Download Protein export elements from Lactococcus lactis

Mol Gen Genet (1992) 234:401-411
MGG
<9 Springer-Verlag
Protein
export
elements
from
Lactococcus
1992
lactis
Gaspar Perez-Martinez*, Jan Kok, Gerard Venerna, Jan Maarten van Dijl,
Rilde Srnith**, and Sierd Bron
Departmentof Genetics,Centreof Biological Sciences,Kerklaan 30, 9751NN Haren, The Netherlands
Received November
21, 1991 / Accepted
Apri114,
1992
Summary. Broad-host-range plasmids carrying a-amylase or 13-lactamasereporter genes lacking a signal
sequencewereusedto selectexport elementsfrom Lactococcuslactis chromosomal DNA that could function as
signal sequences.Fragments containing such elements
were identified by their ability to direct the export of the
reporter proteins in Escherichiacoli. Severalof the selected export elements were also active in Bacillus subtilis
and L.lactis, although the efficienciesdependedstrongly
on the host organism and reporter geneused.The export
elementsAL9 and BLl were highly efficient in L. lactis
in the expressionand secretion of at least two heterologous proteins (Bacil/us licheniformis a-amylase and
E. coli TEM-13-lactamase).AL9 even permitted growth
of this organism on starch as the sole carbon source.
Nucleotide sequenceanalysis of five selectedfragments
indicated that theseencodeoligopeptides with the major
characteristics of typical signal peptides. The putative
expressionsignals had a limited similarity to previously
described expression signals for E. coli, B. subtilis and
L. lactis. Differences in both expressionand export efficiency are likely to underlie the host-specific effects.
Key words: Lactococcus protein export -Expression
nals -Signal peptide -a-amylase -13-lactamase
sig-
Introduetion
Knowledge of the geneticsand physiology of lactic acid
bacteria has rapidly increasedduring the last few years.
In particular, the availability of molecular genetictechniques has contributed in a major way to our knowledge
of theseindustrially important bacteria, which are widely
.Present
Alimentos
address; Instituto
de Agroquimica
y Technologia
(C.S.I.C.), Jaime Roig 11,46010 Valencia, Spain
..Present
address; Central Veterinary
8219 PH Lelystad, The Netherlands
Correspondence to; S. Bron
Institute,
Edelhertweg
de
15,
used in the food industry .Potential new applications of
these organisms are the production of (heterologous)
extracellular proteins and pharmaceuticals.Although, so
far, the natural protein export capacity of lactic acid
bacteria has scarcely been studied, these bacteria are
known to export several proteins, some of which are
secretedinto the growth medium. Thus, lactococci export a cell wall-associatedserine proteinase essentialfor
rapid growth in milk (Kok and Venerna 1988),and they
secrete Usp45, a protein of unknown function (van
Asseldonk et al. 1990). With respect to heterologous
proteins, it has been shown that Lactobacillusplantarum
is able to secreteBacillus stearothermophilusa-amylase
and Clostridium thermocellumcellulase A, using the native signal peptides of these proteins (Scheilink et al.
1989). The secretion by Lactococcus lactis of Bacillus
subtilis neutral protease fused to a lactococcal promoter
was demonstrated by van de Guchte et al. (1990). Escherichia coli TEM-13-lactamase,when joined to lactococcal expression/exportsignals, can also be secretedby
L. lactis (Sibakov et al. 1991).
Exported proteins are initial1y synthesizedas precursors with an N-terminal extension, the signal peptide.
The signal peptide is required to guide the protein into
the export pathway by interacting with components of
the cel1ular export machinery and the membrane (for
reviewsseeSaier et al. 1989; Wickner et al. 1991).Moreover, the presenceof the signal peptide retards the folding of the precursor (Laminet and Pliickthun 1989; Park
et al. 1988), which appears to be important for export
competence (Saier et al. 1989; Wickner et al. 1991).
Although signal peptides from different exported proteins have little sequencehomology , they have a tripartite
structure in common: a short (5-8 amino acids) positiveIy charged amino-terminus (n-region) ; a central hydrophobic core (R-region; 8-15 amino acids); and a short
(5-8 amino acids) more polar c-region that contains the
cleavagesite for signal peptidase (SPaseI; von Heijne
1986,1990).A comparison ofsignal peptidesfrom E. coli
and bacil1i was made by von Heijne and Abrahmsén
(1989). On average, signal peptides from bacil1i are
402
slightly longer in the n-, h- and c-regions than those from
E. coli, and the n-region in bacilli is generally more highly
charged. Analyses of signal peptides from lactic acid
bacteria have only recently begun. In addition to signal
sequences of L. lactis proteinase genes (Kok et al. 1988),
a number of randomly selected lactococcal DNA
fragments have recently beeen described that direct the
export of proteins (Sibakov et al. 1991).
The primary aim of the studies reported here was to
isolate and characterize DNA fragments from the
L. lactis chromosome that could function as signal sequences. Such export elements are valuable for the construction of efficient expression/export vectors for
L. lactis. A considerable number of export elements with
signal sequence function were selected from L. lactis in
a similar way to that described previously for Bacillus
(Smith et al. 1987, 1988, 1989). For this purpose plasmids
were used that encode mature parts of exoproteins but
not the signal peptides. Some of these export elements
wer highly efficient in L. lactis. The selected fragments
permitted a comparison of expression/secretion efficiencies in E. coli, L. lactis and B. subtilis.
Matcrials and mcthods
Bacterial strains, plasmids and media. Table 1 lists the
bacterial strains and plasmids used. TY medium was
used for culturing E. coli and B. subtilis (Bron and Luxen
1985), and MI7 medium supplemented with 0.5% glucose for culture of L.lactis (Kok et al. 1984). Ifrequired,
I % starch was added to the media. Erythromycin was
used at 5 ~g/ml for L. lactis and B. subtilis, and at
200 ~g/ml for E. coli. To score for ~-lactamase export in
E. coli, 5 ~g/ml of ampicillin (Amp) (plus 100 ~g/ml
erythromycin to select for the plasmid) were used in the
initial screening. E. coli was transformed using standard
procedures (Maniatis et al. 1982), B. subtilis by the competent cell procedure (Bron and Luxen 1985), and
pGB14
pGBl8
pGAL(9,
39)
pGBL(I,
11, 13)
L. lactis
1988).
by electrotransformation
(van der
,elie et al
DNA techniques. General procedures for tbe manipulation of DNA were carried out essentially as described by
Maniatis et al. (1982). Restriction enzymes and T4 DNA
ligase, obtained from Boebringer (Mannbeim, FRG), or
New England BioLabs (Beverly, Mass.), were used as
specified by tbe suppliers. Nucleotide sequences of botb
strands of DNA were determined by tbe dideoxy cbaintermination metbod (Sanger et al. 1977) using tbe T7
DNA polymerase sequencing kit (Pbarmacia LKB, Uppsala, Sweden). Eitber M 13mp 1O/mp 11 subclones or
pGA1 4/pGB14 derivatives were used in sequencing.
Mapping oftbe 5' termini ofmRNA by primer extension
witb reverse transcriptase was performed as described by
Calzone et al. (1987). mRNA was isolated from L. lactis
MG 1363 carrying tbe appropriate plasmids as previously
described (van der Vossen et al. 1987).
Enzymatic assays. AII enzymatic activities were determined in exponentially growing cells at an optical density
of 0.6 at 600 nm. 13-Lactamase and isocitrate dehydrogenase activities were measured as described by Smith
et al. (1987). 13-Galactosidase was determined by the
method of Milier (1972), phosphoglucose isomerase by
that of Michal (1984) and a~amylase activity was determined using the procedure of Spiro (1966).
Spheroplasting. The contens of the periplasmic space
were released from E. coli cells by treatment for 30 min
at 37° C with 5 mg/ml of lysozyme in 0.1 M potassium
phosphate buffer, pR 7.5 plus 10 mM EDT A and 0.3 M
sucrose. Under these conditions no /3-galactosidase activity was released into the medium, indicating that the
E. coli cells remained intact during spheroplasting.
803supE supF met rk-mkRNAse Ith, leu thi lac y tonA phx supE vt,
lac p,tP
his np,R2 np,El8 ap,AJ
a-Amylase negative derivative of strain DBIO4
K. Murray
de Vrije et al. (1987)
Raleigh et al. (1988)
Laboratory collection
Kawamura and Doi (1984)
Laboratory collection
a-Amylase-based vector for selection
of export elements;
pWVOI replicon; contains SPO2 promoter; Emr
~-Lactamase-based vector for selection
of export elements; pWVOI replicon; Emr
pGBI4 containing the SPO2 promoter
pGAI4 derivatives containing the AL9 (AL39) inserts directing export of
a-amylase
pGBl4 derivatives containing the BLI (BLil, BLI3) inserts directing
export of ~-Iactamase
This work
This work
This work
This work
This work
403
Results
We define stages in protein export according to Pugsley
and Schwartz (1985): export, the translocation ofprecursor proteins to an extracytoplasmic location, including
the cytoplasmic membrane; processing, the removal of
the signal peptide by signal peptidase; secretion, the special case of export that results in the extracellular accumulation of exported proteins. In the context of the
present studies, we define export elements as signal
sequence-like functions enabling the export (translocation) of reporter exoproteins across the cytoplasmic
membrane. As discussed in the following section, the
presence of a processing site for signal peptidase was not
required for all of the export elements described here.
Selection of protein export elements from
L. lactis DNA
Two plasmids (Fig. I) were constructed that carried
either the Bacillus licheniformis a-amylase gene (pGAI4),
or the E. coli TEM-13-lactamase gene (pGBI4). The reporter genes were 5'-terminally truncated and lacked
their transcription/translation
initiation signals and the
signal sequence, which were replaced by a multiple cloning site (Smith et al. 1987). Consequently, the reporter
proteins are not expressed from these plasmids. The
selection vectors are based on the broad-host-range
lactococcal plasmid pWV01 (Kok et al. 1984), which
enabled the comparison of the effects of cloned DNA
fragments in different bacteria without subcloning. The
copy numbers of this plasmid per chromosome equivalent are about 5 for B. subtilis and L. lactis and about
50 for E. coli (Kok et al. 1984). Cloned DNA fragments
that restore the export of the reporter proteins can be
selected with these plasmids, provided that they are
placed in frame with the reporter genes. In addition, the
selection of export elements with pGB14 requires the
cloning of expression signals. With pGA14, carrying the
SPO2 promoter, which is functional in all three bacteria
used in these studies, the cloning of a promoter is not
essential. The initial selection of export elements was
carried out in E. coli. Since resistance to Amp can only
develop when 13-lactamasehas been translocated into the
periplasm (Kadonaga et al. 1984), this provides a positive
assay for pGB14 clones containing export elements. We
have shown before (van Dijl et al. 1991b) that translocated 13-lactamase that is not processed also renders E.
coli cells resistant to Amp. As a consequence, the
presence of a signal peptidase cleavage site on the selected export elements is not required in this assay. With
pGA14, halo formation around E. coli colonies on
starch-containing plates was used to monitor a-amylase
export. Since this assay requires the diffusion of a-amylase into the agar, processing of the a-amylase precursor
is required in this case.
Chromosomal DNA from L. lactis MG1363 was digested with the restriction enzymes Rsal plus HaeIIl, or
Alul, to obtain fragments of about 0.3 to 1.5 kb. The
digested DNA was ligated into the Smal site of pGA14
and pGB14 and the ligation mixtures were used to transform E. coli BHB2600. Among approximately 50000
erythromycin-resistant
E. coli transformants obtained
with pGB14, 12 were resistant to > 5 ~g/ml Amp. The
selected fragments were designated BL. From approximately 50000 erythromycin-resistant transformants obtained with pGA14, 48 exported a-amylase. The selected
fragments were designated AL.
XboI
~
~dm
pWW1
\lndm
SocIl
Emr
pGB14
5900bps
1'1
BclI-\
,
--"f
Hi1d
m
SalI
\
XboI
SmoI
BomHI
SocI
EcoRI
13-1actamase
+1 +2
a.-amylase
+1
GDPLESTAAAA
GAA TTC GAGC TC GCC C GGGGATC C TC T AGAGTC GACCGCAGC GGCGGCA
~~~
~RI
~~~~~~
Fig.
I. Plasmids
from
the
used
Lactococcus
5'-terminally
Escherichia
the selection
Plasmids
truncated
genes
the
were
and
licheniformis
genes,
described
bacteriophage
of protein
pGA14
Bacillus
coli TEM-[3-lactamase
ed reporter
contains
for
lactis.
by
SPO2
Smith
elements
pGB14
contain
a-amylase
respectively.
promoter
export
et al.
and
The truncat(1987).
upstream
pGA14
of
the
~I
GDPLESTAQACPP
TCC~~CGCC~GCCCCCCA
~i-~~I
~I
~III
truncated a-amylase gene. Both plasmids contain the replication
functions of the broad-host-range plasmid pWVOl (Kok et al.
1984). The plasmids contain the erythromycin resistance marker
from plasmid pE194. The sequencesbelow the plasmids show the
regions of the fusions between the multiple cloning sites and the
reporter genes
404
Table 2. 13-Lactamase activities
Export
function
Escherichia
Ampr
(~g/ml)
BLl
BL4
BLS
BLlO
BLll
BLl2
BLl3
BLl4
AL9
None (pGBl4)
obtained
with export elements from Lactococcus
coli
Activitya
(U/ml)
Exportedb
(%)
10
40
100
10
30
35
53
10
130
100
1580
110
1785
420
10
nd
nd
< 10
nd
5
10
75
30
500
48
26
45
18
16
lactis
Bacillus subtilis
Activitya
Exportedb
(U/ml)
(%)
880
700
150
0
3480
620
1660
730
nd
<10
100
100
100
0
69
100
100
100
nd
nd
Lactococcus
/actis
Activitya
(U/ml)
Exportedb
(%)
18700
160
570
80
1270
160
3660
90
38850
<10
92
100
100
100
24
100
65
100
98
nd
Size insert
(hp)
300
800
750
900
720
800
1300
360
720
One unit of activity (seeMaterials and methods) was defined as the
amount of J:I-lactamasethat in l ml samples raised the absorbance
at 486 nm by 0.001/min at room temperature. Resistance to ampicillum (Amp) was expressed as the maximal concentration of
Amp (in J.lg/ml) at which growth to colonies occurred. AL9, which
had been selected with plasmid pGA14, was inserted into plasmid
pGB14
a Total amount of J3-lactamaseactivity. In the case of E. coli this
Two assays were used to obtain information about the
relative efficiencies of the selected export elements. The
first was the measurement of the levelof resistance to
Amp in E. coli. Since only exported (periplasmic) ~-lactamase causes resistance to Amp (Kadonaga et al. 1984),
this is a measure of export efficiency in E. coli. This assay
cannot be used for the Gram-positive bacteria, because
with TEM-~-lactamase, these do not develop resistance
to Amp. The second assay involved measurement of
export and secretion of a-amylase and ~-lactamase in the
three organisms tested. We have shown previously
(Smith et al. 1988) that not only mature, processed forms,
but also precursor forms of the a-amylase reporter protein are enzymatically active. Therefore, determination
of the activities in cell-associated and extracellular fractions ( B. subtilis and L. lactis) , or those of cell-associated
and periplasmic fractions (export in E. coli), allows a
comparison of the efficiencies of selected export elements, and a comparison of effects associated with the
host bacteria. Although quantitative data are lacking, we
assume that the same holds for ~-lactamase. Since pre-~lactamase (Laminet and Pliickthun 1989) and hybrid
pre-~-lactamases are active (van Dijl et al. 1991a), activity measurements give information about amounts of
protein. A potential problem with this assay is instability
of the hybrid proteins in the three hosts. This would
reduce the enzymatic activities, as has been shown for
hybrid ~-lactamase in B. subtilis (Smith et al. 1987). This
problem was avoided by using exponentially growing
cultures. Prolonged incubation of cell extracts indicated
that no losses of a-amylase and ~-lactamase activity
occurred as a function of time (all three bacterial species
were tested; results not shown).
in E. coli (Table 2). BLl3 and BLll were exceptions:
compared with export elementsisolated from B. subtilis,
the first conferred high levels and the second moderate
levels of resistance.Plasmids containing the BL inserts
were transferred to B. subtilis DBl04 and L. lactis
MG1363, and the total /3-lactamaseactivities were determined. In addition, the fractions of the activities exported into the periplasm ( E. coli) or culture supernatant
( B. subtilis and L.lactis) were determined. Controls with
the intracellular enzymes/3-galactosidase( E. coli) , isocitrate dehydrogenase ( B. subtilis) , or phosphoglucose
isomerase( L. lactis) indicated that in none ofthe species
were detectable amounts of intracellular proteins releasedby cel11ysis(data not shown).
In E. coli the total activities of /3-lactamasevaried
greatly, indicating that different export elements had
been selected. In B. subtilis and L. lactis al1 selected
elements (except BLIO in B. subtilis) directed the expression of /3-lactamase.However, the relative levels of
the activities were different in the three bacterial species.
For instance, BLll was the most efficient in B. subtilis,
BLl3 in E. coli, and BLl in L. lactis. BLll and BLl3
were moderately active in L. lactis, and BLl had only a
low activity in B. subtilis and E. coli. These and other
data from Table 2 indicate that the host had a drastic
effect on the total /3-lactamaseactivity.
Depending on the selectedfragment, the fraction of
exported /3-lactamasealso varied considerably. In E. coli,
BLl was efficient in directing export: all /3-lactamase
activity was located in the periplasm. With all other
elements50% or more ofthe /3-lactamaseremained associated with the cells. In spite of this, some of these elements were so highly efficient in expression (BLll and
BLI3) that, as compared with BLl, large amounts of
/3-lactamaseaccumulatedin the periplasm. A remarkable
observation was that with BLl3 a very high levelof
resistanceto Amp (500 I.1g/ml)was obtained. Since only
periplasmical1y localised /3-lactamasecan cause resis-
Export elements selected with the p-Iactamase gene
Most of the DNA fragments selected with pGB14 conferred rather low levels of Amp resistance ( < 30 ~g/ml)
represents the total activity in pelleted cells; in the caseof B. subtilis
and L. lactis this represents the activities in cells plus culture supernatants
b Fraction (in %) ofthe J3-lactamaseactivity present in the periplasmic space ( E. coli) , or in the culture supernatant ( B. subtilis and
L. lactis ) ; nd, not determined
405
tance (Kadonaga et al. 1984), this suggestedthat, in
E. coli, BL13 permits efficient translocation of /3-1actamase into the periplasm. The low percentage of free
/3-1actamase
in the periplasm (18%) may indicate that
pre-(BLI3)-/3-1actamase
was poorly processedby SPaseI.
Most of the BL elementscausedsecretionof nearly all
/3-1actamase
into the extracellular medium of B. subtilis
and L. lactis. BLll and BL13 were exceptions:with these
export elementsconsiderableamounts of the /3-1actamase
activity were associatedwith the cell fraction. For comparison, the results obtained with AL9 (see following
paragraph) are also shown in Table 2. When placed in
plasmid pGBI4, this fragment appeared to be the most
efficient of all in L. lactis (38850 U/ml /3-1actamase;
98%
secreted).
Export elements selected with the a-amylase gene
Twenty-three of the AL export elements selected with
pGA14 in E. coli were transferred to B. subtilis
DBIO4( amy ) and L.lactis. The total a-amylase activities
were determined, as well as the fractions of the activity
that were exported (Table 3). With these element also, a
considerable variation in total amount of activity was
observed in E. coli, indicating that, again, many different
elements had been selected. The majority of the AL
Table
3.
a-Amylase
activities
obtained
with
export
Export
Escherjchja
function
A CtiVlty.
..
E xporte
(U/ml)
(%)
(J.lg/ml)
33
62
75
52
73
48
49
66
30
50
85
78
56
44
50
56
71
22
32
27
46
62
55
nd
nd
nd
10
AL6
AL9
ALl2
ALl4
AL24
AL25
AL27
AL29
AL30
AL31
AL32
AL33
AL35
AL36
AL37
AL39
AL40
AL41
AL42
AL43
AL46
AL47
AL48
None (pGAI4)
BLI
BLl3
44.9
16.0
20.2
11.9
81.0
10.6
24.6
46.2
84.0
87.4
42.2
106.0
6.8
86.0
>126
>126
63.0
29.8
18.1
31.7
122
63.0
118
<2
nd
nd
colj
elements
from
Lactococcus
Bacjllus
db
AmA pr
10
elements directed low a-amylase production in the
Gram-positive bacteria: in B. subtilis, a-amylase activity
could be measured only with AL9, AL14, AL3l, AL39,
and AL40, while in L. lactis only AL9 was active. This
demonstrates that there is a drastic host-specific effect on
a-amylase production. The fraction of the a-amylase
released into the periplasm of E. coli varied from 22% to
85%. In B. subtilis, AL9 and AL39 appeared to be more
efficient in directing export of a-amylase ( > 88% secreted) than in E. coli (about 60% exported). AL14,
AL31 and AL40 were moderately efficient in both E. coli
and B. subtilis (50%-70% exported). As in B. subtilis,
AL9 was also efficient in the secretion of a-amylase by
L. lactis (90% in the supernatant). The host-specific effects on the amounts of exported a-amylase are illustrated in Fig. 2, which shows halo formation on starchcontaining plates by various E. coli, B. subtilis, and
L. lactis clones.
Several of the DNA fragments selected with the aamylase gene were transferred as SacI-XbaI fragments
into the corresponding sites in pGB18. This placed the
export elements downstream of the SPO2 promoter, in
frame with the 13-lactamasereporter gene. The levels of
resistance to Amp were measured in E. coli (Table 3
column 4). Although all AL elements caused resistance
to Amp, little correlation was observed between a-amylase accumulation in the periplasm ad the levelof Amp
CtlVrty.
..
lactjs
subtjlis
Lactococcus
E xporte
lactis
db
Activitya
Exportedb
(U/ml)
(%)
(U/ml)
(%)
+
13.6
95
11.7
90
9.6
5.0
77
65
nd
200
200
4.2
+
62
3.2
62
10
200
200
20
10
200
10
nd
+
nd
10
200
10
1.4
2.2
88
48
200
10
10
+
+
75
nd
+
100
nd
nd
+
+
Units of activity (seeMaterials and methods) are given in rnillimoles
of glucose equivalents released from starch per hour and per millititre culture. nd, not determined; + , halo formation on starch plates,
but activity too low to be measurable; -, no activity detectable
Size insert
(bp)
300
720
300
720
300
300
300
<300
1200
750
<300
650
600
<300
<300
<300
<300
1200
<300
<300
<300
490
400
300
1300
a. b See Table 2 (here a-amylase activities)
c Levels ofresistance to ampicillin (Amp) (in E. coli) obtained with
pGBI8 plasmids into which the AL fragments had been introduced
406
DNA sequences and expression signals
Fig. 2. Differences in relative a-arnylase activities in Escherichia coli,
Bacillus subtilis, and Lactococcus lactis. Colonies of the three bacterial strains that carried identical pGAL plasmids were toothpicked onto an agar plate containing M 17 medium plus glucose and
1% starch. The plates were first incubated for 18 h at 300 C to permit
growth of L. lactis, foliowed by 24 h at 37° C to provide optimal
growth conditions for E. coli and B. subtilis. Halo formation was
subsequently visualized by staining with iodine solution (Smith et
al. 1988). Upper row, E. coli; middle row, B. subtilis; bottorn row,
L.lactis. Frorn left to right, AL48, AL39, AL37, AL31, AL24, AL9,
and AL6
resistance. For instance, AL37 and AL39 were equally
efficient in the export of a-amylase Cabout 50% in the
periplasm), but only AL39 caused high levels of resistance to Amp. This suggested that, in contrast to
preCAL37)-a-amylase,
preCAL37)-!3-lactamase
was
translocated inefficiently in E. coli. Since in this case
identical expression signals were compared, these results
indicated that the translocation efficiencies were also affected by the mature part of the reporter proteins.
Growth of L. lactis on starch
Of the selected export elements, AL9 and HL 1 were the
most efficient in the production and secretion of both
reporter proteins in L. lactis (Tables 2 and 3). HL I should
contain a promoter, since it was selected in the absence
of the SPO2 promoter. The removal of the SPO2 promoter from plasmid pG-AL9 did not affect halo formation on starch-containing plates (data not shown), also
indicating that AL9 carries transcription/translation
signals. AL9 directed such high levels of a-amylase synthesis
and secretion (Table 3) that L. lactis could grow weil
(OD6oo values of at least 1.2 could be obtained) in M17
medium containing 1% starch as the sole carbon source.
With the aim of identifying expression signals and export
signal functions, five of the fragments were sequenced :
B LI (efficient in L. lactis); AL39 (efficient in E. coh);
BLll and BLl3 (efficient in both E. coli and B. subtilis) ;
and AL9 (efficient in both L. lactis and B. subtilis). The
data (Fig. 3A-E) indicated in all sequenced fragments the
presence of: (1) an open reading frame (ORF) fused in
frame to the reporter gene; and (2) promoter-like sequences upstream of the ORFs. The similarity of the putative promoters to previously described lactococcal promoters (de Vos 1987; Koivula et al. 1991a; van de
Guchte et al 1992 ; van der Vossen et al. 1987) was rather
limited, however. None of the putative promoters described here had a perfect match with the consensus -35
(TTGACA) and -10 sequences (TATAAT).
We consider the sequence denoted b in AL9 as the best candidate
for a functional promoter (spacing 19 bp). In fragment
B LI, the sequences denoted a and al are likely -35 and
-10 promoter sequences (spacing 15 bp). Primer extension assays with mRNA extracted from L. lactis were
used to map the start sites of transcription (results not
shown) in the two most efficient export elements in
L. lactis (AL9 and B LI). These start sites fitted weIl with
the promoter regions deduced from the sequence. The
-35 region ofthe putative promoter on B LI is contained
within a 19 b p (imperfect) inverted repeat sequence,
which can potentially
form a stem-loop structure
(L1G = -14.8 kCal/mol). In AL9, B LI and BLI3, which
contain the most efficient expression signals for L. lactis
(Tables 2 and 3), the region preceding the putative promoters is extremely A/T rich. In addition, in B LI (position 25 to 50, Fig. 3B) and BL13 (position 24 to 76,
Fig. 30), stretches of three or more As are present with
a helical periodicity (about 10 bp). These regions might
facilitate ONA bending.
In four of the fragments (AL9, AL39, BLll,
and
BLI3) potential translation signals are present: a start
codon (AUG), preceded at a short distance (less than 12
bp) by a Shine-Oalgarno (SO) sequence. Some support
for the assignment of the translation start sites was obtained by estimating from SOS-polyacrylamide gel electrophoresis the actual sizes of ~-lactamase precursors
synthesized in vitro. These corresponded weIl with those
predicted from the sequences (results not shown).
The high levelof expression by BLI in L. lactis was
surprising, since no SO-like sequence is present in the
region preceding the single AUG codon (Fig. 3B; position 117) in frame with the reporter gene. Moreover, this
codon is directly adjacent to the transcription start site
(position 116; Fig. 3B), which makes the assignment of
the translational start at position 117 doubtful. However ,
no obvious alternative start codon nor SO sequence is
present downstream from the transcription start point.
Characteristics of the export elements
The hydrophobicity
profiles of the five oligopeptides
encoded by the selected fragments are shown in Fig. 4.
AL9
A
BL11
10
20
30
40
50
60
TCGCCCCTTATTTATAATAAGCAAAAATCCATCTTCTGATTTGTTGGGTATCTTTAAAAC
70
80
90
100
110
120
CATATTTTTTIGATAAGGAATGATAACCAGTTCTCTTGTCAAGATACTCTTTAACAATCT
130
140
150
160
170
180
TTAGTTTAAATTCAAATGTATATTTTGTCATAAAAMATTGACCTCCATTAACTAGACTT
190
200
210
220
230
240
TTTAGTCTAACTTATGGGGGTCATATCATTTTATAGAGGTTTrTTGGTATCTAMAAAGC
250
260
270
280
290
300
CAATGGCATTTATTTTTTATATTGCGATTGATAAAAATAATATAAGTGAAAATACAAATT
-a
310
bi
330
320
--a-340
~
10
20
30
40
50
60
GGAAMG AAATAAAAA TT CGTGTT ATTG CTGCCAA TAAACGAAM GGAACAGTAGATITT
70
80
90
100
110
120
GAACAAAT CGCTCCTGAAAAAAACTA GCAATTTG CTAGTTTTTTT CTGTATGTTTTTTA T
130
140
150
160
170
180
TAGCTAATATCGTCTTTACAGTATATTTTACATAACATAAATTATGTTAGCTGTGTTATT
190
200
210
220
230
240
TTGAGTATAAGGGAGCCTTATTTTTGATTACGAATTGATTTAGGCACGGATATCAGTAGA
250
260
270
280
290
300
AAAATGAAATCAAAGTTTGGTTGATAAAAGATTGTTACTATTTAAATTAAGTTATATAGT
---0--350
360
TATCAATTTTTGTTAGAATAGAGACTATAAATG~AATACATTGACAATTCAAGGAGCGA
C-
(
a-
~
--a--
---6--
310
320
330
340
350
360
TATCAAGCGAATATGCTAGCTTAACAAACTATTTATGTAATAATATAGGTATGGTAATCA
---(--
370
380
390
400
410
420
AAATGGCTACACTTGAATTAAAGTCTTCAGTGGCGATTGAAGATAAAGAGATCTCAAAGG
370
380
390
400
410
420
AAAAAACGCATACATAGATTTAGGGAGGGAACGTATCACTAGTAAAATAAAATCATTGAC
MTSKIKSLT
430
440
450
460
470
480
AGTCAACCTAGTCATCAATACCAATGAAATTCATGCAATTATGGGACCTAACGGAACTGG
430
y
490
500
510
520
530
560
570
580
590
610
620
630
640
650
670
680
690
460
470
480
L
F
I
V
G
I
500
E
I
I
510
G
G
L
520
S
G
F
F
530
A
540
I
I
K
E
I
550
y
N
N
560
L
I
L
P
P
L
A
P
P
D
y
570
TTTATTTGGAATTGTTTGGGGGGATCCTCTA
L
AAATGAGAGCATTMTGT1"rCTACMCGCTTTTTAGCMCMTMTTGAT1"rMTCATTG
M
R
A
L
M
F
L
Q
R
F
L
A
T
I
I
D
L
490
600
GTTCTTTTTGATGGTCAAAATGTTTTAGAGATGGAAGTTGATGAAAGAGCTGGAGGGAGA
450
GGGAATTATCAAGGAAATTTATAATAATCTCATACTTCCGCCGCTGGCTCCGCCAGATTA
G
550
L
540
TAAATCGACTTTATCGGCAGCGATTATGGGGAACCTAATTATACCGTCACTGAGGGAGAA
440
ATATCTTCTATTATTTATTGTGGGAATCGAAATTATTGGTGGCTTATCTGGTTTCTTTGC
F
G
I
V
W "g"""!1,PL
660
L
I
I
BL13
D
10
20
30
40
50
60
GGTTAAAGGGGTTTTTGGCMCTAAAAGTAAAAAAACACTGAAAATATGATAAAATATGA
700
T1"rAT1"rACCAGTT1"rACTTCTTGTTCAGGGGGATCCTCTA
VYLPVLLLVQGDPL
«««,«««««««««,"
70
T AAAA
BL1
B
10
20
30
4()
50
80
TT AA GAA G AAA
190
I
7()
80
TT C TTTTTG
A TTT
90
C G TGAA
TTTTTT
100
110
C A TT AA TTTTG
100
AAG A C AAAAA
110
G AA C TT AAAGM
120
T AAAGA
TAG
130
140
150
160
170
180
AAATCGAGAAGGAAAATCAAGCGTTACGTCACAGGTTTTAATGGATTACGGACTATTGGA
MKRYVTGFNGLRTIG
-60
CTAAAGAACTTCTAAAGATGAACTAAAACAAAATACAAAAAAATAAGAAAGAGCCTATTG
G C TTT
90
G C G TAG T A G TTTT
~120
200
210
220
GTTTTAACGGTTATTCTTTATCACCTCTGGGGGGATCCTCTA
CT A T AA TT AA TC TT AATGA
V
L
T
V
I
L
y
H
L
W ,9,0,!!,0,~~
M
a
a1
130
ACTT
N
CTTT
F
140
AAAAA
F
K
C ACGA
N
T
190
150
160
T AA TTTTT1-rAAAAACAA
I
a2
I
F
200
L
K
T
170
T AAAAA
C GAAA
I
T
210
K
K
220
180
C CG CTT GA TTTTG
p
L
D
230
F
AL39 E
10
20
30
40
50
60
TTGCTACCGCCAGCGGGTTCTAATCGGCAACTATGCCAAAGTGCTCGGCCTGGGCAAAAC
240
TTATCGTTTTCGTGTTATTTCTTGCTTCCTTTTCAACGATATTCCTTTTCGCCACTGGTT
V
I
V
F
250
V
L
F
L
260
A
S
F
S
T
I
F
L
F
A
T
G
70
80
90
100
110
120
TATCTCTTCACCTGGTTGCGTGATAACGGAATTCGAGGCTCGCCCCCATATACTCACGTT
270
CTACTGGAGCCAAAGCCGAGGGGGATCCTCTA
STGAKAEGDPL
ili:ili;;::::1ii*t:::\::\:)\t;:
130
140
150
160
170
180
TAGGAAATCCAACCAACGATGTTTTTGAAGCACGCATCGCAGCTCTTGAAGGTGGGAGTG
MFLKHASQLLKVGV
190
200
210
220
CAGCGCTTGGTGTTGGTTCTGGCTGTGCGGGGGGATCCTCTA
Q p L V L V L A L R "g»»»~»w»r",,"~,,"
Fig. 3A-E. Nucleotide sequenceand deduced amino acid sequence
of fragments AL9 (A); BL1 (B); BLl1 (C); BL13 (D); and AL39
(E). Putative promoter sequencesare underlined ( -35 regions) or
indicated with interrupted lines ( -10 regions); Shine-Dalgarno
sequences are indicated with dots underneath the nucleotides.
Heavy arrows show the transcription start sites in AL9 (position
334) and BLl (position 116). An inverted repeat in BLl is indicated
by overlining arrows. Amino acids encoded by the multiple cloning
site are given in boldface (stippling)
408
Fig. 4. Hydrophobicity profiles of peptides encoded by fragments BLl, BLll, BLl3, AL9, and AL39. Hydrophobicities
were predicted according to Kyte and Doolittle (l982).
Hydrophobic and hydrophilic regions are indicated below
and above the dotted lines, respectively. Vertical arrows indicate potential cleavage sites for SPase I (according to von
Heijne 1986; heavy arrows indicate the most likely sites).
Charged amino acids are indicated by + (arg and lys), or (glu and asp)
These show the tripartite structure typical of natural
signal peptides(von Heijne and Abrahmsén 1989): (1), a
short n-region containing two or three positive1ycharged
amino acid residues; (2), an h-region varying from 14
(BL13) to 24 amino acids (BL11); and (3), potential
SPaseI cleavage sites C-terminally from the h-region,
usually at 4 to 8 amino acids distance (von Heijne 1986).
AL9- and AL39-encoded peptides lack potential SPase
I cleavagesjtes.This does not representa problem, however, since such sites are present in the amino acids
specifiedby the multiple cloning site at a short distance
from the cloned fragments. Three export elements,AL9,
BLI and BL11, appearedto contain a negatively charged
amino acid residue in or near the h-region.
Discussion
In previous work (Smith et al. 1987, 1988, 1989) the
selection of a great variet y of B. subtilis DNA fragments
that directed the export of the reporter proteins a-amylase (fiom B. licheniformis) and TEM-/3-1actamase (from
E. coli) was described. In the present work we extended
this approach to L. lactis in order to study properties of
export signal functions frorn this organisrn and to exploit
its potential for the production of extracellular (heterologous) proteins. The initial selection of L. lactis export
elernents was carried out in E. coli. Although a possible
lirnitation of this procedure is that sorne functional export elernents for L. lactis and B. subtilis rnay not be
active in E. coli and, therefore, rnay not be selected,
several functional export elernents for the two Grarnpositive bacteria were obtained.
Several of the protein export elernents selected frorn
B. subtilis (Srnith et al. 1987, 1988, 1989) appeared to
contain highly efficient expression/secretion functions for
(heterologous) proteins. In addition, this set of export
elernents proved to be extrernely valuable in the cloning
of the B. subtilis SPase 1 gene and studies on the properties of its product (J.M. van Dijl, G. Venerna, S. Bron;
to be published). In the present study a great variet y of
protein export elernents was sirnilarly selected frorn
L. lactis. The large nurnber of different export elernents
obtained could weIl rnean that functions other than natural signal peptides, such as rnernbrane insertion signals
409
of integral membrane proteins, have also been selected.
Such functions could be active as export elementsin the
present study since, particularly in assaysinvolving the
/3-lactamasereporter protein, the presenceof an SPaseI
processing site was not required. Several of the export
elements selectedin E. coli (AL9 and most of the BL
elements)appearedto be active in B. subtilis and L. lactis.
In addition, the B. subtilis export function A2 (Smith et
al. 1987, 1988) was also efficient in L. lactis (data not
shown). These results indicate that the protein export
machinenes of E. coli, B. subtilis and L. lactis are sufficiently similar to enable the export of at least a number
of the hybnd proteins in an active form. This conclusion
is supported by the observation that in L. lactis the
hybrid pre(AL9)-a-amylase precursor was processedin
vivo to a mature product of the expectedsize. This was
demonstrated by pulse-labelling and subsequent immunoprecipitation with specific antibodies (results not
shown). Another indication that the export machineries
of L. lactis, B. subtilis, and E. coli share similanties was
also obtained by Koivula et al. (1991b). These authors
demonstrated that L. lactis contains a genehomologous
to the secy gelles of B. subtilis and E. coli. The E. coli
SecY protein forms part of the protein translocase
(Wickner et al. 1991).
Most of the selectedlactococcal export elementshad
the typical tripartite structure of natural signal peptides
(von Heijne and Abrahmsén 1989).However, in someof
the hybrid export proteins the most likely processingsite
for SPaseI was present in the amino acids specified by
the multiple cloning site. All peptides encoded by the
sequencedfragments were different from known natural
lactococcal signal peptides and also from the export
elements selected by Sibakov et al. (1991). Whereas
about half of the export elements selectedby the latter
authors contain potential lipoprotein signal peptidase
cleavage sites, such sites were not found in the export
elementsstudied in the present work. The length of the
h-regions (12 to 24 amino acids) of the selectedexport
signals was relatively large. A similar conclusion can be
drawn from the work of Sibakov et al. (1991). These
observationsare in accordancewith the view (von Heijne
and Abrahmsén 1989) that h-regions in signal peptides
from Gram-positive bactena are generally larger than
those from E. coli (average 12 amino acids). A negative
chargewas presentin or near the h-region of three of the
export signals. Whether this is related to signal peptide
function in lactococci is not known. Negative chargesat
these positions are rare in signal peptides from other
bacteria that have been analysed so far .
The present results indicated that, in addition to the
selectedexport elements,at least two other factors affected the production of exported proteins, namely the nature of the mature part of the reporter proteins and the
host bacterium used. Effects of the mature part of a
protein on its export have also been reported for E. coli
(Lehnhardt et al. 1988; Park et al. 1988), and were also
observedwith export elementsfrom B. subtilis (Smith et
al. 1989; van Dijl et al. 1991a).Severalexplanations for
these observations can be entertained. First, the signal
peptide is involved in maintaining an export-competent
state of the precursor (Park et al. 1988; Saier et al. 1989 ;
Wickner et al. 1991). In that respect, different signal
peptides may have different efficiencies. Second, the signal peptide and the mature part have to interact correctly
with the components of the cellular export machinery
(Cunningham and Wickner 1989; Lill et al. 1990; Wickner et al. 1991). Third, in several of the hybrid proteins
the likely SPase I cleavage site was located in the amino
acids specified by the multiple cloning sites, which are
different in pGA14 and pGBI4.
At least three factors could be involved in the
phenomenon of host-specific efficiency of the selected
DNA fragments. The first is a species-specific requirement for transcription/translation
initiation
signals.
L. lactis promoters generally have the same consensus
sequence as that used by the major RNA polymerase of
E. coli and B. subtilis (de Vos 1987; Koivula et al. 1991a ;
van de Guchte et al. 1992; van der Vossen et al. 1987).
Several putative promoter sequences from the present
work had, however, only a limited similarity with
previously described lactococcal promoters. Since, in
general, E. coli exhibits less stringent requirements for
promoter specificity than B. subtilis (Hager and Rabinowitz 1985) and L. lactis (de Vos 1987; van de Guchte
et al. 1992; van der Vossen et al. 1987), differences in
transcription efficiencyare likely to occur in the three
hosts. Of special interest are the promoters on AL9 and
BL1, which have low activity in E. coli but are efficient
in L. lactis. These fragments appeared to be extremely
A/T-rich in the sequence upstream ofthe -10 promoter
box, a property commonly observed with promoters
from Gram-positive bacteria (Moran et al. 1982; van der
Vossen et al. 1987). Fragment BLl showed two other
properties that can explain its host dependence. First, it
contains a region potentially capable of bending, and
second, part of its putative promoter is contained within
an inverted repeat sequence. Both regions could be under
regulatory control of functions specific for L. lactis.
Appropriate translation initiation signals could be detected in fragments AL9, BL11, BL13, and AL39. BLl
was an exception: no SD sequence could be found and
transcription appeared to start one nucleotide upstream
ofthe AUG codon fused in frame with the reporter gene.
Although examples offlanking transcription and translation start sites in the absence of SD sequences are known
for the Gram-positive bacterium Streptomyces (Horinouchi et al. 1987; Lopez-Cabrera et al. 1989), this
phenomenon has not yet been reported for L. lactis.
The second factor possibly, involved in the observed
host-specific effects is a difference in export efficiency. As
already discussed, the h-region and the c-region (containing the SPase I cleavage site), are in general shorter in
E. coli than in the Gram-positive bacteria (von Heijne
and Abrahmsén 1989). Moreover, we have recently
shown (J.M. van Dijl, G. Venema, S. Bron to be published) that the B. subtilis SPase I is rather different from its
E. coli counterpart. This and other host-specific properties of the host export machinery are likely to affect
host-specific export efficiencies.
The third possible factor determining host-specific
export efficiency is a difference in the stabilities of the
410
exported products. With regard to this factor, it is noteworthy that proteolytic degradation of exported
heterologousproteins, such as TEM-~-lactamase (Smith
et al. 1987), is a serious problem in B. subtilis. In the
present experiments, proteolytic degradation has not
played an important role, however, since exponentially
growing cells were used to avoid this problem. At
present, little is known about product stabilities in
L. lactis. Our data (not shown) and those presentedby
Sibakov et al. (1991) suggestthat TEM-~-lactamase is
relatively stabIe in this organism.
In summary, we have shown that a great variety of
protein export elementscan be selectedfrom the L. lactis
chromosome.Thesemay have useful applications and be
of value in fundamental studies. Two of the export elements (AL9 and BLI) are ofparticular interest, as these
were highly efficient in directing the export of at least two
heterologous proteins in L. lactis. The export element
AL9 fused to a-amylase is an example of a potential
application: the large amounts of secreted a-amylase
causeda change in the growth requirements of this fastidious bacterium so that it can grow on starch as the
sole, cheap, carbon source.
Acknowledgements. This work was partially financed by a Senior
Scientist Training Contract from the European Commission to
G.P.M. We thank Anne de Jong for skilful technical assistanceand
Henk Mulder for preparing the photographs.
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