plasmid, termed pCM100, both Klebsiella strains as well as E. coli RR28 gained the ability to hydrolyze starch.

(b) Expression and excretion of CGT by E. coli

Immunoblotting experiments were carried out in order to analyze whether the starch-hydrolyzing activity conferred by pCM100 is due to the produc- tion of CGT. Fig. 1 gives the result: Transformation of E. coli RR28 by plasmid pCM100 confers to E. coli the capacity to synthesize and secrete a poly- peptide cross-reacting with anti-CGT antibodies. This polypeptide exhibits the same apparent M, (of 68 kDa) as that produced by K. pneumoniae M5al.

1 A 0

2 3 4 5 6 7 6 kDo

- 170 --974

-, r CI -

-55

-36

-20

Fig. 1. Immunoblotting analysis of cgf expression by K.pneumoniae and E. co/i. Cultures of K.pneumoniae M5a1, E. coli RR28 and E. coli RR28[pCMlOO] were grown in minimal medium containing 1% partially hydrolyzed starch (Noredux 150B) to a density of 10s cells/ml. 0.5 ml of the total culture (cells plus supernatant) and of the supernatant alone, respectively, were lyophilized; the freeze-dried material was taken up in 50 ~1 sample buffer (Laemmli, 1970) and subjected to immunoblotting analysis using the method of Howe and Hershey (198 1) with the modifications given by Schmid and Bock (1984) alter SDS-PAGE in 12.5 % gels (Laemmli, 1970). (Panel A) Cells plus supernatant. Lanes: 1, K. pneumoniae MSal; 2, E. coli RR28; 3, E. coli RR28[pCMlOO]; 4, purified CGT. (Panel B) Super- natant alone. Lanes: 5, E. coliRR28[pCMlOO]; 6, purified CGT; 7, K. pneumoniae M5al; 8, E. coli RR28.

In view of the fact that E. coli does not normally secrete proteins this finding may be evidence that it is an intrinsic structural property of the CGT which directs the export. It is interesting in this context that the acquisition of CGT secretion by E. coli provides the organism with the ability to hydrolyze starch but not with the capability to grow on this carbon source. The CD which accumulate in the medium (un- published results) apparently either cannot be taken up into the cells and/or they cannot be linearized in the cytoplasm.

(c) The nucleotide sequence of the cgt structural gene

Fig. 2 gives the restriction map of plasmid pCM100; the size of the PstI insert in vector pHE3 is 5.4 kb.

To confirm that the insert of the recombinant plasmid pCMlO0 was derived from K. pneumoniae DNA, a 2.55-kb EcoRI-EcoRV fragment of pCMlO0 was labelled with [ 32P]dATP by nick- translation and used as a probe for hybridization experiments with genomic DNA digested with &I. A hybridization signal was obtained with a 5.4-kb fragment of K. pneumoniae M5al DNA. PstI digests of DNAs from E. coli RR28, K.pneumoniae

KAY2026 and K. pneumoniae RClO did not exhibit any hybridization signals.

The PstI fragment was shortened by subcloning with appropriate restriction enzymes. The shortest fragment which still promoted the expression of active CGT was a 2.9-kb HpaI-EcoRV fragment (Fig. 2). Recloning of the 5.4 kb PstI-fragment in vector pHE3 with the opposite direction showed that the expression of the gene is independent of the orientation of this insert indicating that transcription starts from the homologous promoter. Plasmid pCM100 was used for the determination of the nucleotide sequence of the cgt gene. The sequencing strategy and restriction sites used for labellmg of fragments are depicted in Fig. 2. Within the sequenced segment of 2809 bp there is an ORF of 1965 bp (Fig. 3) corresponding to a protein of 655 aa residues.

The putative CGT protein contains a highly biased aa composition (Table I); for example, the per- centage of phenylalanine plus tyrosine is lo%, and that of serine plus threonine is almost 16%. Many of

212

, lkb I

PstI Hpal EcoRl Bgll Rll

L

I

Cyclodextrin-glycosyl- transferase

Fig. 2. Restriction map of the 5.4-kb insert of pCM 100 encoding the cgr gene. The expanded map gives that part for which the nucleotidl sequence was determined. Arrows indicate starting point and direction of sequence readings. (0) 5’ labelling; (a) 3’ labelling. Position corresponding to the C-terminus and N-terminus of precursor-CGT are also given (aa pos. 1 and 655, respectively).

TABLE I

Codon usage in gene cgt”

F TTT 26 F TTC 12 L TTA 10 L TTG 7

(5.79)” s S

(6.55) S S

TCT TCC TCA

TCG

8 (7.62) Y 3 Y

10 *

4 *

TAT TAC TAA

TAG

22 (4.27) C 6 C 1 *

0 W

TGT

TGC TGA

TGG

3 (0.46) 0 0

13 (1.98)

CCT ccc CCA CCG

(3.35) H H

Q Q

CAT CAC CAA CAG

9 (1.52) R 1 R

12 (3.20) R 9 R

CGT 12 (3.96) CGC 3 CGA 1 CGG 4

L CTT 7 L CTC 4 L CTA 8 L CTG 7

11

2 5 4

I ATT 23 (6.10) T ACT I ATC 7 T ACC I ATA 10 T ACA M ATG 11 (1.68) T ACG

20 (8.08) N AAT 8 N AAC

15 K AAA

10 K AAG

53 (10.21) s AGT 17 14 S AGC 8 20 (3.96) R AGA 4

6 R AGG 2

V GTT 19 (5.34) A GCT V GTC 3 A GCC V GTA 8 A GCA V GTG 5 A GCG

18 (6.71) D GAT

10 D GAC 9 E GAA I E GAG

39 (7.16) G GGT 26 (7.93) 8 G GGC 6

18 (3.96) G GGA 13 8 G GGG 7

a Numbers in parentheses give the percentage of occurrences of the respective amino acid. Other numerals specify the number c occurrences.

273

1

39

134

229

324

419

AMTATAC

514

566

656

730

802

Ikt L 6 Ar Am Arg Pha Phe hsn Thr Scr Ala Ala Ile Ala Ile Sar 11s Ala Leu Am Thr Phe *e C l ATG AiA AOR MC CGT TFTTTT MT ACC TCG GCT GCTATT CCC ATTTCG ATTGCA TTA AAT ACTTTT- T&r

SW blot Gin Thr Ile Ala Ala Glu Pro Glu Glu Thr 'K

r Leu As! Phe As LJ; @ZZ; mh ;g T& z K AGCATGCAGACGAlTGCTGCTGAACCAGAAGAA ACTT TCTT GA TTTC

t

Phe L=" A6! B AT Phe Ser Asp Gly Asp Pro Sar hsn hsn hla Gly Phe Aan Sor Ala Thr Tf$ ;;g .P& fis

TTC CFTGA CG TTCAGCGATGGA GATCCA AGT AAT AATGCA GGG TCT MTTCTGCA ACC T

i%%i %ti %T ACT GGK GGi GAbTC CGG GGG %=I% ATTMT dA CTACCC %K:&:%= i%% I Thr G1 Gl As Leu Arq Gly Lsu Ile Asn L 6 Leu Pro

Val Thr Ser Ile Trp Ile Thr Pro Pro 11s Asp Asn Val Asn Asn Thr Asp Ala Ala Gly Am Thr Gly Tyr GTTA~TCAA~n;GA~A~CCCCCAA~GAThATGn:MTMTACFGAT~CCTCCCMT~OCATAT

074

946

::: ii% 2; % i% :a %,P %y: !i? iii8 ATA CAT GAA CAT TTT Gd 11~ h6p Glu ills Phe Gl Es g ;A$ As! g L& g &

Thr Ssr Lau Met His Ser Pro Asp Tyr Asn Mat Lys Leu Val Leu Asp Tyr Ala Pro Asn llis 6ar Asn Ala ACT AGT !lTG ATG CAT AGT CCT GAT TAT AAT An; Ahh Cl’G GTT CTT GAT TAT CCC CCC MT CAT TiX AAT GCT

1018

1090

It62

1234

Ann Asp Glu Asn Glu Phe Gl MTGATGAAAATGAATTT GG4

Ala Leu Tyr Arg Asp Gly Val Phc Xle Thr Asp TK

r Pro Thr Asn Vsl Ala GCA CPA TAT CGT GhT GGT GTG TTT ATT ACT CAT T T CCT ACG AAT GH GCC

Ala Am Thr Gly Tr Tyr Ilis His Asn Gly Gly Vd Thr hsn Trp Aan Asp Phe Phc Cln Val L 6 Aan ills GCCAATACG GGC d TAT CATCACAATGGT GGG GTh ACG MCTGG AATGAT lTC TTCCAA G'lG AXG MTCAT

Am Leu Phe Asn Leu Sor As L&u Asn Gln Scr Asn Thr As Val T r Asn MT CFA TFC AAT CTA TCA GA e CTC MTCAA TCC hhT ACT GA

$j 1 ~rbeuLeuA8,G&~~ GTCT CCAG T C TTG TTG GA

Phe Trp Ile Asp Ala Gly Val Asp Ala Ile Arg Ile Asp Ala Ile Lys 1118 hat Asp Lys Ser Phe Lie Gln TTTTGG ATC GATGCTGGTGTG GATGCTATC AGG ATTGATGCC ATCMC CAT An; GACAAGTCTTl'T ATACAG

1306

1376 Phe Gly Ala Ser Ala Asn Thr Thr Thr Gly Val hsp Gly Ann Ala Xle Asp Tyr Ala Asn Thr Ser Gly Ser 'ITTGGTGCCAGTGCG MT ACTACAACAGGTGTTGATGGTMTGCTATCGATTACGCCAACACTTCCUXWA

1450

1522

1594

f666

Ala Leu Leu As F GCG TTG CTG GA

Phe Gly Phe Arg Asp Thr Leu Glu Arg Val Leu Val Gly hrg Ser Gly Asn Thr Rut L 6 TTT GGA TTC CGC GAT ACT TTA GhA AGA GTT TTG GTA GGA CGT AGC GCA MT ACA An; x

Thr Leu Am Ser r Leu Ile L 6 At Gin Thr Val Phe Thr Ser As ACG TTA MThGT %TCTG ATA & Ad CAA ACA GTC TTF ACC AGTGAf'%f%:k% gPke$i&

Asn His As P MC CATGA

Het Ala Arg fle Gl Thr Ala Leu Arg Ser Asn Ala Thr Thr Phe Gly Pro Gly Am Asn Glu ATGGCACGCATT & ACCGCTCTG CGTTCAAACGCCACTACT~GGTCCTGGA MTMTGM

ACCd63; Thr Gl GI Ser Gin Scr Glu Ala Phe Ala Gln Lys Arg Ile Asp L+su Gly Leu Val Ala Thr Uet Thr Vsl

AGTCAG AGTGAA GCTTTTGCTCAG AAA CGTATA GACCTCGGTCTGGTTGCGACA ATCACTGTA

1736 CG! Ar Gly Ile Pro Ala Ile Tyr Tyr Gly Thr Glu His Tyr Ala Ala Asn Phe Thr Ser Asn Ser Phe Gly Gln

GGTAm CCTGCCAmTATTAT GGTACT GM CAT TATGCC GCTMC TTT ACC TCTAAC AGTYYYGGTCAA

1810

1882

Val Gl Ser As Pro Glu L B Met Pro Cl Phe As Thr Glu Ser Glu Ala Phe Scr fle Ile GTTGG$AGTGAkCd%~~~ GAG A ATG CCA GGjl T'lTGAg ACG GAAAGT GAG GCFlTCTCCAYTAR

Lys Thr Leu sly Asp teu Arq Lys Ssr Ser Pro Ala Ilc Gln Asn Gly Thr Tyr Thr Clu Leu 'Frp Val Asn AAAACA CTG GGT GAC CTAAGG AM AGTAGC CCG GCA ATT CM MTGGA ACTTAT ACl'GAA CTA XGGTTAAT

1954

2026

As GA P

Asp Ile Leu Val Phe Glu Ar Arg Ser Gly Asn Asp Ile Val Ile Val Ala X&u Asn Arg Cly Glu Ala GAT ATA TTA GTAlTTGAG CG! CGTTCT GGG AhC GATATTGTTATT GTTGCA ClTAATCGTGGTGAG GCT

Am Thr Ile Aan Val Lys Asn Ile Ala Val Pro Asn Gly Val Tyr Pro Ser ku Ile Cly Asn Asn Ser Val MC ACA ATTAATGTPAAA MTATA GCG GTTCCT MT GGGGTA TAT CCG AGTTTG ATTCGG MT MT AGTtXT

2090

2170

Ser Val Al6 Am Lys Ar Thr Thr Leu Thr Leu Met Gin Ann Glu Ala Val Val Ile Ar Ser Gin Sar Asp TCA GTAGCAAATAAA CG8 ACA ACA CTA ACA CTT ATG CM MTGAACCT GlTGTC ATNG&CACAA TCA GAT

Asp Ala Glu Ann Pro Thr Val Gln Ser Ile hen Phe Thr Cys Asn Aen Gly Tyr Thr Ile Ser GLy Gin Ser CAT GCG GAG MCCCTACA GTA CAAAGCATA AAC TTCACA TGT MTMCGGT TATACG A'rl'TCAGGTCiiA AGT

2242

2314

2386

2456

Val TK

r 110 11s 01 Asn Ile Pro Gln Leu Cl GTTTTATTATT Gd NTATACCTCAG lTA GG r

Gly Trp Asp Leu Thr Lys Ala Val I s Ile Sar Pro Thr GGT TGG GACTTA ACT MA GCG GTA AIA ATA TCA CCG ACA

Gln Tyr Pro Gln Trp Ser Ala Ser Leu Glu Leu Pro Ser Asp Leu hsn Val Glu Trp Lys Cya Val Lys Ar CAA TAT CCA CAA TGO AGT GCG AGC TTA GAG CTT CCT TCT GAC TTA MT GTTGM TGG MC TGTGTG MA td

Asn Glu Thr Asn Pro Thr Ala Asn Val Glu Trp Gln Ser Gly Ala Am Am Gln Phe Asn Ser Asn Asp Thr AAT CM ACC MT CCC ACG GCT MT CTT GAG TGG CAG TCT GGT GCA MT MC CAG TTC AAT AGC MT GAC ACA

Gln Thr Thr Asn Gly Ser Phe l ** TTCGThCTCCGGCCATMTT.~T CAAACA ACG MT GGCTCG Tl'TTAA

lTAAAA_ --

2546 TTTTGACTAATACTCTTACAAATTTTCAACCTAGGCTGATGTGhCTACATATTPl’TG CTGTACGCGGACCTGGTCGTCTGCGTCTATTThGAGTC

2641 AAGA~ATATCCA’ITTCACCCKIACGAAATA~MATAG~CGCChAhTAGTAGA~A~~~GAA~CAGCCCGGATTATGCChTCIY;A

2736 ‘FCAATCCCCAAACCMCCATCACCMCCGGnCTAnGCGATGTCGATCATAGCACCAATACCCCGTACCGhAC

Fig. 3. Nucleotide and amino acid sequences of the K. pneumoniae M5al cgr gene. The sixfold repeated hexanucleotide 5’-TTGTAG-3’

(mentioned in the text) is underlined, the CAP-binding site is marked by a dotted line. Transcription initiation and putative ribosome-

binding sites are indicated (boxed and S/D, respectively). The cleavage site of the signal sequence is given by an upward arrow. The

palindromic sequence downstream from the ORF is indicated by horizontal arrows.

214

the arvmatic aa residues are clustered. The refevance of these structural features for the export process is not yet knvwn.

The cgt gene has a codon composition in which rare cvdons frequently occur. The f. index (Ikemura, 1981) was calculated to be 0.47, which is that of a weakly expressed gene in E. cofi. It is also striking that the percentage of G + C within the cgt gene is 397; compared to 52 to 56% for total chrvmvsomal DNA from Kleb~iellu. The unusual codvn usage and G + C-content and the fact that the cga gene vnly occurs in strain M5ai of R. ~~~~~~~ffe may indicate that this gene could have been acquired by lateral transfer from some other vrganism.

The N-terminal aa of purified CGT from K. pneumoniae M5al were determined in order to correlate primary structures of the protein and of the nucleotide sequence derived polypeptide. The aa positions at the CGT N-terminus which could be determined unambiguously, were H,N-ala-glu-pro; this N-terminus is separated from the start point of

the ORF by 30 aa which bear all the characteristic features of a signal peptide (Perlman and Halvorson, 1983). This indicates that CGT is synthesized as an M, 72 916 precursor protein of 655 aa residues with a 30-aa signal peptide which is processed between two alanine residues. A precursor of this size can be detected by immunoblvtting experiments in strains in which cgt expression has been placed under the control of the hybrid fat promoter (unpublished results).

Mature native CGT from K. ~ne~rnon~~e as well as

SDS denatured enzyme display an apparent Mr of 68000 (Bender, 1977). This is consistent with a M, of 69029 calculated from the aa composition and demonstrates that the enzyme consists of a single polypeptide.

Nuclease Sl mapping of the start point of tran- scription was performed to provide the structural basis for regulation studies. As shown in Fig. 4, a signal was obtained only with RNA prepared from K. pneumoniae MSal grown at the expense of starch. Upon growth on glucose the transcriptivn of egg is repressed. Transcription starts within a region between nt 150 and 155 upstream from the ATG of the ORF (boxed in Fig. 3).

Fig. 4. happens of the in viva start point of ~~sc~pt~on of the cgl gene. Bulk RNA was prepared from cultures ofK. pnetcmoniae

MM grown in minimal medium at the expense of starch and glucose, respectively, as carbon sources. Cells were harvested in the late exponential growth phase. The RNA was hybridized (45°C) with 3 405bp BgflI-EcoRI fragment of plasmid pCUlO0 (Fig, 2), labelled with 32P at its 5’-BglII-end. The hybridization mixture was treated with nuclease Sl (With et al., 1986) and el~~ophore~ca~y analyzed by PAGE in a 64/, gel conta~ing 7 M urea. Lane 1: Labelled 405 bp-fragment; lane 2: RNA pre- pared from K. pneumoniue M5al cells grown at the expense of starch; lane 3: RNA prepared from K.pnemnoniae M5al cells grown on glucose.

Upstream From the start point of transcriptivn there is no sequence motif corresponding to the - 10 and -35 consensus boxes of E. co/i promoters (Rosenberg and Court, 1979). Within a distance of 90 nt, however, a classical ~AP-b~~g motif 5’-TGTGA-3’ (De Crombrugghe et al., 1984) occurs. The existence of an E. co&type CAP binding site and the fact that c@ expression is subject to cat&o&e repression both in E. co& (~pub~sh~ results) and K. pneumoniae, suggest that the repres- sion systems of the two organisms are identical.

CGT K.pn. 49 AMY B.st. 20 AMY B.am. 17 AMY B.su. 16 AMY A.or. 36 AMY Rat 15 AMY Hog 15 AMY H.s. 18 AMY Bar. 20

CGT K.pn. 103 AMY B.st. 76 AMY B.am. 73 AMY B.su. 72 AMY A.or. 92 AMY Rat 71 AMY Hog 71 AMY H.s. 74 AMY Bar. 76

CGT K.pn. 149 RDGVFITDYPTNVAANTGWYHHNGG AMY B.st. 130 ISGTYQIQAWTKFDFPGRGNTYSSFKWRWYHFDGVDWDESRKLSRIYKFRGIGKA AMY B.am. 127 TSEEYQIKAWTDFRFPGRGNTYSDFKWHWYHFDGADWDESRKISRIFKFRGEGKA AMY B.su. 117 IPNWTHGNTQ AMY A.or. 137 FKPFSSQDYFHPFCFIQNYE AMY Rat 116 GSYFNPMNREFSAVPYSAWYFNDNKC NGE AMY Hog 116 GSYCNPGNREFPAVPYSAWDFNCNGKKTASGG AMY H.s. 119 GSYFNPGSRDFPVVPYSGWDFNDGKCKTGSGD

AMY Bar. 130 CRDDTK

CGT K.pn. 174 VTNWNDFFQVKNHNLFNLSDLNQSNTDVYQYLLDGSKFWIDAGV AMY B.st. 185 !iDWEVDTENGNYDYLMYADLD?lDHPEWTELKSWGKWYVNTTNI AMY B.am. 182 WDWEVSSENGNYDYLMYADVDYDHPDWAETKKWGIWYANELSL AMY B.su. 127 IKNWSDRWDVTQNSLLGLYDWNTQNTQVQSYLKRFLDRALNDGA AMY A.or. 157 DQTQVEDCWLGDNTVSLPDLDTTKDVVKNEWYDWVGSLVSNYSI AMY Rat 145 INNYNDANQVRNCRLSGLLDLALDKDYVRTKVADYMNNLIDIGV AMY Hog 148 IESYNDPYQVRDGCQVLLLDLALEKDYVRSMIADYLNKLIDIGV AMY H.s. 151 IENYNDATQVRDCRLSGLLDLALGKDYVRSKIAEYMNHLIDIGV AMY Bar. 121 FEGGT AMY Bar. 144 YSDGTANLDTGADFAAAPDIDHLNDRVQRKLKEWLLWLKSDLGF

CGT K.pn. 231 SFIQKWTSDIYDYSKSIGREGFFFF SANTTTGVDGNAIDYANTSG AMY B.st. 242 SFFPDWLSDVRSQTGKPLFT V DINKLHNYIMKTNGTMSLFD AMY B.am. 239 SFLRDWVQAVRQATGKEMFT V NAGKLENYLNKTSFNQSVFD AMY B.su. 184 PDDGSYGSQFWPNITNTSAEFQ Y SASRDAAYANYMDVTASHYG AMY A.or. 213 KDFWPGYNKAAGVYC I DPAYTCPYQNVMDGVLNYPI AMY Rat 202 GDIKAVLDKLHNLNTKWFSQGSRPFI F GGEAIKGSEYFGNGRVTEFK AMY Hog 205 GDIKAVLDKLHNLNTNWFPAGSRPFI F GGEAIKGSEYFSNGRVTEFK AMY H.s. 208 GDIKAILDKLHNLNSNWFPEGSKPFI Y GGEPIKSSDYFGNGRVTEFK AMY Bar. 200 PEMAKVYIDGTSPSLA V MATGGDGKPNYDQDAHRQNL

CGT K.pn. 202 SALLDFGFRDTLERVLVGRSGNTMKTLNSYLIKRQTVFTSDDWQ AMY B.st. 289 APLHNKFYTASKSGGTF DMRTLMTNTLMKDQPTLA AMY B.am. 286 VPLHFNLQAASSQGGGY DMRRLLDGTVVSRHPEKA AMY B.su. 233 Ak?Y A.or; 254

HSIRSA LKNRN LGVSMISHYASDVSADKL YYPLLNAFKSTSGSMDD LYNMINTVKSDCPDSTLL

AMY Rat 255 YGAKLGTVIRKTNGEKM SYLKNWGEGWGFVPTDRA AMY Hog 258 YGAKLGTVVRKWSGEKM SYLKGPLKGWGLMPSDRA AflY H.s. 261 YGAKLGTVIRKWTGEKM SYLKNWEEGWGFMPSDRA AMY Bar. 243 VNWVDKVGGAASAGMVFDFTTKGILNAAVEGEL!~RLIDPQGKAPGVMGW~lPAKA

CGT K.pn. 326 AMY B.st. 323 AMY B.am. 321 ANY B.su. 262 Ab!Y A.or. 290 AMY Rat 290 ANY Hog 293 AMY H.s. 296 AMY Bar. 297

GGDLRGLINKLPYLKSLGVTSIbIITPPIDNWiNTDAAGNT GYHGYWGRDYFRID GTLWTKVANEANNLSSLGITALWL PPAYKGTSRSDVGYGVYDLYDLGEFNQKGAVR GQHWKRLQNDAEHLSDIGITAVWI PPAYKGLSQSDNGYGPYDLYDLGEFQQKGTVR NWSFNTLKHNMKDIHDAGYTAIQT SPINQVKEGNQGDKSMSNWYWLYQPTSYQIGN KYCGGTWQGIIDKLDYIQGMGFTA IWITPVTAQLPQDCAYGDAYTGYWQTDIYSLN HLFEWRWADIAKECERYLAPKGFG GVQVSPPNENIIINNPSRPWWERYQPISYKIC~ HLFEWRWVDIAKECERYLGPKGFG GVQVSPPNENVVTGNPSRPWWERYQPVSYKLC HLFEWRWVDIALECERYLAPKGFG GVQVSPPNENVAINNPFRPWWERYQPVSYKLC GFNWESWKQSGGWYNMMMGKVDDI AAAGVTHVWLPPPSHSVSNEGYMPGRLYDIDA

EHFGNLDDFKELT SLMHSPDYNMKLV NANDENEPGALY TKYGTKAQY LQ AIQAAHAAGMQVY TKYGTKSE LQDAIGSLHSRNVQVY RYLGTEQEF KE MCAAAEEYGIKVI ENYGTADDL KA LSSALHERGMYLM SRSGNENEF KD MVTRCNNVGVRIY TRSGNEDEF RD MVTRCNNVGVRIY TRSGNEDEF RN MVTRCNNVGVRIY CGNAVSAGTSSTC SKYGNAAEL KS LIGALHGKGVQAI CADYKDSRGIYCI

Fig. 5. Comparison of amino acid sequences of CGT and various a-amylases. Organisms are abbreviated as: K. pn., Klebsieilu

pneumoniae; B. St., Bacillus stearothennophilus; B. am., Batik amyloliquefaciens; B. su., Bacillw subtilis; A. or., Aspergillm oryzae; H.,

Human; s., saliva; Bar., Barley. Alignment of or-amylases is adopted from Nakajima et al. (1986). CGT sequence is aligned to give the best tit and intervals between neighbouring amino acids are extended, whenever necessary for this purpose. Only the region of high homology is shown, and regions l-4 containing highly conserved sequences are boxed.

275

216

Upstream from the initiation codon there is a putative E. c&type RBS (Stormo et al., 1982) which may contribute to the excellent expression of cgt in E. c&L

The most striking feature of the region flanking the cgc gene at the 5’ side is the .occurrence of a sixfold repeated 5’-TTGTAG-3’ sequence between the start point of ~~sc~ption and the Shine-Dalgarno motif, Its functian in the expression of cgt is not yet known.

Downstream from the ORF there is a palindromic sequence which displays the characteristics of a

p-independent transcription termination signal (Rosenberg and Court, 1979).

(e) Homology to ez-amylaws

The CGT sequence was compared with the pri- mary structures of eight diEerent cr-amyiases com- piled recently by Nakajima et al. (1986) (Fig. 5). Restricted homologies were found between aa 77 and 36.5 of CGT and sequence motifs of ot-amylases assumed to participitate in substrate binding and/or catalysis. Amongst g-amylases, homology occurs at four di&rent segments of the primary structure (Nakajima et al,, 1986); two of them - regions 2 and4- are clearly present in the CGT sequence (Fig. 5). The homology to regions I and 3 is limited, although the spacing of the four regions in the sequences of CGT and a-amylases is similar. The homology of sites important for catalysis in a-amylases, with equivalent regions in CGT, sug- gests a common evolutionary derivation of the two classes of enzymes.

After submission of this manuscript Takano et al, (1986) published the sequence of 3~~~il~ ~~~~~

CGT. A comparision with the primary structure of K. pneumoniue CGT showed an overall homology of about 30 % _ The highly conserved boxes described in this paper can also be seen in the aa sequence of the B. macerans enzyme.

(1) G + C-content and codon usage of the cgt gene of ~~~~e~~oni~e MSal indicate that it might have been acquired by Klebsielh via lateral gene transfer. Sequence homologies between CGT and

a-amylases indicate that they are derived from a common precursor.

(2) The level of expression of cgt in E. coli is

compar&&z to that in K. ~~e~~~~e, It is subject to catabolite repression in both organisms.

(3) CGT is secreted into the medium by E. coli,

although to a lower extent as by K. pneumoniae. The

CGT signal peptide does not display any con- spicuous differences to those of proteins exported into the periplasm or integrated into membranes. Secretion, therefore, seems to be mediated by an intrinsic property of the protein.

ACKNOWLEDGEMENT

We thank GA Heller for her excellent technical assistance and G. Sawers for critically reading the manuscript. We are greatly indebted to M. Geier for editorial work.

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Communicated by K.F. Chater.