Vol. 134, No.

2, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

January 29, 1986 Pages 624-631

S E C R E T I O N A C T I V I T I E S OF .Bacillus subtilis e - A M Y L A S E SIGNAL

PEPTIDES OF D I F F E R E N T L E N G T H S IN E s c h e r i c h i a coli CELLS

Kiyoshi N a k a z a w a l, T o s h i y a Takano 2, Akira Sohma I and Kunio Yamane l

ZInstitute of B i o l o g i c a l Sciences, U n i v e r s i t y of Tsukuba,

Sakura, I b a r a k i , a n d 2 N a t i o n a l Food R e s e a r c h Institute,
Yatabe, Ibaraki, 305 Japan

Received December I0, 1985

T h e B. subtilis e-amylase promoter and signal peptide are

f u n c t i o n a l in ~. coli cells. DNA fragments coding for signal p e p t i d e s
with different lengths (28, 31, 33 a n d 41 a m i n o a c i d s f r o m the
t r a n s l a t i o n initiator Met) were p r e p a r e d and fused with the E. c o l i
B - l a c t a m a s e s t r u c t u r a l gene. In B. s u b t i l i s cells, the sequences of
31, 33 a n d 41 a m i n o a c i d s w e r e a b l e to s e c r e t e B - l a c t a m a s e i n t o the
surrounding media, b u t t h e 28 a m i n o a c i d s e q u e n c e w a s not. In
contrast, a l l of the four sequences were a b l e to export ~ - l a c t a m a s e
into the p e r i p l a s m i c space of E. c o l i cells. Thus, the r e c o g n i t i o n of
t h e B . s u b t i l i s e - a m y l a s e s i g n a l p e p t i d e in E. c o l i c e l l s s e e m s to be
d i f f e r e n t from that in B. subtilis cells. ©1986AcademicPress,lnc.

The gene expression system in G r a m - p o s i t i v e bacteria is d i f f e r e n t

from that of G r a m - n e g a t i v e bacteria. Many genes for extracellular

enzymes of bacilli have been cloned using Escherichia coli vector

systems, and some have been expressed by t h e i r own promoters in the

host cells (I-5). But E. c o l i genes are not by themselves

expressed in B. subtilis cells (6). In contrast, mechanisms for

secreting proteins are considered to be similar among Gram-positive

bacteria, Gram-negative bacteria and eukaryotic cells. The signal

peptides of secreted proteins are N H 2 - t e r m i n a l stretches of amino acids

which are similar in length, contain highly hydrophobic central

sequences and are removed by specific enzymes to form mature

proteins (7,8).

B. s u b t i l i s is w e l l known to s e c r e t e a large number of s o l u b l e

proteins into the surrounding medium. The e-amylase gene of an e-

amylase hyper-producing strain, B. s u b t i l i s NA64, was cloned in a B.

subtilis plasmid pUB110 (9). A c o m p a r i s o n of the nucleotide sequence

0006-291X/86 $1.50
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 Vol. 134, No. 2, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the s-amylase gene and the amino acid sequence of the NH2-terminal

region of B. subtilis extracellular a-amylase indicated that the

precursor protein of s-amylase contained a 41 amino acid signal

peptide. This s i g n a l peptide is l o n g e r than those of o t h e r e x p o r t e d

proteins. Therefore, the e f f e c t s of l e n g t h and s t r u c t u r e of the ~-

amylase signal p e p t i d e was s t u d i e d by a n a l y z i n g the s e c r e t i o n of a

fused E. coli 6-1actamase in B. subtilis cells (10).

In order to compare the secretion mechanisms in Gram-positive and

Gram-negative bacteria, four plasmids, which encoded different lengths

of the B. subtilis a-amylase signal peptide followed by the E. coli

~-lactamase structural gene, were transferred into E. c o l i cells.

Localization of B - l a c t a m a s e synthesized in E. c o l i and B. s u b t i l i s

cells was compared.

MATERIALS AND METHODS

Bacterial strains and plasmids : B. subtilis 207-25 (m~68 hsrM recE4
amyE07 aroI906 !ys21 leuA8) and M15-I (amyE15 metB5 recE4 aroI906) are
d e r i v a t i v e s of B. subtilis Marburg 168. B. subtilis plasmid pTUB4 is a
pUB110 derivative w i t h an i n s e r t i o n of a 2 . 3 k b D N A f r a g m e n t
c o n t a i n i n g amyR2 and amyE + from B. s u b t i l i s NA64 (9). B. s u b t i l i s
p l a s m i d s pTUB231, p T U B 2 4 9 , p T U B 2 5 6 and p T U B 2 2 8 c o n t a i n e d the B.
subtilis a-amylase promoter followed by sequences specifying different
l e n g t h s of the B. s u b t i l i s a - a m y l a s e s i g n a l peptide. U s i n g H i n d I I I
linkers, the E. c o l i B - l a c t a m a s e s t r u c t u r a l gene was fused 28
(pTUB231), 31 (pTUB249), 33 (pTUB256) and 41 (pTUB228) a m i n o acids
d o w n s t r e a m from the i n i t i a t o r Met. The ~ - l a c t a m a s e s t r u c t u r a l gene,
w h i c h was d e r i v e d from p B R 3 2 2 and l a c k e d its own p r o m o t e r and s i g n a l
p e p t i d e c o d i n g region, was p r o v i d e d by Dr. I. P a l v a ( U n i v e r s i t y of
Helsinki, SF-00380, Helsinki 38, Finland). E. coli plasmid pTUE20 is a
~ - l a c t a m a s e - d e f i c i e n t d e r i v a t i v e of pBR325, p B R 3 2 5 was d i g e s t e d by
PstI, treated with $I nuclease and religated. New plasmids described
here (pTUE23, pTUE24, pTUE28, pTUE31, pTUE33 and pTUE41) were
transferred into E. coli C600 (F- thi thr leuB lacY tonA supE I-).
Preparation of plasmids : Plasmids were prepared by the rapid a l k a l i n e
m e t h o d of B i r n b o i m and D o l y (11) and p u r i f i e d by CsCI e q u i l i b r i u m
centrifugation in the presence of ethidium bromide.
F r a c t i o n a t i o n of c u l t u r e s of E. c o l i c e l l s : The e x t r a c e l l u l a r ,
periplasmic and c e l l u l a r (intracellular and membrane) fractions were
prepared by the method of Chan et al (12).
Western blot : After electrophresis in 7.5% SDS-polyacrylamide gels
(13), m-amylase from E. coli and B. subtilis was visualized by western
b l o t a n a l y s i s (14) u s i n g a r a b b i t a n t i s e r u m a g a i n s t B. s u b t i l i q a-
a m y l a s e and the 1 2 5 I - l a b e l e d F(ab')2 f r a g m e n t of d o n k e y a n t i s e r u m
against rabbit IgG (Amersham, England).
Enzyme assay : s-amylase activity was measured by the modified blue
value m e t h o d of Fuwa u s i n g 0.5% s o l u b l e s t a r c h as the s u b s t r a t e (15).
~ - l a c t a m a s e a c t i v i t y was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y using
nitrocefin by the m e t h o d of O ' C a l l a g h a m et al (16). A l k a l i n e
p h o s p h a t a s e a c t i v i t y was a s s a y e d by the m e t h o d of S c h l e s i n g e r and
Barrett using ~-nitrophenyl phosphate (17).

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Enzymes : Restriction enzymes, bacterial alkaline phosphatase, $I

n u c l e a s e and T4 DNA ligase were p u r c h a s e d from Takara Shozo Co., Ltd.
(Kyoto, Japan). E n z y m e s w e r e u s e d a c c o r d i n g to the m a n u f a c t u r e r ' s
specifications.

RESULTS

In o r d e r to v e r i f y that t h e B. s u b t i l i s s-amylase promoter and

signal peptide were functional in E. c o l i cells, a Bg!!I-digest of

pTUB4 was inserted into the BamHI s i t e of p T U E 2 0 , a n d the r e s u l t a n t

chimeric plasmid was transferred into E. coli C600. Chloramphenicol-

resistant, tetracyclin-sensitive, amylase-positive transformants were

selected on L - a g a r plates containing 10 ~ g / m l chloramphenicol and I%

soluble starch. Two p l a s m i d s were identified, pTUE23 and pTUE24, which

possessed opposite orientations of pTUB4 with respect to the vector.

Extracellular, periplasmic and cellular fractions were prepared from

cultures of ~. coli harboring these two plasmids (E. c o l i [ p T U E 2 3 ] and

E. coli[pTUE24]), and s-amylase and alkaline phosphatase activities

were assayed. It w a s r e v e a l e d that more than 80% of t h e two enzyme

activities was localized in the periplasmic fractions. The same

localization of s - a m y l a s e proteins was ascertained by w e s t e r n blot

analysis (Fig. I). A l t h o u g h the molecular weight of extracellular e-

amylase from B. subtilis M15-I containing pTUB4 (B. subtilis MI5-

I[pTUB4]) was 60,000, two e-amylases, with m o l e c u l a r weights of 56,000

and 45,000, were detected from the periplasmic fractions of E.

coli[pTUE23] and E. c o l i [ p T U E 2 4 ] . The p r o d u c t i o n of s - a m y l a s e by these

E. coli strains was approximately I% of that of B. subtilis MI5-

I[pTUB4]. No band was d e t e c t e d in the fraction from E. coli[pTUE20].

Since it w a s a s c e r t a i n e d that B. s u b t i l i s s-amylase and signal

peptide functioned in E. coli cells, the effects of length and

structure of the B. s u b t i l i s s-amylase signal peptide on the s e c r e t i o n

of E. c o l i ~-lactamase in ~. coli cells was compared with that in

B. subtilis. The BglII-digests of pTUB231, pTUB249, pTUB256 and

pTUB228 were separately inserted into the BamHI s i t e of p T U E 2 0 . The

constructed plasmids were designated pTUE28 (pTUB231-pTUE20), pTUE31

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 Vol. 134, No. 2, 1986 BIOCHEMICAL
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•- o

_E.col~ ~1~
fraction
~ ~ M

-68,000

000

000

300

Fig. I. W e s t e r n b l o t a n a l y s i s of a - a m y l a s e s p r o d u c e d by E.
coli[pTUE24] and B. subtilis M15-I [pTUB4]. E. coli[pTUE24] was
cultured in L-broth containing 10 zg/ml of chloramphenicol at 37°C for
12h, at which time the maximum production of e-amylase was observed.
The culture was separated into extracellular (E), periplasmic (P) and
c e l l u l a r (C) fractions. The 5% TCA precipitates from each fraction
were analyzed. The extracellular a-amylase of B. subtilis M15-1[pTUB4]
(M) was a n a l y z e d as the control. Bovine serum a l b u m i n (mol. wt.,
68,000), o v a l b u m i n (mol. wt., 43,000), carbonic anhydrase (mol. wt.,
30,000) and cytochrome C (mol. wt., 12,300) were used as the molecular
weight markers.

(pTUB249-pTUE20), pTUE33 (pTUB256-pTUE20) and pTUE41 (pTUB228-pTUE20)

(Fig. 2). E. coli transformed by these plasmids were both

chloramphenicol- and ampicillin- resistant. More than 90% of t h e ~-

lactamase synthesized by these E. c o l i transformants was located in

the periplasmic fractions, as w a s t h e c a s e f o r a l k a l i n e phosphatase

(Table I). In contrast, the B. subtilis transformants containing

pTUB249, pTUB256 and pTUB228 were able to secrete B-lactamase

extracellularly; however, a B. s u b t i l i s transformant harboring pTUB231

was not (Table I). The production of ~-lactamase directed by the

plasmids encoding 28 a m i n o acids of the B. s u b t i l i s s-amylase signal

peptide was low in b o t h E. coli and B. subtilis cells.

pTUB4 was also inserted into another plasmid YEp13, a n E. c o l i -

Saccharomyces c e r e v i s i a e shuttle vecter, and transferred into S.

cerevisiae SHY3. However, the B. subtilis d-amylase gene was not

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table I. Activity and localization of B-lactamase in E. coli and B. subtilis

cells harboring plasmids

B-lactamase activity
Host cells Plasmids Extracellular Periplasmic Cellular
Units/ml (%) Units/ml (%) Units/ml (%)
E. coli C600 pTUE20 0.2 0 2.5
pBR325 243.8 (25) 730 (73) 19.7 (2)
pTUE28 1.4 (5) 28.1 (95) 0.i (0)
pTUE31 12.2 (5) 258.8 (95) 0.i (0)
pTUE33 12.2 (7) 148.7 (93) 0.i (0)
pTUE41 5.0 (2) 216.2 (96) 3.9 (2)

Extracellular Cell-bound
Units/ml (%) Units/ml (%)
B. subtilis pTUB231 0 (0) 32.8 (i00)
207-25 pTUB249 566 (98) 9.5 (2)
pTUB256 692 (99) 7.1 (i)
pTUB228 296 (98) 5.1 (2)

E. coli strains harboring pTUE20, pBR325, pTUE28, pTUE31, pTUE33, or pTUE41

were grown in L-Broth containing i0 ~g/ml of chloramphenicol at 37°C for 6h,
at which time the maximum B-lactamase activity was observed. The cultures
were separated into extracellular, periplasmic, and cellular fractions
according to the method of Chan et al (12). B. subtilis strains containing
pTUB231, pTUB249,pTUB256, or pTUB228 were grown in LG-medium (i0) containing
I0 ~g/ml of kanamycin at 37°C for llh, at which time cells were separated
from extracellular fractions by centrifugation.

expressed in ~. cerevisiae, based on a m y l a s e activity and western blot

analysis.

DISCUSSION

Compared with other exported proteins, the precursor of

B. subtilis m-amylase has an u n u s u a l l y long NH2-terminal extension of

41 amino acids; and although it has been shown that AIa-X-AIa most

frequently precedes signal peptidase cleavage (18), this signal

peptide is cleaved between Glu (codon position 41 from the initiator

Met) and Leu (42). ~-Lactamases from pTUB249, pTUB256 and pTUB228 in

B. subtilis cells were secreted into the culture media. In c o n t r a s t ,

the e n z y m e was not secreted when preceded by the short, 28 a m i n o acid

signal peptide specified pTUB231. Therefore, we speculate that in B.

subtilis the precursor of m-amylase will be cleaved first between

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 Vol. 134, No. 2, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

positions 31 (Ala) and 32 (Ser) (Fig 2), and subsequently between

positions 41 (Glu) and 42 (Leu) to form the e x t r a c e l l u l a r enzyme.

In the case of E. coli, all ~-lactamases from the d e r i v a t i v e s of

the four Bacillus plasmids were translocated in equal propotion to

• pTUE 41
H

pTUE 33
pTUE 31
pTUE 28

I t,..~

%%
/
/
I 2 3 4 5 6 7 8 9 10 Ii 12 13 14 15 16 17 18 19 20 21 22
pTUE41
pTUE33 Met Phe Ala Lys Arg Phe Lys Thr Ser Leu Leu Pro Leu Phe Ala Gly Phe Leu Leu Leu Phe Tyr
pTUE31 ATG TTT GCA AAA CGA TTC AAA ACC TCT TTA CTG CCG TTA TTC GCT GGA TTT TTA TTG CTG 'TTT TAT
pTUE28

23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Leu Val Leu Ala Gly Pro Ala Ala A1 er Ala Giu Thr Ala ASh Lys Ser Asn Glu Gln Ala Cys P{o Pro
pTUE41 TTG GTT CTG GCA GGA CCG GCG GCT GCG AGT GCT GAA ACG GCG AAC AAA TCG AAT GAG CAA GCT TGC CqC CCA
I =

pTUE33 Leu Val Leu A1 a~G ly Pro Ala Ala Ala~ Ser Ala Gln Ala Cys P~o Pro Glu Thr Leu Val Lys Val Lys Asp
TTG GTT CTG GCA GGA CCG GCG GCT GCG AGT GCT CAA GCT TGC C~C CCA GAA ACG CTG GTG AAA GTA AA~ GAT
• ~-lactamase structural gene

Leu Val Leu AIa~GIy Pro Ala Ala Ala Gln Ala C{,s Pro Pro Glu Thr Leu Val Lys Val Lvs Asp Ala Glu
pTUE31 TTG GTT CTG GCA GGA CCG GCG GCT GCG CAA GCT TGC Cq~ CCA GAA ACG CTG GTG AAA GTA ~ GAT GCT GAA
-- ~-!actamase structural gene

pTUE28 Leu val Leu Ala~Gly ProlGln Ala Cvs P{o Pro Glu Thr Leu Val Lys Val Lys As~ Ala Glu Asp Gln Leu
TTG GTT CTG GCA GGA CCG[CAA GCT TGC C ~ CCA GAA ACG CTG GTG AAA GTA AAA GAT GCT GAA GAT CAG TTG
I .. • 3-1actamase structural gene

Fig. 2. Physical map of pTUE28, pTUE31, pTUE33 and pTUE41; and DNA
nucleotide sequences of the junction regions of the B. s u b t i l i s e-
amylase signal peptide coding regions and the E. c o l i 8-1actamase
structural gene. The fused ~-lactamase starts at codon position two
of the mature enzyme. The HindIII linker regions are boxed. I I ,
pTUE20 D N A ; i....~....!....... , p T U B 4 DNA; ~ ,probable cleavage sites of the
signal peptides b y B. s u b t i l i s signal peptidase; ~ ,assumed cleavage
sites of the signal peptides b y E. c o l i signal peptidase; E, EcoRI
site; H, H i n d I I I site. The numbers in the lower part of the figure
indicate the codon positions from the translation initiator Met. The
curved arrow indicates the direction and extent of the DNA nucleotide
sequences determined.

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 Vol. 134, No. 2, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the periplasmic space. The difference observed between E. coli and B.

subtilis in the localization of ~ - l a c t a m a s e preceded by the 28 amimo

acids signal peptide m a y be d u e to d i f f e r e n c e s eitherin the substrate

specificities of the two signal peptidases or some other membrane

constituents involved in removing the signal peptides. It is

conceivable that the signal peptidase of B. subtilis cleaved between

positions 31 (Ala) and 32 (Ser), while the E. coli enzyme acted closer

to the initiator Met, possibly between positions 26 (Ala) and 27

(Gly). However, we h a v e not yet d e t e r m i n e d the N H 2 - t e r m i n a l sequences

of B-lactamase secreted into the periplasmic spaces of these

transformed E. coli; nevertheless, the lengths of most signal peptides

of proteins exported by E. coli are shorter than those of

extracellular enzymes produced by b a c i l l i (19). The appearance of the

smaller molecular weight a-amylases in E. c o l i compared with the

extracellular s-amylase of B. subtilis is likely caused by d i f f e r e n t

processing of the s-amylase precursor protein in E. coli cells.

These results indicate that much of the secretion mechanism of B.

subtilis will be s i m i l a r to that of E. coli, but that minor d i f f e r e n c e s

will exist between them in areas such as the substrate specificities

of their signal peptidases or in other proteins relating to secretion.

ACKNOWLEDGEMENTS

We are g r a t e f u l to Dr. I. P a l v a for p r o v i d i n g the p l a s m i d pKTH74.

T h i s w o r k w a s s u p o r t e d by G r a n t - i n - A i d for S p e c i a l Distinguished
R e s e a c h from the M i n i s t r y of Education, Science and Culture of Japan.

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